Antifreeze Proteins Enhance Survival of Cells in Cryopreservation - Substituting DMSO With RmAFP#1 in Cryopreservation of Cells 1.2

Antifreeze Proteins Enhance Survival of
Cells in Cryopreservation
Substituting DMSO with RmAFP#1 in cryopreservation of cells
Picture on front page: Rat ovarian tissue and the difference in intact follicles when adding an AFP from cold ocean Teleost
fishes. On the left side is rat ovarian tissue after vitrification and warming (upper picture), and after vitrification, warming
and transplantation (lower picture). On the right side is rat ovarian tissue after vitrification and warming (upper picture),
and after vitrification, warming and transplantation (lower picture). The difference in between the two sides is that on the
right side 20mg/mL type III AFP is during the vitrification. It was concluded based on these pictures that the AFP from cold
ocean Teleost fishes increased the amount of intact follicles. Picture is modified from (Lee et al., 2015).
Resum
In some situations, it is necessary to preserve living cells and biological tissues for a period of time,
preferably without losing living cells or the function of the tissue. This can be done by
cryopreservation, which is any method where the storage temperature of the cells or tissues is at or
below 0oC. Preserving cells or biological tissues is, among other, used in assisted reproduction in
humans, in animal breeding programs and in preserving endangered species of animals and plants.
Simply freezing cells or tissues without any protection would result in loss of living cells and a high
loss of function in the tissue. This is due to the high concentration of water (~80%) in cells and
tissues. When cells or tissues are cooled to or below 0oC the cooling of water leads to ice crystal
formation, which is damaging to cells and tissues. Damage caused by freezing of cells and tissues is
called freezing damage, and can result from several different factors. Two factors dominate the
mechanisms leading to freezing damage; mechanical damage due to the formation of ice crystals or
recrystallization during thawing and changes in the intra- and extracellular osmolarity. To avoid
freezing damage a number of variables, such as the cooling rate, heating rate and intracellular
osmolarity should be taken into consideration.
The cooling rate is the rate (oC/min) at which the specimen is cooled to the desired final
temperature. A slow cooling rate leads to an increase in extracellular osmolarity, since ice crystals
are forming slowly and pushing solutes in front of the surface of the growing ice crystal. The solute
concentration in the remaining non-frozen liquid outside the cell increase and this leads to an efflux
of water from the cell. If too much water leaves the cell, it will result in lethal dehydration. A fast
cooling rate leads to a quicker extra- and intracellular formation of ice crystals and a lower efflux
time, which hinders lethal dehydration. However, this increase in intracellular water leads to a high
degree of intracellular ice formation (IIF), which leads to loss of living cells.
To increase the survival rate of cells cryoprotective agents (CPAs) are often added to the
cryopreservation medium. CPAs can be divided in two groups; the non-permeating CPAs and the
permeating CPAs. Non-permeating CPAs are big molecules, often sugars or even bigger molecules.
Permeating CPAs are small, aprotic molecules that are able to penetrate the cell membrane.
Science Bachelor Project
Roskilde University
Spring semester 2015
Dimethyl sulfoxide (DMSO) is a permeating CPA, and widely used in the biological science fields.
This aprotic and amphiphilic molecule can easily penetrate the cell membrane and enter the interior
of the cell, where main mechanism of action takes place. DMSO readily forms hydrogen bonds and
as some water molecules bind to DMSO other water molecules becomes excessive. These excessive
water molecules flow out of the cell, thus the internal osmolarity is increased and the degree of IIF
is decreased. This leads to a higher cell survival rate. However, DMSO is cytotoxic to most cells. Its
cytotoxicity depends on concentration (optimal between 1-15%), temperature (as cool as possible)
and the time the cells are exposed to DMSO. This is why DMSO is usually added just before
cryopreservation, where the temperature of the specimen is low, or is lowered right after adding
DMSO.
Organisms living in sub-zero environments has to protect themselves against freezing damage.
Organisms are divided into two groups, endotherms and ectotherms, depending on how they create
their body temperature. Mammals, including humans and birds are endotherms. They produce their
own body heat through metabolic processes in the body, and maintain the temperature say, by
having fur, feathers or wearing clothes. Ectotherms, on the other hand, cannot produce their own
body heat through their metabolism, so their body temperature is depending on the temperature of
its surroundings. Reptiles, amphibians and most fishes and insects are ectothermic.
This means, that ectothermic organisms living in sub-zero environments are at great risk of
freezing. Such organisms can be either freeze-avoiding or freeze-tolerant. Some of these organisms
express certain proteins that help them to survive at low temperatures. These proteins are antifreeze
proteins (AFPs) which are proteins, that hinder either nucleation or recrystallization, or both. AFPs
hinder the recrystallization and/or nucleation in a non-colligative manner; they lower the freezing
point but not the melting point of the liquid. The gap AFPs cause in between the melting point and
freezing point is called the Thermal Hysteresis (TH), and the activity of AFPs is usually deemed
based on its maximal TH.
There are several types of AFPs (I-IV) but they appear to act in the same way, hindering
recrystallization and/or nucleation through the same mechanism, although there are multiple
theories on how they do so. The most accepted theory is the adsorption-inhibition model. This
theory suggests that AFPs bind to the surface of the ice crystal in such a way so that the growth is
restricted to be in between two AFP molecules. This increases the curvature of the surface of the ice
crystal. This stabilizes the ice crystal as, according to the Kelvin effect, an increase in surface
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curvature leads to a decrease in vapour pressure, which is the main reason for the expansion of ice
crystals. This hinders recrystallization, and because the surface of the ice crystal is changed,
nucleation is also hindered.
It is clear to see, that AFP from fishes and AFP from insects work in different ways, when looking
at the shape of the crystals they have inhibited. Ice crystals inhibited by fish AFP create hexagonal
bipyramids when the temperature is in its TH gap. Below the TH gap the ice crystal expands
quickly through the tips of the bipyramid and creates spicules. Insect and other hyperactive AFPs
create hexagonal ice crystals when the temperature is in its TH gap. Below the TH gap the ice
crystal expands quickly and forms a hexagonal bipyramid.
The AFP used in this project comes from the beetle Rhagium mordax. This beetle produces several
isoforms of its hyperactive AFP. The different kinds are all called RmAFP#1-8, and it is the
RmAFP#1 which is used in the project.
The aim of this project is to investigate whether partially substituting DMSO with RmAFP#1 could
lead to an increase in cell viability. The hypothesis is that the collected stress caused by the intraand extracellular recrystallization as well as the IIF reaches a stress threshold, which the cells are
killed by. Stress includes mechanical stress by ice formation, long or short water efflux times due to
changes in the intra- and extracellular osmolarity, and pressure changes around and in the cells
caused by recrystallization.
This study shows that adding RmAFP#1 to cryopreservation media increases cell viability. For a
heating rate of ~17oC/min, adding RmAFP#1 to a cryopreservation medium with 10% DMSO
results in a 1.5-7 fold increase in amount of intact living cells, a 2.5-9 fold increase in the total
amount of intact cells, and a 2-9 fold increase in the survival rate, compared to a cryopreservation
medium which has 10% DMSO and no RmAFP#1. Furthermore, it is shown, that there is a lower
limit (0.69-1.74 mg/mL) and an upper limit (1.74-2.08 mg/mL) for the beneficial concentration of
RmAFP#1.
For a heating rate of ~10oC/min Adding 2.08 mg/mL RmAFP#1 to a cryopreservation medium with
7.5% DMSO leads to the same total amount of intact cells, and the same amount of living cells, as a
cryopreservation medium with 10% DMSO and no RmAFP#1, but it leads to a 2 fold increase in
survival rate. Furthermore, there is no great difference in the survival rate of the sample having 10%
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DMSO, the sample with 7.5% DMSO and 2.08 mg/mL RmAFP#1 and the sample having 2.08
mg/mL RmAFP#1 and no DMSO.
Based on this, it is concluded to be beneficial adding RmAFP#1 to a cryopreservation medium. This
is likely due to the hindrance of recrystallization during the thawing.
Abstract
Cryopreservation is a useful method for preserving living cells and biological tissues. Dimethyl
sulfoxide (DMSO) is considered the most effective cryoprotective agent (CPA) used in
cryopreservation. DMSO helps to reduce ice crystallization within the cell and thus preventing cell
death during the freezing and thawing process.
However, DMSO has toxic effects on cells which are not only concentration dependent, but also
temperature dependent. In this study, DMSO was substituted with an insect antifreeze protein
(RmAFP#1) in various amounts, in order to investigate if a media with both DMSO and RmAFP#1
in a certain ratio could increase cell viability. The main function of antifreeze proteins is to bind to
small ice crystals and inhibit their growth. Adding RmAFP#1 to the cryopreservation media leads to
a 1.5-9 fold increase in the total amount of intact cells, amount of living cells and survival rate. This
increase depends on the heating rate and the concentration of DMSO and RmAFP#1 in the media.
Adding RmAFP#1 to cryopreservation media proves to be beneficial for the cryopreservation.
Furthermore, samples with 7.5% DMSO and 2.08 mg/mL RmAFP#1 had the same total amount of
cells, amount of intact cells and cell survival rate, as conventional cryopreservation media with 10%
DMSO and no RmAFP#1, indicating that DMSO can be substituted with RmAFP#1 to some degree.
Acknowledgements
We would like to thank Hans Ramlv for guidance and for the use of lab as well as equipment.
Dennis Friis for guidance and help in the lab. Anne Lise Maarup for help in the lab and guidance in
lab procedures. And Marianne Lauridsen for lending us equipment as well as giving us the A6 cell
culture needed in our experiment.
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Table of contents
Preface .................................................................................................................................................. 7
Hypothesis ............................................................................................................................................ 7
Problem Formulation ....................................................................................................................... 7
Definitions for selected words ............................................................................................................. 8
Osmolality or osmolarity ................................................................................................................. 8
Antifreeze proteins or Ice structuring proteins ................................................................................ 8
Cryopreservation .............................................................................................................................. 8
Nomenclature list and abbreviations .................................................................................................... 8
Delimitation ....................................................................................................................................... 10
Transformation of strain................................................................................................................. 10
Cell culture ..................................................................................................................................... 10
Counting method ............................................................................................................................ 10
Bacterial contamination testing ...................................................................................................... 10
Freezing of samples ....................................................................................................................... 10
Introduction ........................................................................................................................................ 11
Cryopreservation ............................................................................................................................ 11
Cryoprotective agents .................................................................................................................... 13
A6 Cell line .................................................................................................................................... 16
Ice crystallization and nucleation ................................................................................................... 17
Antifreeze Proteins ......................................................................................................................... 19
Ice crystallization and AFPs ..................................................................................................................... 21
RmAFP#1.................................................................................................................................................. 25
Theoretic summary and elaboration on the hypothesis .................................................................. 26

Materials and methods ....................................................................................................................... 29
E. coli strain ................................................................................................................................... 29
Production and Purification of RmAFP#1 ..................................................................................... 30
Experimental methods for the Cryopreservation experiments ....................................................... 30
Part 1: Nucleation test ............................................................................................................................. 30
Part 2: Heating rate ................................................................................................................................. 31
Part 3: Maximal amount of RmAFP#1 ..................................................................................................... 32
Part 4: DMSO variation ............................................................................................................................ 33
Part 5: RmAFP#1 concentration and DMSO concentration ..................................................................... 33
Cell viability determination ........................................................................................................... 34

T-test .............................................................................................................................................. 35
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Cryopreservation: experimental outline for RmAFP#1 and DMSO experiments.......................... 36

Results ................................................................................................................................................ 37
Production and purification of RmAFP#1 ...................................................................................... 37
Nucleation experiment ................................................................................................................... 37
Heat rate experiment ...................................................................................................................... 39
Maximal RmAFP#1........................................................................................................................ 39
Varying DMSO constant RmAFP#1 .............................................................................................. 41
Variation of DMSO and RmAFP#1 ............................................................................................... 42
Discussion .......................................................................................................................................... 44
AFP production and purification.................................................................................................... 44
Nucleation experiment ................................................................................................................... 44
Heat rate experiment ...................................................................................................................... 45
Cell data reliability ......................................................................................................................... 46
Maximal concentration of RmAFP#1 experiment ......................................................................... 46
Total amount of intact cells ..................................................................................................................... 47
Amount of living cells............................................................................................................................... 49
Cell survival rate ...................................................................................................................................... 49
Illustration of P-values ............................................................................................................................. 50
DMSO variation experiment constant RmAFP#1 .......................................................................... 51

DMSO and RmAFP#1 variation experiment ................................................................................. 54

Amount of living cells............................................................................................................................... 54
Sample 0 vs. sample 1.............................................................................................................................. 54
Cell survival rates ..................................................................................................................................... 55
Conclusion ......................................................................................................................................... 57
Overall conclusion.................................................................................................................................... 58
Perspective ......................................................................................................................................... 59
References .......................................................................................................................................... 60
Appendices ......................................................................................................................................... 65
Appendix #1: Calculations protein purification ............................................................................. 65
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Appendix #2: Solutions and standard procedures .......................................................................... 67

Appendix #3: Detailed lab protocol ............................................................................................... 69
Protein purification .................................................................................................................................. 69
Experiment days ...................................................................................................................................... 72
Appendix #4: Data from experiments ............................................................................................ 76

Data from maximal RmAFP#1 experiment .............................................................................................. 76
Data from variation of DMSO, constant RmAFP#1 experiment .............................................................. 78
Data from DMSO + RmAFP#1 variation experiment ............................................................................... 83
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Preface
Cryopreservation is a method of preserving biological tissues and cells at sub-zero temperatures. A
cryoprotective agent (CPA) is a substance which is used to protect biological tissue from damage
during cryopreservation. One kind of CPA is dimethyl sulfoxide (DMSO) which is added to the
cryopreservation media primarily to reduce intracellular ice formation (IIF). Thereby it prevents cell
death during the freezing and thawing processes of cryopreservation. Prolonged exposure to DMSO
is toxic for cells and might be the reason why cell viability is low or varying in an unpredictable
manner.
To improve the process of cryopreservation, it is beneficial to look into how organisms in nature are
overcoming the challenge of sub-zero temperatures. Ectothermic organisms in sub-zero
environments can either be freeze tolerant or freeze avoiding. Freeze avoiding organisms use
different mechanisms to avoid freezing altogether, where freeze tolerant organisms can survive their
body fluids freezing. Both kinds can use antifreeze proteins (AFPs) as a CPA, to prevent freezing
damage. AFPs are a group of diverse proteins expressed in fish, insects, plants, fungi and bacteria.
The main function of AFP is to bind to ice crystals and inhibit their growth.
RmAFP#1 is an AFP from the beetle Rhagium mordax, the black-spotted pliers support beetle. It is
used in this project to test whether it can enhance viability of cells during cryopreservation, when it
is added to cryopreservation media together with DMSO in different ratios.
Hypothesis
Partially substituting DMSO with RmAFP#1 will result in an increase in cell viability.
Problem Formulation
By substituting DMSO with RmAFP#1, in various amounts, it might be possible to find a
DMSO/RmAFP#1 ratio that could increase the viability of A6 cells. The cell line is derived from
kidney cells from the frog Xenopus laevis (X. laevis). Even though antifreeze proteins cannot enter
the cell, the amount of recrystallization around the cell is lowered with antifreeze proteins, thus
increasing the chance of A6 cell survival when the cryopreservation is finished.
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Definitions for selected words

Osmolality or osmolarity
Using osmolality (mol/kg) or osmolarity (mol/L) to define the osmotic concentration is a widely
discussed issue within the scientific community. To avoid any misunderstandings this project will
use the word osmolality for any concentration that is measured by an osmometer, and osmolarity for
any other concentration. The osmotic gap will be considered in situations where the concentration is
given in osmolality and needs to be converted into osmolarity.
Antifreeze proteins or Ice structuring proteins

Clarke et al proposed in 2002 that the name antifreeze protein was changed to ice structuring
protein, due to the fact that the proteins do not prevent freezing, but inhibits growth of ice crystals
by re-structuring ice crystals (Clarke et al., 2002). The name ice binding proteins was suggested
by Davies (2014) since the protein, based on the newest research, does not directly re-structure the
ice crystal. Instead the restructuring happens because of the thermodynamically changes that
happen, when the protein bound to the ice crystal (Davies, 2014). Since the largest part of the
references used in this project refers to the proteins as antifreeze proteins this word is used
throughout the report.
Cryopreservation
The word cryopreservation describes every type of method where cells, or tissue, are preserved
for longer or shorter periods of time at sub-zero temperatures, where sub-zero temperatures refer to
temperatures at or below 0oC. Since some methods for preserving tissues are not below 0oC the
word cryopreservation is used for every method where cells, or tissue, are preserved at
temperatures below the freezing temperature of the cell or tissue, which most often is 0oC or below.
Nomenclature list and abbreviations

Aprotic molecule: a molecule with neither basic nor acid properties.
Cooling rate: The rate (oC/min) at which cell solutions are cooled to the desired storage
temperature.
Freezing damage: Damage which is caused by the natural effects occurring when cells are cooled to
or below 0oC.
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Glycoside: a compound formed from a simple sugar and another compound by replacement of a
hydroxyl group in the sugar molecule.
IIF: Intracellular Ice Formation is the formation of ice within the cell during the cooling stage of a
cryopreservation. IIF is considered to be deadly to cells and tissue since the ice can damage the cells
leading to unwanted apoptosis and loss-of-function.
Melting point: the temperature at which the last small ice crystal present in the liquid disappears
(melts).
Normothermia: the normal living temperature of the cells and tissue.
PenStrep: PenStrep is a mixture of Penicillin and Streptomycin and is used in cryopreservation of
cells to hinder contamination by bacteria.
RCSB.org files; these files are throughout this project indicated by a parenthesis, the four character
identification number, and an end parenthesis, for example (2Y1J). It will not be mentioned that
these are .gb files from RCSB.org in the text.
Sample/replica: when the word sample is used, it is to refer to the different test samples, with
varying amounts of RmAFP#1, DMSO, or both. When the word replica is used it is one, or more,
of the replicas within the same sample. There are 6 replicas for each sample, unless anything else is
stated.
Solution effects: effects that can cause cell injury as a result of the concentration of solutes.
Vitrification; a process by which a liquid solidifies without its components (the molecules) forming
a crystal. That means, the molecules are randomly distributed and as no pattern can be recognised
the substance is said to be amorph. The higher the viscosity and the faster the cooling rate the more
likely is the liquid to vitrify.
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Delimitation
Transformation of strain
Due to the time limit and because a usable strain was at hand in the lab freezer, we did not make our
own RmAFP#1 producing strain. The strain used was made in 2011 and has been logged in the lab
strain list by Dennis S. Friis.
Cell culture
It was not possible to make our own A6 cell culture, due to time limitations. The cells were
provided by lab. technician Marianne Lauritzen from Associate professor H. Bjerregrds laboratory.
Counting method
A Burker-Turk counting chamber was used to count the cells, after Trypane staining, since it was
possible to take pictures of the cell samples and count them later, ensuring that the time in between
the samples was as low as possible. Furthermore, this method was used in a previous study to assess
cell survival of A6 cells, and was proven to be useful. It might have been proven easier and quicker
to use a Coulter Counter, and a more objective method, but this was not possible.
Bacterial contamination testing

A small contamination test was made, but since the medium had twice the amount of PenStrep in it
than used in the final cryopreservation media, this test was not successful. This test was excluded
since there were no time to do another testing of the contamination possibilities and since the cells
were not cultured after the preservation.
Freezing of samples
The samples were in standard 250L Eppendorf tubes and not in cryopreservation tubes, since it
was not possible to get a hold on enough cryopreservation tubes before the start of the experiment.
An Eppendorf tube can tolerate temperatures down to -80oC so the temperature was not a problem.
But the well in the Mr. Frosty used has the radius of a cryopreservation tube, and since the radius is
bigger than that of a 250L Eppendorf tube. The size difference was assumed to be irrelevant since
the temperature within the Mr. Frosty wells are the same. The lack of direct contact was estimated
to be such a small factor in the cryopreservation method that it was not considered further.
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Introduction
Cryopreservation
Cryopreservation is a method used to preserve living cells and biological tissues at sub-zero
temperatures, with the aim to keep cells structurally intact and viable. This method can be defined
as the storage of cells and tissues at temperatures at or below 0 C. Nitrogen vapour (-130C) and
liquid nitrogen (-196C) is often used in cryopreservation, and at the temperature of the latter, all
enzymatic and metabolic activity is suspended (Bakhach, 2009). At this temperature (-196 0C) it is
not long term storage that damages the cells, but progression to this temperature and back to
normothermia (Higgins, 2008;Mullen and Critser, 2007). Therefore, it is vital to understand the
intracellular and extracellular processes during cooling and thawing (Higgins, 2008).
Simply freezing cells or tissues without protection is normally lethal due to freezing damage. Cells
and tissues consist of around 80 % water, and when cooled below 0 C, the freezing of water
dominates the biological effects (Pegg, 2007). Several things might cause the freezing damage:
Mechanical damage due to ice crystal formation and the change in composition of both the intraand extracellular liquid during freezing due to osmotic flow. Extracellular ice formation can result
in the dehydration of the cells. When the extracellular liquid freezes and ice crystals start to form,
the solutes of the liquid are displaced by the growing ice crystals (Karlsson and Toner, 1996). This
causes the concentration of solutes in the remaining, non-frozen extracellular liquid to increase,
which causes the osmotic efflux of water from the cell (Higgins, 2008). This osmotic efflux is
dependent on the rate of ice formation. When the cooling rate is decreased the rate of ice formation
is also decreased, thus the amount of water leaving the cells increase (Bakhach, 2009). It is
considered to be the most crucial parameter of cryopreservation (Rubinsky, 2003).
Mazur et al. (1972) suggested a two-factor hypothesis of cryoinjury in their study, theorizing that
the cell damage occurs from solution effects at slow cooling rates, whereas cell damage at fast
cooling rates results from intracellular ice formation (IIF) (Mazur et al., 1972). With slow cooling
rates, cell dehydration will maintain chemical equilibrium between intra- and extracellular
solutions. During fast cooling rates, cell dehydration is too slow to maintain chemical equilibrium
and thus intracellular cooling occurs and this may also lead to lethal IIF (Baust et al., 2009;Mullen
and Critser, 2007). If the cooling rate is too slow the cells will become lethally dehydrated, but if
the cooling rate is too fast the risk of IIF increases. Both scenarios will lead to a lower survival rate,
and the mechanisms must be considered before cryopreserving tissues or cells (see Figure 1).
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Figure 1: When the temperature of a cell suspension is cooled below its equilibrium freezing point, extracellular ice formation
occurs. Due to extracellular ice formation water is forced out of the cell by an osmotic gradient across the cell membrane.
During slow cooling (cell A) plenty of water leaves the cell and IIF does not occur. Rapid cooling (cell B) results in limited cell
dehydration, larger ice crystals and less IIF. Very rapid cooling (cell C) results in no cell dehydration, small crystals and a
great amount of IIF. If the very rapidly cooled cells are slowly heated, recrystallization happens, where where smaller crystals
melt and larger crystals grow. Therefore, it is also critical to prevent IIF during cryopreservation (Mullen and Critser, 2007).
Picture modified from (Mullen and Critser, 2007).
Cryopreserving living cells or tissues is essential in a variety of areas, such as in assisted

reproduction where oocytes, semen and embryos can be cryopreserved. In human assisted
reproduction, cryopreservation of gametes or embryos is used when pregnancy has to be postponed,
either by choice or when the fertility is decreased. Ovarian cryopreservation and transplantation are
considered to be promising and beneficial options for female cancer survivors (Lee et al., 2015).
Cryopreservation is also used on gametes from animals and plants, in breeding and in conservation
of endangered species.
Cryobiological research studies have revealed that antifreeze proteins (AFPs) are effective
cryoprotective agents for oocytes, embryos, and spermatozoa (Lee et al., 2015). The mechanism of
action, structure and classification of AFPs will not be discussed in this part, for this see the
Antifreeze Proteins section. The reason why AFPs could be beneficial for cryopreservation is that
these proteins inhibit recrystallization (Knight et al., 1995;Wang, 2000) and maintain the super
cooled state of body fluids by preventing nucleation (see Ice crystallization and nucleation
section) (Gauthier et al., 2005;Knight and Devries, 1989;Raymond and DeVries, 1977;Wang,
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2000). Therefore it has been suggested these proteins can protect the plasma membrane of cells
(Beirao et al., 2012; Fletcher et al., 2001;Wang, 2000).
Cryoprotective agents
Cryoprotective agents (CPAs) are agents that protect biological tissues and living cells during
cryopreservation. CPAs are divided into two groups, permeating and non-permeating CPAs. The
permeating CPAs are small, aprotic molecules that are able to permeate the cell membrane.
Members of this group are dimethyl sulfoxide (DMSO), ethylene glycol and glycerol, although
some cells have aquaporins for the transport of glycerol (aquaglyceroporins) (Hara-Chikuma and
Verkman, 2006). The non-permeating CPAs are usually bigger molecules, compared to the
permeating CPAs. They are often sugars such as sucrose and trehalose, or even bigger molecules
like the polymeric hydroxyethyl starch or the non-polymeric antifreeze proteins. They contain one
or more polar moieties and thus do not easily penetrate the cell membrane, for most parts.
Dimethyl sulfoxide (DMSO) is widely used in biological sciences. Some of its applications are as a
solvent, drug and CPA. DMSO has the chemical structure (CH3)2SO, see Figure 2. The molecular
weight is 78.13 g/mol. The two non-polar methyl groups and the polar sulfoxide groups make
DMSO an amphiphilic molecule. Being both amphiphilic and aprotic makes it possible for DMSO
to enter the interior of the cell, one of the crucial properties of DMSO that makes it a highly
effective CPA.
Figure 2: The structure of Dimethyl Sulfoxide (DMSO). The

stick ball presentation of DMSO shows the configuration of the
CH3, S and O atoms in a single DMSO molecule. The figure is
made in the freeware Avogadro program, based on its molecular
formula.
One of the most important factors for maintaining cell viability in cryopreservation is the avoidance
of IIF. DMSOs readiness to form hydrogen bonds makes it suitable for replacing water inside the
cell. As some water molecules bind to DMSO other water molecules become excessive and tend to
flow out of the cell, until equilibrium is established. Another factor that causes water to flow out of
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the cell is the formation of extracellular ice crystals. The formation of extracellular crystals
increases the concentrations of the solutes of the extracellular fluid this leads to an osmotic out flux
of water from the inside of the cells which again leads to an increase in the intracellular osmolarity,
and thus the freezing point is decreased and IIF is postponed. This prolongs the time the efflux is
possible, therefore the amount of intracellular water is lowered leading to fewer and smaller ice
crystals. By a mechanism that still remains to be elucidated, DMSO makes the cell membrane more
permeable to water. It is suggested that DMSO forms pores in the cell membrane. The non-polar
parts of DMSO can bind to the lipids in the lipid bilayer of the cell membrane and thereby
separating them. The polar part then forms a pore through which water can flow. According to
Gurtvenko et al. (2007) this process is highly dynamic since the pores exist only in a few
picoseconds. However, it should be emphasized that the results are based on a computer simulation,
not on actual experiments (Gurtovenko and Anwar, 2007).
It is possible that DMSO also partly acts by inducing vitrification, that is, the formation of a glass
like structure within the cell. Vitrification is more prone to occur when the concentrations of solutes
are high or when the solution is viscous. The most important factor is the cooling rate since the
amount of vitrification increases with the cooling rate; a low cooling rate leads to a low amount of
vitrification and vice versa.
Despite the fact that DMSO is considered one of the safest permeating CPAs, it still has some
disadvantages. DMSO has a toxic effect on cells. The optimal concentration of DMSO ranges in
between 1-32% and depends on the type of cell to be preserved. In general the concentration should
be kept below 15%. The toxic effects of DMSO are not only concentration dependent but depend on
the temperature as well. The higher the temperature the more toxic is DMSO. The time of exposure
to DMSO is also important and should be kept as short as possible (Hubalek, 2003). Therefore,
addition of DMSO, when the temperature is close to 0oC, is standard procedure in many companies.
The measured outcome parameter to estimate toxicity is usually cell viability. However, this
parameter does not reveal anything about the underlying mechanisms, but it can reveal the overall
toxicity. These mechanisms, therefore, are still poorly understood. One of the mechanisms is
DMSOs interaction with the lipid bilayer. This interaction enhances viability when the
concentration is low. By higher concentrations, however, the interaction becomes detrimental to the
cell. The simulations, mentioned earlier, done by Gurtvenko et al. (2007) showed that by
concentration of 20 % the bilayer becomes so affected that they refer to it as a borderline bilayer.
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By concentrations of 40 % the bilayer becomes isotropic (an intermix of water, DMSO and lipids)
(Gurtovenko and Anwar, 2007).
DMSO is one of the most widely used permeating CPA. This due to the fact, that despite its toxicity
it is still less toxic than many other permeating CPAs. The toxicity of DMSO and glycerol is
comparable. For some organisms DMSO is less toxic than glycerol for others it is the other way
round (Hubalek, 2003).
Many of the non-permeating cryoprotectants, such as the polymeric ones, are acting by some of the
colligative properties; they increase the viscosity of the extracellular fluid, thus slowing down the
movement of water, or even trapping it, and thereby delaying the growth of ice crystals. This means
that the extracellular osmotic pressure also grows at a reduced rate and the osmotic stress on the cell
is diminished. These cryoprotectants are seldom used alone but often in combination with a
permeating CPA. They are most often used with high cooling rates in order to obtain vitrification.
The combination of trapping water and a high cooling rate effectively hinders ice crystallization.
The water molecules do not have the possibility to arrange themselves in the crystalline form and
thus the solution solidifies in an amorphous state (Mullen and Fahy, 2011).
Antifreeze proteins (AFPs) may be used as a kind of non-permeating, or permeating,
cryoprotectants depending on which kind is used. Their main purpose would be to inhibit uneven
heterogenous nucleation during cooling, and to inhibit recrystallization during thawing. AFPs
cannot, in most cases be used as the only CPA, since most kinds of AFPs will not inhibit IIF.
Lee et al. (2015) revealed in their study that using AFP, from cold ocean Teleost fishes, as a CPAsupplementation improved the survival of ovarian tissue after cryopreservation and subsequent
transplantation. Furthermore supplementation with AFPs, have also shown favourable effects in
bovine, ram and chimpanzee spermatozoa as well as mouse oocytes (Lee et al., 2015; Prathalingam
et al., 2006;Younis et al., 1998). However, studies have shown that high concentrations of AFP
have a toxic effect on cells and tissues. For example, Carpenter et al.(1992) showed that low
concentrations (5-150 g/mL) of AFP, from winter flounders, enhanced the survival rate of red
blood cells, and that high concentrations (1.54 mg/mL) were associated with a reduction in the
survival rate (Carpenter and Hansen, 1992). Tomczak et al. (2001) discovered that the cytotoxicity
of an AFP depends on which kind it is. This was accomplished by comparing the instability of
thylakoid membranes after subjecting them to two types of AFP and an AFGP from fish. They also
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showed that the cytotoxicity is concentration dependent, and that it is not possible for all AFPs to
determine whether it would be cytotoxic before testing it (Tomczak et al., 2001)
Besides cells, it is also possible to cryopreserve uniform (non-composite) tissues, such as skin or
cartilage. Cryopreservation of organs, which are composed of multiple types of tissues, is more
complicated which is why long-term preservation of organs is not applicable yet (Bakhach, 2009).
Amir et al. (2004) showed that AFPs from Arctic fish (15 mg/mL) improved post-transplant
viability of rat hearts, which was cryopreserved for 18-24 hours. This was achieved by extending
the time before damage due to ischemia occurs, when the heart was kept at -1.3oC. Furthermore, the
use of AFP hindered nucleation within the heart tissue in the presence of nucleating agents, thus the
amount of apoptotic cell death and damage to myocytes was reduced. They concluded that the
functional recovery of hearts is considerably improved by using a cryopreservation temperature of 1.3oC and AFPs as a CPA (Amir et al., 2004).
If it becomes applicable to long-term cryopreserve entire organs, it would revolutionize organ
transplantation, allowing organs to be stored until a suitable match is found, and transporting them
over long distances. The use of AFPs in cryopreservation may be a giant leap forward in the field of
organ preservation and transplantation.
A6 Cell line
Various animal cells can be used for assays in biological experiments. It can prove difficult to
define an experiment in a way that one cell line, which fits perfectly for a given experimental setup,
can be singled out. In order to single out a cell line eliminating factors need to be defined. The cell
line chosen is primarily based on four eliminating factors; availability, the ability of the cell to
survive cryopreservation, readiness to be re-suspended and elimination of variables related to the
experiment.
The A6 cell line consists of renal epithelia cells from the South African clawed toad (Xenopus
laevis) (Danilchick et al., 1991). The cell membrane has a large number of ion channels, which is
reflected by the amount of studies that have used the cell as a model for different kinds of ion
channel research (Eaton et al., 2004;Faurskov and Bjerregaard, 2002;Hill et al., 2002;Mauricio and
Ferreira, 1999;Thit et al., 2013). The large number of ion channels makes it easier for the cell to
maintain its cytoplasmic osmolarity since it can absorb or emit ions through the channels. This
makes the cell line easily applicable for cryopreservation, since the cell can adjust its cytoplasmic
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osmolarity at a much higher rate, than other cells with fewer ion channels. This high adjustment rate
makes A6 cells more easily adaptable to the osmolarity of the fluid around the cell. As a result, the
A6 cells are less susceptible to damage caused by deviations in osmolarity in relation to other cells
with a smaller number ion channels. A6 cells are readily re-suspended within the cryopreservation
fluid, since they can be divided into single cells by trypsination. This lowers the possibility of
clustering when the sample is preserved and/or heated. Clustering within a sample would lead to
large differences in-between different samples, since the fluid contact within the same sample
would be uneven. Thus, clustering leads to different uncontrollable environments within the sample.
Another argument for using the A6 cell line is that it can be sub-cultivated making the cost of
testing the cell line low, since the cells do not have to be bought more than once. A final argument
for using the A6 cell line is that, since it was used in a former study of RmAFP#1s effect on the A6
cell survival rate after cryopreservation (Friis, 2010), a large amount of variables can be removed
from the current study making it possible to complete the experiments within the time limit.
Ice crystallization and nucleation

Several different factors are important in the formation of ice crystals within liquids. The first factor
is the melting point. Often the freezing point and melting point is the same, however, lowering the
temperature of a liquid below its melting point is not always enough to turn the liquid into its solid
state. This is due to super cooling which happens because activation energy is needed to start the
phase change from liquid to solid (Kornyushin, 2000).
The phase change from liquid to solid is termed nucleation. The activation energy needed for
nucleation, and thereby ice growth to occur is lowered by ice embryos, or nucleators. Ice embryos,
and nucleators, promote the organization of water molecules into an ice crystal lattice. The
nucleation is either homogenous or heterogonous depending on whether the nucleation has been
activated by an ice embryo or a nucleator respectively (Zachariassen and Kristiansen, 2000).
Homogenous nucleation is nucleation that is activated by an ice embryo, which is stable enough to
maintain its structure (Zachariassen and Kristiansen, 2000), and has been observed at -35oC and
below (Jeffery and Austin, 1997). It is obtained because the polar parts of water molecules
electrostatically attract each other, and thus tend to form an ice crystal structure. This leads to
aggregation of water molecules around the structure creating an ice crystal lattice, since the
temperature restrict their movement away from each other. If the temperature where ice crystals are
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formed is below the melting point of the solution this is called the hysteresis freezing point. The
decreased movement of water molecules leads to a larger cluster of water molecules that, at some
point, form a stable ice embryo (Zachariassen and Kristiansen, 2000). A homogenous nucleation
happens at very low temperatures (in highly super cooled water) because there is not anything
present which can promote the formation of an ice crystal lattice.
Matsumoto et al. (2002) showed that formation of a big and stable ice crystal in a water mixture
started nucleation (see Figure 3.a-e). Furthermore, they showed that the freezing of water can be
divided into four stages. In the first phase, named the long quiescent period by Matsumoto et al.
(2002), the energy change is not significant (red line in Figure 3.f) because the small ice embryos
formed are unstable, and disappear quickly. In the second phase, the slow energy decreasing
moment, the stable ice embryo is formed at the beginning. This is seen as an energy decrease (see
beginning of the green line in Figure 3.f). At one point, the ice embryo starts the nucleation, thereby
leading to the third phase. The nucleation is indicated by a sudden energy change between phase
two and three (see end of green line, start of the blue line in Figure 3.f). This third phase is termed
the fast energy-decreasing period. The last phase is not shown here, but is the phase where the
energy is constant once more, indicating that the solid phase has been reached (Matsumoto et al.,
2002).
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Figure 3: Depicts the formation of ice embryos at different times and the nucleation. A) Ice embryos form during the first
208 nanoseconds (ns) but are not stable enough to keep their structure and nucleation is not initiated. B) At t = 256 ns; a
sufficiently stable ice embryo is formed and the surrounding water molecules are affected. C) The ice embryo has initiated
the nucleation and the surrounding water molecules are re-organizing. D) t = 320; the ice lattice is formed although it is not
perfectly hexagonal yet. E) t = 580 ns; the re-organizational period, which follows nucleation, has completed a perfect
hexagonal ice crystal lattice. F) Depiction of the energy change during the periods A, B and C. The periods are indicated by
different colours: The red colour is A: The long quiescent period, where aggregation of water molecules leads to formation
of several unstable ice embryos. In the second period, B, the slow energy decreasing moment, the stable ice embryo is
formed at the beginning, seen as a slow decrease in energy. When the ice embryo initiates nucleation, the third period, C,
starts. The nucleation is indicated by a sudden energy change between the phase B and C. The third phase, the fast energydecreasing period, is the phase where water molecules aggregate quickly due to the nucleation. The figure was modified
according to (Matsumoto et al., 2002).
A heterogeneous nucleation is a nucleation where a nucleator is introduced to the liquid causing the
water molecules to aggregate spontaneously around the nucleator, initiating nucleation. The
heterogeneous nucleation usually happens at a higher temperature (less super cooled) than the
homogenous nucleation, since the ice embryo does not have to be formed, before the nucleation
initiates (Zachariassen and Kristiansen, 2000).
Recrystallization is a term for the formation of bigger ice crystals by eliminating smaller ice
crystals. Recrystallization happens due to the Kelvin effect which states that there is a curvatureinduced effect on vapour pressure. When the temperature is increased the vapour pressure inside the
ice crystals increases, and the surface becomes convex. To decrease the pressure, smaller ice
crystals are engulfed in bigger ones, to form a more concave surface, which is thermodynamically
favourable (Kristiansen and Zachariassen, 2005).
Antifreeze Proteins
Cold endurance is essential for organisms living in areas with sub-zero temperatures, since the body
temperature can drop below the freezing temperature of the body and liquids within. Different kinds
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of organisms have developed different kinds of ways to survive the cold temperatures. Endothermic
organisms create their own heat by their metabolism, and keep the heat within the body by having
insulation such as fur and/or a layer of fat. The body temperature of ectothermic organisms depends
on the temperature of its surroundings, since they do not have the same kind of mechanisms as
endothermic organisms. Thus, ectothermic organisms are much more sensitive to changes in
temperature. These organisms have developed several different ways to survive sub-zero
temperatures, including the accumulation of AFPs. The other ways these organisms use to survive
sub-zero temperatures will not be discussed any further.
Ectothermic organisms can be divided into two groups; Freeze avoiding organisms and freeze
tolerant organisms. Freeze avoiding organisms cannot tolerate freezing of their body fluids. At
temperatures below the melting point of their body fluids, these are constantly in a metastable super
cooled state. In this state, one tiny ice crystal from outside the body is enough to start a
heterogeneous nucleation, which would lead to an ice crystal lattice forming throughout the entire
body. The freeze avoiding organisms need a method to prevent nucleation caused by ice crystals in
the fluids, or ice that have penetrated the body cavity in other ways. Freeze tolerant organisms can
survive ice within their tissues. They have to avoid recrystallization of ice crystals, which are
already present within the organism, to avoid damage to the interior lining of their body cavity
(Ramlov, 2000). Synthesis of AFPs is an adaptation that some of these organisms have evolved, to
avoid the different issues they face by living in the sub-zero temperatures (Davies et al., 2002).
How AFPs can help the organisms survive, is discussed further below.
AFPs and antifreeze glycoproteins (AFGPs) were first identified in Antarctic teleost fishes about 5
decades ago (DeVries et al., 1970). These fishes can survive in sub-zero temperatures, which can
reach as low as -1.9 C, even though the equilibrium body fluid freezing temperature of a typical
marine teleost is -0.8oC (DeVries, 1983;Yeh and Feeney, 1996).
Harding et al (2003) did a comparison of the known types of AFPs and AFGPs, with focus on
AFGPs. This comparison led to the identification of some key features for the different kinds of
AFPs and the AFGPs, leading to a clear distinction between AFGPs and AFPs based on the
structure, as well as composition, of the protein (see Figure 4).
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Figure 4: General descriptions of the four known types of AFPs and one known type of AFGP. The AFGP and Type IV AFP
representative structure is from (Harding et al., 2003). The type I AFP representative structures are from (solution structures,
and ice growth inhibitory activity of peptide fragments derived from an Antarctic yeast protein) to the left is an illustration of
an AFGP. In the middle is three different types of type I AFPs (from left to right); winter flounder AFP (1WFB) and mutated
winter flounder AFP (1J5B). On the right hand side of the mutated winter flounder AFP, is the type II AFP. The
representative structure is from RCSB.org (2PY2) and is from a herring. The type III AFP representative structure is from
RCSB.org (1HG7), and is from Macrozoarcesamericanus (Ocean pout). The figure is modified based on (Harding et al., 2003).
AFGP is a term for at least eight structurally related glycoproteins that is found in different types of
Arctic and Antarctic fish. All the glycoproteins consist of (Ala-Ala-Thr)n repeats with minor
variations in the sequence. The hydroxyl oxygen of the Thr group has the disaccharide -Dgalactosyl(13)--N-acetyl-D-galactosamine (see Figure 5) joined as a glycoside. AFPs do not have
a disaccharide attached anywhere in their structure, which is the defining difference between the
two groups (Harding et al 2003).
Figure 5: Molecular structure of disaccharide -D-galactosyl(13)--Nacetyl-D-galactosamine.
Ice crystallization and AFPs

Almost all types of AFPs are fundamentally different from one another in their primary, secondary
and tertiary structure, thus they are stated to be non-homologous. Even though the different nonhomologous AFPs and AFGPs are diverse in sequence and structure, they appear to prevent
recrystallization of ice crystals and nucleation within an organism through the same mechanism
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(Davies, 2014). AFPs and AFGPs prevent growth of ice, and nucleation, in a non-colligative
manner, since they prevent ice growth without affecting the melting point. They are therefore
evaluated based on the thermal hysteresis (TH), which is the temperature difference between the
melting point and the hysteresis freezing point in a specific solution of antifreeze proteins
(Zachariassen and Kristiansen, 2000). It has to be mentioned that the concentration dependence of
TH has an upper limit. For RmAFP#1 this limit is around 0.1mM, where the TH is approximately
4.5oC (Kristiansen et al., 2012).
There are several theories on how AFPs and AFGPs cause this thermal hysteresis. The currently
accepted model is the adsorption-inhibition model by Raymond and Devries (Drori et al., 2014),
and it fits with the concentration dependence of the TH. The adsorption-inhibition model suggest,
that AFPs binds to the surface of ice crystals and inhibit their growth by increasing the surface
curvature, since the ice growth is restricted to the gaps in between the AFPs, or AFGPs (Raymond
and DeVries, 1977), thus the concentration of AFPs is crucial since the number of AFPs should fit
with the size of the crystal. Furthermore, the model can be supported by the Kelvin effect (see the
A6 Cell line section), since the alteration of the ice crystal surface curvature changes the vapour
pressure, thus making the thermodynamically unfavourable state, favourable (see Figure 6). As a
result, the hysteresis freezing temperature is lowered, without affecting the melting point.
Figure 6: An illustration of the ice crystal structure when AFPs are bound to the surface of the prism plane (the side of the
crystal), based on the adsorption-inhibition model. Ice crystal A has a stable vapour pressure (P). When the temperature
increases, as in ice crystal B, P increases. Normally this would lead to growth throughout the prism plane, but this is
inhibited by the AFP bound to the surface of the prism plane, leading to a curved surface of the prism plane, as seen in ice
crystal C. The increased curvature of C stabilizes the pressure inside the ice crystal and it will not expand anymore.
The adsorption-inhibition model assumes, that AFPs binds to the ice crystal surface irreversibly,
which has been widely discussed in the scientific community (Drori et al., 2014). A recent study by
Celik et al (2013) showed, that AFPs from Tenebrio molitor did not detach from the surface of an
ice crystal, even though the medium was changed, thus proving that this AFP, and possibly all
AFPs, binds irreversibly to the surface of the ice crystal. Furthermore, it fits with the observation
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that the concentration of AFP has an effect on the thermal hysteresis, until a certain point
(Kristiansen et al., 2012); the point of saturation, based on the amount of ice crystals in the liquid.
AFPs are commonly separated into two groups based on their activity; moderate active AFPs and
hyperactive AFPs. The hyperactive AFPs can be 10-100-fold more active than the moderate AFPs,
and are often found in insects, whereas the moderately active AFPs are often found in fish
(Kristiansen et al., 2012). Scotter et al. (2006) suggested that, the difference in the activity is partly
based on which planes of the ice crystal the AFP bind. Fish AFPs bind to the prism plane, whereas
insect AFPs bind to both the prism and basal plane (see Figure 7).
Figure 7: Difference in binding of fish and insect

antifreeze proteins to ice crystals. On the far left,
the ice crystal without AFPs attached is
visualized. On the top is the binding of fish AFP
shown, on the bottom the binding of insect AFP is
shown. The dark blue parts represent the basal
plane, and the light blue parts represent the prism
plane. The red dots are AFPs.
All fish AFPs (type I-IV) and AFGP shape the ice crystals into hexagonal bipyramids, which results
from AFPs binding to a single plane of ice as shown in Figure 7, left ice crystal in the top part of the
figure. These bipyramids have weak points, at the two pyramid tips, for containing the ice crystal.
In contrast, insect AFPs presumably can attach to both prism and basal planes on the surface of the
ice, to form a hexagonal plate as shown in Figure 7 (bottom part left most crystal) (Scotter et al.,
2006). When the temperature exceeds the TH the ice crystals grow explosively. The ice crystals
covered with AFPs tend to form hexagonal bipyramid ice crystals, whereas AFGPs form hexagonal
spicules which are pointier ended than bipyramids (Bar-Dolev et al., 2012).
The exact molecular knowledge behind how an AFP would recognize and bind to the surface of the
ice crystal is not fully understood. In earlier studies, researchers postulated that the binding
mechanisms depended completely on a hydrogen bond match between the antifreeze protein and the
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ice surface. According to this theory, hydroxyl groups could possibly make additional hydrogen
bonds (Knight et al., 1993). This theory is based on AFPs and AFGPs being hydrophilic which was
proven not to be the case (Garnham et al., 2008;Sonnichsen et al., 1996).
Based on the hydrophobicity of AFPs, another hypothesis arose; the entropy driven AFP binding
theory (see Figure 8.A). It suggests that water molecules are constrained on the surface of the AFP.
When the AFP is near an ice crystal surface, the AFP releases the water molecules and binds to the
ice crystal. This leads to an increase in entropy (disorder) within the systems, since the water
molecules that detached from the AFP is released to the surrounding water, and the AFP will
interrupt with the stable ice crystal surface (Davies, 2014).
Another theory, based on several modelling studies (Nutt and Smith, 2008;Smolin and Daggett,
2008;Yang and Sharp, 2004) suggests, that the AFPs organize water molecules into a pattern that
resembles the nearly-liquid layer of water, which is commonly found next to the ice crystal surface
under normal conditions. Then the two layers, the nearly-liquid layer of water organized by AFP
and the naturally occurring nearly-liquid layer of water next to the ice crystal surface, merge and
turn into ice. Hereby the AFP is incorporated in the ice crystal surface (see Figure 8.B) (Davies,
2014).
Figure 8: Illustration of two of the ice crystal growth
inhibiting mechanism theories. The red molecule
structure represents the hydrophobic AFP ice binding
site. The dark blue dots are surface restrained water
molecules (in figure A) and the nearly liquid layer of
water around AFP, and the ice crystal surface, in figure
B. The light blue
A) The entropy driven ice crystal growth inhibiting
mechanism suggests that water molecules are
constrained on the surface of the AFP. When the AFP is
near an ice crystal surface the AFP releases the water
molecules and binds to the ice crystal, thus increasing
entropy (disorder) within the ice crystal surface. B) The
Ice-like layer driven ice crystal growth inhibiting
mechanism suggests that the AFPs organize water
molecules into a pattern that resembles the nearly-liquid
layer of water, which is commonly found next to the ice
crystal surface under normal conditions. Then the
nearly-liquid layer AFP has organized and the naturally
occurring nearly-liquid layer, next to the ice crystal
surface, merge and turn into ice and hereby the AFP is
incorporated in the ice crystal surface. The figure is
modified based on (Davies, 2014)
Although there are different theories regarding the binding mechanisms of AFP, the theories have
one thing in common; AFP has a specific site where ice crystal interaction takes place. This site is
called the ice binding site. Davies (2014) made a comparison of the known ice binding sites and
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commented on the similarities between them. Davies (2014) suggests in his article that the ice
binding sites of AFPs (referred to as Ice Binding Proteins in his article) are extensive, relatively flat,
hydrophobic, contains none or few charged residues and often contain repeating motifs (Davies,
2014). Kristiansen et al. (2012) proposed that the flatness of the insect AFPs binding site was a
result of a coiling of the peptide chain (Kristiansen et al., 2012). Since the flatness of the binding
site has been observed in several different AFPs it would seem like the flatness is important for the
binding, and not just a result of the peptide chain coiling.
RmAFP#1
The AFP used in this study is from the longhorn beetle Rhagium mordax (R. mordax), a close
relative of the longhorn beetle Rhagium inquisitor (R. inquisitor). They belong to the coleopteran
superfamily the Chrysomeloidea. R. mordax is a freeze avoiding beetle native to northern Europe
and can be found hibernating under the bark of dead broad leaf trees in areas where the winter
temperature drops well below the equilibrium body fluid freezing temperature (Kristiansen et al.,
2012). R. mordax antifreeze protein (RmAFP) has several isoforms of a hyperactive AFP
(RmAFP#1-8) (Kristiansen et al., 2012).
Kristiansen et al. (2012) showed in their study that the sequence similarities between RiAFP and the
isoforms of RmAFP, including RmAFP#1, ranged from 75% to 82%. This similarity, and the fact
that other hyperactive insect AFPs characterized had either been shown or proposed to have helical configurations, was used to prepare an architectural presentation of the RmAFP#1 (see
Figure 9). The architectural presentation was confirmed by further analysis, but it was not possible
to decide whether the peptide chain coils in the right hand or left hand direction. Though, due to its
close relationship with the RiAFP, it is assumed that the RmAFP#1 would most likely possess a lefthanded -helical structure.
Figure 9: The molecular model of the RmAFP#1.
A) Rhagium mordax B) Front view of the protein,
which shows the ice binding site. C) Showing the
flattened shape down the helix. D) Tilted view of the
protein. Figure modified from (Kristiansen et al.,
2012).
The structural analysis of the RmAFP#1 gave additional insights about the structure of the protein.
It is composed of parallel -strands of various lengths, arranged side by side, forming a very flat
surface. RmAFP#1 is rich in the amino acid Threonine. Threonine is assumed to be crucial for the
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ice binding properties of insect AFPs. This was proposed by Kristiansen et al. (2012), since all
insect AFPs had the amino acid Threonine as the principal interactive residue within their ice
binding motifs (Kristiansen et al., 2012). Marshall et al. (2004) suggested that the Threonine
residues are arranged in such arrays that they match the spacing of oxygen atoms in the ice lattice
and the Threonine arrangement (Marshall et al., 2004). This indicates that Threonine promotes an
effective ice binding of the protein which fits with the previously discussed theories concerning the
ice binding mechanism.
The ice binding site of RmAFP#1 contains an ice binding motif of six recognizable repeats TxTxTx,
where T is Threonine. The repeats are separated by non-repeat regions that vary in length from 13
to 20 amino acid residues. The x can be any amino acid, but most often it is Alanine (A) or T. The
TxTxTx motifs have earlier been regarded as the ice binding motifs of RiAFP (Kristiansen et al.,
2012). These motifs appear to be the extended versions of the TxT triplet earlier identified as the ice
binding motif of several other insect AFPs (Marshall et al., 2004). Further examination of the
RmAFP#1 surface also revealed that the protein is amphipathic and that the recognized ice binding
site has greater hydrophobic characteristics than the opposite side of the protein molecule
(Kristiansen et al., 2012).
Theoretic summary and elaboration on the hypothesis

As discussed previously, cells, and tissues, are under the influence of chemical and physical
changes, both intra- and extracellular. These changes can cause lethal damage to the cells.
Cryopreservation has been proven successful enough to be implemented widely, both in the
scientific world as well as in the industry. Despite this, cryopreservation of certain cell types, and
complex tissues, has been proven difficult. The main part of the intracellular damages by IIF is
hindered by DMSO. However, the chemical is cytotoxic, and not applicable in every type of
cryopreservation. Furthermore, DMSO cannot hinder the extracellular recrystallization, which has
been proven to be a major factor in disappointing cell survival during cryopreservation. Ectothermic
organisms living in sub-zero temperatures have evolved ways to avoid heterogeneous nucleation
and recrystallization, including the production of AFPs. These kinds of proteins have been proven
to increase the cell survival rate, and tissue survival rate, when used during cryopreservation. The
AFPs used are for the most part moderately active AFPs, and AFGPs, not hyperactive AFPs. Since
the activity of an AFP is crucial when studying its impact on cell survival, a hyperactive AFP was
chosen, specifically the RmAFP#1. This proteins has been used in a previous study (Friis, 2010),
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where the results showed that it might be beneficial to add RmAFP#1 to cryopreservation medias.
This might be due to that RmAFP#1 can inhibit recrystallization in a large enough amount, so that
the cell membrane is not compromised, which would lead to a decrease in the cell survival rate.
Furthermore, the nucleation would be expected to be sudden and even, since the RmAFP#1 hinder
nucleation until a certain point where the ice crystal surface cannot be hindered anymore.
This has led to the project hypothesis; partially substituting DMSO with RmAFP#1 could lead to an
increase in the cell survival rate. This hypothesis partly rely on that the intra- and extracellular
recrystallization, as well as the IIF, collectively leads to a stress threshold, which the cells are killed
by. The word stress is used as a common term for mechanic stress due to ice crystals pushing on the
membrane or tearing it, increases in vapour pressure, and thermic stress from sudden bursts in
energy as in homogenous nucleation. If this is true then adding RmAFP#1 would lower the
extracellular stress, making it possible to increase the intracellular stress, by lowering the amount of
DMSO, without reaching the point where the cells are killed due to the overall stress level. This
hypothesis is illustrated in Figure 10.
Figure 10: Intra- and extracellular pressure in a cell experiencing recrystallization. Image A illustrates the stress (red
arrows) on the cell and membrane, with DMSO but no RmAFP#1. Image B illustrates the intracellular stress on the
cell and membrane when DMSO is not present, but also the lack of extracellular stress, since RmAFP#1 is present.
Image C shows the lack of stress when both DMSO and RmAFP#1 are present. Image D shows the stress when neither
DMSO nor RmAFP#1 is present.
The heterogeneous nucleation inhibition by RmAFP#1 might also improve the survival rate, since
the sudden and even nucleation, which happens when the ice crystal surface is not hindered any
more, would lower the chance of differences in osmolarity (See the Cryopreservation section).
This is illustrated in Figure 11.
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Figure 11: The effect of RmAFP#1 on nucleation and osmotic

gradients. Without RmAFP#1: When nucleation happens
solutes are pushed in front of the ice crystals forming, which
increases the extracellular osmolarity. If extracellular
osmolarity increases too much, and the efflux time is too long,
the cells will dehydrate. With RmAFP#1: RmAFP#1 hinders the
nucleation caused by ice embryos for w few degrees, until
reaching a certain threshold, where RmAFP#1 cannot hinder
nucleation any longer, and nucleation will happen with an
explosive rate. This allows ice embryos to become more stable
before nucleation, which might lead to a more even nucleation,
and decreased extracellular osmolarity.
Furthermore, it is reasonable to assume that the cell survival rate will be increased by lowering the
amount of DMSO in the media, since it has been proven that the cytotoxicity of DMSO is
concentration dependent. The last part of the hypothesis relies on the growth pattern of ice crystals
when these are inhibited by AFPs. Fish AFGPs have been used in cryopreservation experiments,
without an impressive success rate. But these types of AFPs form bi-pyramidal hexagonal ice
crystal structures or thin hexagonal spicules which has pointed ends, depending on the
concentration, whereas RmAFP#1 does not. The pointed ends of the hexagonal bipyramid ice
crystals might influence the membrane in a negative way, since it might puncture the membrane.
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Materials and methods

E. coli strain
For the production of RmAFP#1 the most common used strain in the lab is HR012. The strain was
transformed using the pQE-2 plasmid (see Figure 12) and the E. coli BL21 strain (for genotypic
specifications, see Table 1), which is commonly used to produce recombinant proteins. The HR012
is known to produce about 10 mg RmAFP#1 per litre batch culture (Friis, 2015).
Figure 12: The pQE-2 plasmid. The T5 promoter. The lac O is the
lac operator. RBS is the ribosome binding site. ATG is the start
codon. 6xHis the his-tag (it is a seven his tag in this one). MCS is the
multiple cloning site. Stop codons (3 reading frames) ColE1 (origin
of replication). Ampicillin (amp resistance gene). Laclq (repressor
gene) Modified from (UCLA).
Table 1: Genotypic information for E. coli HR012. Modified according to (Open WetWare, 2015).
Genotype
FompT
lon
hsdSB(rB-mB-)
gal
dcm
Function
Does not carry the F plasmid and cannot conjugate
Mutation in the outer membrane which reduce proteolysis of expressed
proteins
The lon protease is inactivated leading to a higher outcome of recombinant
proteins
Deletion of restriction and methylation of certain sequences resulting in that
un-methylated DNA can be introduced without being degraded
Mutants with this gene cannot metabolize galactose
The existence of a cytosine methylation at second C of CCWGG sites
The plasmid has been constructed so that the 7xHis-tagged RmAFP#1 is placed in-between the
ATG and Stop Codon in the Multiple Cloning Site (MCS) (see Figure 12). The 7xHis-tag is at the
N-terminal end of RmAFP#1 (see Figure 13). Before and after the His-tag Methionine and a Lysine
are placed. This is a cleavage site which makes it possible to remove the His-tag if it has to be, due
to experimental circumstances.
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Figure 13: The 7xHis-tag RmAFP#1 sequence. The green outlined amino acids are the 7xHis tag and the blue coloured
amino acids are the RmAFP#1 amino acid sequence.
Production and Purification of RmAFP#1

For the purification procedure the standard laboratory protocol is used, with a few modifications.
The exact production and purification protocol can be seen in Appendix #3: Detailed lab protocol;
Protein purification. The different solutions used can be seen in Appendix #2: Solutions and
standard procedures. The following is an overview of the procedure used the production and
purification of RmAFP#1.
The E. coli batch culture was French pressed to disrupt the cell membrane, and centrifuged to
remove the membrane from the cytoplasm. Since RmAFP#1 can almost completely refold to its
native state after being exposed to 70oC (Friis et al., 2014), the cell cytoplasm was heat treated and
centrifuged to remove a large part of unwanted proteins. This was done to hinder that unwanted
proteins would affect the following KTA Fast Protein Liquid Chromatography (FPLC) by
blocking the beads or break the bond between the nickel on the bead and the His-tag. When the
unwanted proteins are removed the solution is run on a NiNTA column and the bound protein
solution was collected. The bound protein solution was dialysed against a 1000MW Cut-off
membrane against MilliQ water overnight. This was done to remove salts, such as the TRIS used in
the collecting solution. Then the solution was freeze dried overnight, and the dry protein powder
was re-suspended in a known amount of liquid, since the dry protein is almost impossible to weigh.
The last step was doing a Bicinchonic Acid Assay (BCA) to determine the protein concentration,
and a SDS PAGE to determine the protein purity, as well as verify the concentration determined by
the BCA. Protein purity and concentration can be seen in the Results section under Production
and purification of RmAFP#1.
Experimental methods for the Cryopreservation experiments

Part 1: Nucleation test
Nucleation is a stochastic event that happens at different temperatures. Uncontrolled nucleation will
lead to uneven nucleation temperatures between the samples. Since uneven nucleation temperatures
will result in an uneven increase in the osmolarity in the cryopreservation media, this will affect
cells in different stages of maturation in different ways (see Figure 14). This could lead to a high
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degree of variation in amount of cells between the samples. Thus, different lid models were tested
to achieve the model, which causes the most even heterogeneous nucleation. The experiment is
done with clean water instead of a medium with RmAFP#1, since the linearity of the nucleation rate
would not be affected by the protein. The experiment will test four different types of lids; intact lid,
without a lid, without a lid but with a 0.2 M pore sized membrane and a lid with a small hole in it.
Figure 14: Homogenous nucleation
leading to different vapour pressures
outside the cells. The first picture shows
three different types of cells within the
media; dead cells (black), living cells
(black but white in the middle), and
living cells with a compromised
membrane (thin round line). These three
cells are all affected differently by the
extracellular vapour pressure. The living
cells will have the highest chance of
staying intact, many of the dead cells will
disintegrate, since their membrane is
compromised, and the living cells with
compromised membranes will be at the
highest risk of disintegrating. When
homogenous nucleation happens, the
different solutes are moved around due
to an increase in pressure. The solute
concentration around the cells change,
and some of the cells will be destroyed,
since the higher concentration of solutes
outside the cells will lead to a hypertonic
cell, if the ion transporters are not
efficient.
Part 2: Heating rate

Since a slow heating rate will lead to a great amount of recrystallization, a heating rate experiment
is done to determine the heating rate. The heating rate is tested by putting the samples in different
environments with different temperatures and logging the time it takes for the samples to start
changing phases. The 17oC/minute heating rate (which was tested in a former study by Friis,
Dennis), and a new heating rate of 10oC/minute (by putting the samples in a cooling cabinet with a
temperature of 10-14oC) was tested. The samples were defined as thawed when there was a visible
layer of liquid water, which happens when the sample temperature is around 0oC. The heating rate
was calculated by dividing 80 with the amount of minutes it took to reach a thawed sample, since
the samples are -80oC when the thawing starts and will be thawed around 0oC.
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Part 3: Maximal amount of RmAFP#1

Based on Kristiansen et al.s article (2012), the highest achieved TH is reached at a concentration of
RmAFP#1 around 0.1mM (Kristiansen et al., 2012). This experiment is to test in what concentration
the RmAFP#1 should be added, and the concentrations used are based on the concentration at which
the highest TH was achieved (0.1mM). This concentration can be calculated to mgs, making the
calculation much easier:
() # . :
(0.0001 0.001 1 13895.16
) 1000 = 1.388 /
In the study by Kristiansen et al. (2012) the TH is plotted against different concentrations of
RmAFP#1 (see Figure 15) (Kristiansen et al., 2012). Since the curve is not completely linear at a
concentration of 0.1mM the experiment in this study tested the survival of cells at 150% and 125%
of the concentration where the maximal TH was achieved; 2.08mg/mL and 1.74mg/mL.
Figure 15: TH concentration dependency. As illustrated on
the figure, the TH increases with the concentration of
RmAFP#1, but reaches a threshold around 0.1 mM.
For all of the RmAFP#1 concentrations in the samples see the table below. The different
concentrations should be ample enough to decide the amount of RmAFP#1 which should be added
in part 2 and 3.
Sample name
Concentration of RmAFP#1
(mg/mL)
Control
Exp. 50%
0.69
Exp. 75%
1.04
Exp. 100%
1.39
Exp. 125%
1.74
Exp. 150%
2.08
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The heating rate will be 17oC/minute since this heating rate was proven to be an adequate one, in
regards to the survival of A6 cells (Friis, 2010). The DMSO concentration will be the same in each
sample (10%), since a different amount of DMSO would influence the outcome and thereby making
it impossible to conclude at which concentration the RmAFP#1 should be.
Part 4: DMSO variation
Different cells require different amounts of DMSO. The A6 cells can tolerate 10% DMSO in its
Ringer media (Friis, 2010). As described in the Cryoprotective agents section the DMSO is added
to inhibit IIF. Due to this, the addition of RmAFP#1 could reduce the extracellular recrystallization
during the thawing period. This would minimize the stress on the membrane and thereby reduce the
amount of DMSO needed in the media, since the collected stress of recrystallization and IIF would
be below cell membrane threshold. To test this, RmAFP#1 is kept at a constant concentration (0.521
mg/250L based on the previous experiment), while the DMSO concentration is varied. The control
does not have any RmAFP#1. To ensure that the effect of RmAFP#1 is clearly visible, the heating
rate was lowered to 10oC/minute. The concentration of DMSO in each sample can be seen in the
table below:
Sample name
Concentration of DMSO %
of total media amount
Control
10%
Sample 1
10%
Sample 2
7.5%
Sample 3
5%
Sample 4
2.5%
Sample 5
0%
Part 5: RmAFP#1 concentration and DMSO concentration

This experiment is performed to either discard or confirm the hypothesis; Partially substituting
DMSO with RmAFP#1 will result in an increase in cell viability. The heating rate in this experiment
was 17oC/minute for one of the 10% DMSO 0% RmAFP#1 samples, and 10oC/minute for another
10% DMSO and 0% RmAFP#1 sample as well as the rest of the samples. This was to see, whether
the two chosen heating rates would result in a difference in the amount of cells, and survival rate.
The different concentrations and heating rates can be seen in the table below.
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Sample name
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Concentration
of Concentration
of Heating
DMSO % of total media RmAFP#1 (mg/mL)
rate
(XCo/minute)
amount
Control
10%
17
Sample 1
10%
10
Sample 2
7.5%
0.52 (25% of 2.08)
10
Sample 3
5%
1.04 (50% of 2.08)
10
Sample 4
2.5%
1.56 (75% of 0.521)
10
Sample 5
0%
2.08
10
Cell viability determination

The cells for the cryopreservation experiment were provided to us by Marianne Lauridsen, the head
of Henning F. Bjerregaards laboratory at Nature, Systems and Models (NSM, Roskilde University,
Denmark). The concentration of the cell suspension received was 106 cells per mL. To determine
the survival rate right after the cryopreservation, the cells were stained with Trypane blue and
counted in a Burker-Turk counting chamber. The stained sample is applied to the counting chamber,
which is then put in a microscope. The microscope is linked to a computer, and pictures are taken
with 100x magnification, of each of the four counting areas. When using a Burker-Turk counting
chamber the counting area has to be defined for every sample (see Figure 16).
Figure 16: Burker-Turk counting chamber. On the left side is an actual picture from one of the samples. The red line
represent the area within cells are counted, the red circle is around a cell which is counted as dead and the black circle is
around a cell which is counted as alive. On the right side is an illustration of the entire Burker-Turk chamber, the upper left
area is the same as the picture on the left.
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T-test
The T-test is a statistical method to find out whether or not the experimental results obtained from
different samples differ and whether or not this is due to chance, or if it is statistically significant.
The T-test is used to compare two sets of data that are both normally distributed. The nullhypothesis assumes that the mean of the data sets are equal. If the test shows significance the data
sets can be assumed to be drawn from two different populations, id est the null-hypothesis can be
rejected (with a confidence of more than 95%, for level of significance of 0.005).
A T-test is based on two values; the difference between two data sets (f-value) and the P-value. The
f-value indicates how close the data obtained from different samples are to each other, and the Pvalue gives the chance of the two data sets being different due to chance or not (see Figure 17). The
T-test executed in this report is two-tailed, which means that the P-value is assumed for both ends
of the data set, as seen in the figure. Two-tailed T-tests count on that the variance of the data can be
in both ends, whereas a one-tailed T-test assumes that the variance can only change in one end of
the data set.
The significance level () of the T-test is chosen by those using the data. In this report = 0.05
since this is the most widely accepted in non-medicinal studies. dictates the maximal value for P,
which in this case is 0.05. Every difference between data with a P-value below 0.05 is considered
significant, and meaning there is a difference between the samples that is not due to chance. Every
difference or similarity in between data with a P-value above 0.05 is considered insignificant, since
it is assumed that any difference is likely due to chance.
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Figure 17: Three different scenarios when doing a T-test. The t-value is the variance between the highest part of the two
curves, and the p-value is the area under the curve for the high part of the curve. For two data sets with medium variability
the t-value for the two data sets will be unequal, and the P-value will be below 0.05, but not necessarily far below. For two data
sets with low variability, the t-value for each data set will be very unequal, and the P-value will be far below 0.05. For two data
sets with a high variability, the t-vale for each data set will either be equal or a bit unequal, and the P-value would be far
above 0.05.
The T-test was applicable since the data used are normally distributed and there are more than ten
data points for each sample, but less than thirty. If there were more than thirty data points a Z-test
would be the most reliable statistical test.
Cryopreservation: experimental outline for RmAFP#1 and DMSO experiments

Figure 18: Flow chart for each experiment, the Xs are values given in our protocol
(not shown), and depends on the amount of cells in each sample, since the perfect
amount of cells in each corner of the Burker-Turk is 30-100. The maximal RmAFP#1
experiment differs since the media in the samples is not exchanged in between the
thawing and the staining with Trypane blue. In the two following experiments the
media was changed right after thawing. Since the heating rate in the maximal
RmAFP#1 experiment and DMSO + RmAFP#1 variation experiment is
17oC/minute, these were thawed at ~20oC. The DMSO variation experiment was
thawed at ~10oC, since the heating rate should be 10oC/minute. For the exact
protocol for each experiment see Appendix #3: Detailed lab protocol; Experiment
days.
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Results
Production and purification of RmAFP#1
Based on several BCAs and gels the first protein purification procedure led to a batch containing
approximately 7.6 mL of RmAFP#1 in MilliQ solution, with a concentration of 2.89 mg/mL (SD =
0.221). This batch is referred to as AFP P. 1 throughout the remainder of the report. This is the
only RmAFP#1 solution which was used in the experiments, so the BCA and final gel for AFP P.
2 and AFP P. 3 will not be shown. AFP P. 2 and AFP P. 3 are a re-run of the unbound
solutions from the NiNTA run done on AFP P. 1.
To determine the purity a SDS PAGE was run with the samples, and the samples appeared to be
uncontaminated with any proteins, see Figure 19.
Figure 19: Silverstained SDS PAGE with the Mark12 unstained ladder,
RmAFP#1 Freeze Dry precipitate from the 1st purification in a 5g
concentration based on the BCA (AFP P. 1 (5)), RmAFP#1 Freeze Dry
precipitate from the 1st purification in a 10g concentration based on
the BCA (AFP P. 1 (10)), RmAFP#1 Freeze Dry precipitate from the 2nd
purification (Freeze dry flask #2) in a 10g concentration based on the
BCA (AFP P. 2 (10)), RmAFP#1 Freeze Dry precipitate from the 2nd
purification (Freeze dry flask #3) in a 10g concentration based on the
BCA (AFP P. 3 (10)), and the NyDapase100 positive control in a 10g
concentration.
It is clearly seen that based on this gel the previously performed BCA
was wrong, since the concentration of the samples that contains 10g
(based on said BCA) is not as strong as the band for the 10g positive
control. It is also clear that the samples does not contain any other
protein than RmAFP#1 (lies in the green marked area)
The gel has been modified since there was some errors in the running of
the gel which resulted in a large discoloration in the last part of the gel.
The unmodified picture can be seen in Appendix #3: Detailed lab
protocol; Protein purification
For the calculations the concentrations are based upon, see Appendix #1: Calculations protein
purification. The calculation of the concentration in the different wells is based on a previous
BCA, since the gel was used to confirm the BCA. For the exact protocol see Appendix #3:
Detailed lab protocol; Protein purification.
Nucleation experiment
This experiment was designed to ensure an even heterogeneous nucleation within the cell samples,
since homogeneous nucleation, or uneven heterogeneous nucleation, might lead to a lethal osmotic
gradient across the cell membrane. An even heterogeneous nucleation will be seen as a straight line
compared to a homogenous nucleation, since the energy, and thus heat, released when nucleation
happens will be lower in a heterogeneous nucleation.
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Figure 20: Nucleation experiment. To find a lid type that ensures a heterogeneous nucleation, different types of lids were
tested. Homogenous nucleation will appear as a peek in temperature due to the sudden change in the energy needed for the
nucleation to happen, as seen in the nucleation theory part for the homogenous nucleation. A heterogeneous nucleation will
be a gently curved line since the nucleation is happening at a higher temperature, where the energy within the system is
higher. The difference in energy within the system and the energy released by the nucleation will then be smaller, leading
to a smaller deviation in temperature. The temperature (oC) is on the Y-axis, and time (seconds) is on the X-axis. The three
different coloured lines are three different replicas, notice that the 0.2 M membrane experiment is missing a replica.
When a normal Eppendorf tube lid is used during the cryopreservation the nucleation (increase in
temperature) happens at around -30oC and approximately 200-300 seconds in. At this temperature
and time, it is implausible that it is a homogeneous nucleation. In the sample with no lid, the
nucleation happens in between -23oC (blue replica) and -30oC (red and green replica). When a 0.2
m membrane is used as the lid it is apparent that there is a gentle increase in temperature around
100 seconds in, and that the temperature increase starts when the water is approximately -24oC (see
Figure 20). When a 1 mm hole is made in the lid, the nucleation happens around -23oC (red replica)
and -30oC (blue and green replica), exactly the same as for the sample with no lid. The data is
discussed further in the Discussion under Nucleation experiment.
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Based on this it is assumed that the samples that are most likely to go through heterogeneous
nucleation are those with no lid, and with a 1 mm hole in the lid. Since it is easier to keep out
contaminants and avoid spilling with a lid, all the samples in the following experiments had a 1 mm
hole in the lid.
Heat rate experiment

A heating rate of 17oC/minute was previously obtained by (Friis, 2010) by letting the samples thaw
in the lab with a room temperature (~20oC). This heating rate was tested and it was discovered that
the samples had thawed about 5 minutes after start of the thawing process, which results in a
heating rate of ~16oC/minute. Samples put in a cooling cabinet, which had a temperature that
fluctuated between 10-14oC, was thawed after approximately 10 minutes. This leads to a heating
temperature of ~8oC/minute.
Maximal RmAFP#1
This experiment was designed to test if there is an upper limit for the concentration of RmAFP#1
based on the survival of the cells. This test had 6 varying concentrations of RmAFP#1 but the
concentration of DMSO was the same. The cell samples were kept in the freezer for one week. The
heating rate was approximately 17oC/minute. The results indicate that adding RmAFP#1 would be
beneficial for the cells (see Figure 21 and Figure 22).
Figure 21: Total amount of intact cells, dead and alive, in each sample. This figure shows the difference in the amount of
intact cells in the samples. The only varying factor is the amount of RmAFP#1, DMSO is kept constant at 10%. The data
can be seen in Appendix #4: Data from experiments; Data from maximal RmAFP#1 experiment.
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From Figure 21 it is apparent that the amount of intact cells increase with the amount of RmAFP#1,
since the samples with RmAFP#1 has a higher total amount of intact cells, than the control. This
indicates that the recrystallization is hindered since the concentration of RmAFP#1 is the only
factor that could lead to this difference. A further discussion of this concentration dependence can
be seen in the Discussion under Maximal concentration of RmAFP#1 experiment.
Figure 22: Percentage of survived cells within the different samples. The survival rate has been calculated by dividing the total
amount of living cells with the total amount of cells, within the different samples. The data can be seen in Appendix #4: Data
from experiments; Data from maximal RmAFP#1 experiment.
From Figure 22 it is apparent that the sample with the highest survival rate is sample 1, and the
samples with the second highest survival rate are sample 2 and 3. But the samples different survival
rates do not differ greatly from each other. The importance of this is discussed in the Discussion
under Maximal concentration of RmAFP#1 experiment.
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Varying DMSO constant RmAFP#1

This experiment was designed to determine whether the DMSO concentration could be decreased
when 2.08 mg/mL RmAFP#1 is added to the cryopreservation medium. The cell samples were kept
in the freezer for approximately eight days. The heating rate was ~10oC/minute. The results indicate
that adding RmAFP#1 would be beneficial for the cells (see Figure 23 and Figure 24).
Figure 23: Total amount of intact cells, both dead and alive, in each sample. This figure shows the difference in the amount of
cells in the samples. All the samples had the same amount of RmAFP#1 (0.521mg/250L) but the amount of DMSO varied.
The data is found in Appendix #4: Data from experiments; Data from variation of DMSO, constant RmAFP#1 experiment.
From
FigureData
23 itfrom
is apparent
theconstant
amountRmAFP#1
of intact
cells increases when RmAFP#1 and DMSO
, in the section
variation ofthat
DMSO,
experiment.
are present together in the media, at a high concentration. A further discussion of this concentration
dependence can be seen in the Discussion under DMSO variation experiment constant
RmAFP#1.
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Figure 24: Percentage of survived cells within the different samples. The survival rate has been calculated by dividing the
total amount of living cells with the total amount of cells, within the different samples. The data are found in Appendix
#4: Data from experiments, in the section Data from variation of DMSO, constant RmAFP#1 experiment
From Figure 24 it is apparent that the survival rate differs slightly in between the samples and that
the samples with a low amount of DMSO (2.5% and 0%) have the highest survival rate. For a
further discussion on this DMSO concentration dependence, see the Discussion under DMSO
variation experiment constant RmAFP#1.
Variation of DMSO and RmAFP#1

This experiment was designed to conclusively prove or disprove the hypothesis that RmAFP#1
could substitute DMSO, based on the survival of the cells. This test had 5 varying concentrations of
RmAFP#1 and DMSO. The cell samples were kept in the freezer for one week and the heating rate
was approximately 17oC/minute. One of the control replicas (10% DMSO) were missing on the
final day, but since it was only one this has not been considered further.
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Figure 25: Total amount of intact cells, both dead and alive, in each sample. This figure shows that there is a difference in the
total amount of cells but not in the amount of living cells. The data can be seen in Appendix #4: Data from experiments; Data
from DMSO + RmAFP#1 variation experiment.
From Figure 25 it is apparent that the difference in the amount of intact cells in the samples depends
on the amount of both DMSO and RmAFP#1. The samples differed in both DMSO and RmAFP#1.
The purpose with sample 0 was to show whether or not the heat rate made a difference in the
amount of cells. The heating rate was in fact the same for all the samples. Both the result and
discrepancies in the heating rate method will be discussed in the Discussion under DMSO and
RmAFP#1 variation experiment.
Figure 26: Percentage of survived cells within the different samples. The survival rate has been calculated by dividing the
total amount of living cells with the total amount of cells, within the different samples. The data can be seen in Appendix #4:
Data from experiments; Data from DMSO + RmAFP#1 variation experiment.
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From Figure 26 it is apparent that the sample with the highest survival rate is sample 1, and the one
with the second highest survival rate is sample 2. The importance of this is discussed further in the
Discussion under DMSO and RmAFP#1 variation experiment.
Discussion
AFP production and purification
The production and purification of RmAFP#1 was done following the standard protocol in the lab.
According to personal communication it was expected that following the protocol it would be
possible to produce 10 mg RmAFP#1 per litre batch. This was not the actual production outcome.
The actual outcome was 2 mg RmaFP#1 per litre batch, but it was 9.60 mg RmAFP#1 per litre
overnight culture. The outcome is based on the calculated outcome for AFP P.1, and it should be
noticed that the previously mentioned samples (AFP P. 2 and AFP P. 3) had some RmAFP#1 in
them (DATA NOT SHOWN) but not enough to make a difference in the calculations, so these are
left out. This low production outcome might be because the protease inhibitor solution added to the
centrifuged cells contains ethylenediaminetetraacetic acid (EDTA). EDTA is used in the stripping
solution for the NiNTA columns used when catching the His-tag. 50 mL of 50 mM EDTA is run
through the column in the stripping step, when cleansing the column, and since every French
pressed solution contains 10 mM EDTA, 250 mL can be run on the NiNTA before the same amount
of EDTA is reached. This could affect the catching of the His-tag, since the amount of Nickel
decreases when the column is stripped, and in the end a lot of the RmAFP#1 might end up in the
unbound solution, which is treated as waste. When this was noticed, a gel was run with samples
from the different unbound solutions, but since the amount of RmAFP#1 it would be possible to
gather, was in a very small concentration it would take too much time to collect it.
Nucleation experiment
The nucleation experiment was designed to determine which lid achieved the most evenly
distributed heterogeneous nucleation in the cryopreservation samples. The control (standard lid)
should have shown a homogeneous nucleation happening, but this was not the case, since the water
was not super cooled enough to reach the temperature where a homogeneous nucleation is most
commonly observed. A reason for this could be that the samples were not completely still during
the experiment, due to closing of the freezer lid, the cooling unit starting or something else. Moving
of the samples would result in a heterogeneous nucleation in the control due to the energy in the
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moving. Another reason could be that the interior surfaces of the Eppendorf tube is not entirely
smooth, which would lead to an increase in the aggregation of water molecules and result in a
heterogeneous nucleation. The possibilities are endless, but the end result is the same; a
homogeneous nucleation was not achieved.
Nucleation was achieved at about -24oC in both replicas when using a 0.2 m pore membrane. The
third replica was left out due to misleading data (it showed 20oC when all the other sample replicas
were showing -10oC). This indicates that it is possible to achieve a heterogeneous nucleation when
using a 0.2m pore membrane as a lid, but since the mounting of the membrane was time
consuming this model was excluded for the experiments. If it were possible to make the mounting
of the membrane easier, quicker and with less risk of contamination, it might be a better way to
achieve the heterogeneous nucleation. This is because the two replicas were very similar, indicating
that the nucleation in between the replicas are more even, than in those without a lid or with a 1mm
hole in the lid.
Nucleation was achieved in between -23oC and -30oC in the samples without a lid and a 1 mm hole
in the lid. This signifies that exterior nucleators can cause the same heterogeneous nucleation in a
sample with a small hole in the lid, as in a sample with no lid. Leading to the assumption that the
nucleators are small ice crystals floating around in the air, thus they are not the moving of the
samples or internal aggregation of water (as have been suggested for the control). Since the risk of
contamination increases with the exposure of the medium to the surroundings, it was decided to use
the model with a 1 mm hole in the lid.
Heat rate experiment

The reliability of the heating rates is not that great. This is due to the simple method used to
determine the heating rates. Other methods were considered but these were not applicable. A
thermo-element was tried out instead of just guessing the temperature of the sample, but the
temperature measurement was not usable. This is probably due to the sensitivity of the instrument
which leads to a high degree of fluctuation in the temperature measurements, thus the exact
temperature in the sample is difficult to estimate on the basis of the measured temperature within
the samples. But since the experimental data shows a difference in between the control in the
DMSO variation experiment and the control in the DMSO and RmAFP#1 variation experiment (see
Figure 23 and Figure 25), it is reasonable to assume that the 10oC/minute heating rate was slow
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enough to increase recrystallization. This would theoretically lead to a lower amount of intact cells
in the control exposed to the slower heating rate, as is apparent when comparing the two samples.
Cell data reliability

In the Results section the number of total intact cells, the number of survived cells and the number
of dead cells were presented along with the survival rate. The survival rate was calculated as the
number of survived cells divided by the total number of cells. The total number of cells varied,
however, considerably more than expected. Ideally, since it comprises both living and dead cells,
the number for each sample should have been the same. For some samples, it may be that some
cells are lost during the process. It is not possible to know exactly what happened to them but it is
reasonable to assume that they are dead. Since the number of cells at the start was equal for all
samples, the direct comparison of the number of survived cells can be used as a measure for the
survival rate as well, this in contrast to the method mentioned in the Materials and methods; Cell
viability determination section. This survival rate differs from the other but if the assumption that
the lost cells are dead it might be a more accurate measure.
Some data show a high standard deviation (SD). The cells were counted manually. Due to time
limitation the replica within the same sample were split up and counted by different persons. None
of the replica was counted by more than one person. Sometimes the decision whether a cell was
alive or dead was difficult to make. This could be due to the staining being diffuse, aggregation of
cells or other artefacts. These are factors that can have contributed to the variation in the
distribution of the samples and, thus, the standard deviation.
Maximal concentration of RmAFP#1 experiment

The maximal concentration of RmAFP#1 experiment was performed to find the maximal beneficial
concentration of RmAFP#1. Every sample had 10% DMSO in it since it is needed to ensure survival
of cells, according to the theory. The parameter that varied was the amount of RmAFP #1, which
varied between 50%-150% of the maximal TH concentration (1.388 mg/mL), which has previously
been observed by (Kristiansen et al., 2012). Based on the hypothesis presented in the Theoretic
summary and elaboration on the hypothesis section, it is expected that there is an increase in
survival rate, with increasing amounts of RmAFP#1. At some point the survival rate does not differ
anymore, this is either the lowest or highest beneficial concentration, depending at which end of the
spectrum it lies.
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Total amount of intact cells

When adding RmAFP#1 to the media, the total amount of intact cells present increases substantially
(see Figure 21 in the Results under Maximal RmAFP#1). This increase in intact cells might be due
to RmAFP#1s ability to inhibit recrystallization during the thawing. This reduces the risk of ice
crystals expanding which might stress the membrane enough to disrupt it. The stress is not reduced
in the control and this might be the reason why the total amount of cells is low (see Figure 27). The
heating rate was 17oC/minute for all samples and the amount of DMSO in the media was the same
in all of the samples. Therefore each sample has been equally exposed to damage caused by IIF and
toxic effects from the DMSO. Since the concentration of RmAFP#1 is the only varying factor the
results support the idea that the increase in the amount of intact cells is due to RmAFP#1 inhibiting
recrystallization (see Figure 10).
Figure 27: Theory for how the
concentration of RmAFP#1 has an effect
on the total amount of cells, and thus
indirectly on the survival rate. The first
picture shows three different types of
cells within the media; dead cells (blue),
living cells (blue but white in the
middle), and living cells with a
compromised,
or
non-mature,
membrane (thin round blue line). 1st
picture: The cells are randomly spread
in, or in-between, the ice crystals in the
crystal
lattice.
2nd
figure:
the
recrystallization causes some crystals to
expand and some to decrease. This
changes the pressure (stress) around the
cells which leads to disruption if the cell
membrane is compromised, or nonmature. Another reason could be that
the membrane is permeated partially by
the expanding ice crystals, thus leading
to a disruption of the weakest
membranes (the dead cells membrane
and the membrane of the living cells
with compromised, or non-mature,
membranes). 3rd picture: at the end the
cells that are the fittest will survive and
be intact, leading to misleading survival
rates.
The increase in total amount of cells is somewhat correlated with the amount of RmAFP#1. The
concentration dependence in the total amount of intact cells, observed is coherent with the fact that
there is a correlation between the concentration of an AFP and the activity of the AFP (see Ice
crystallization and AFPs in the Antifreeze Proteins section). It should be recalled that the activity
of AFPs are measured as its ability to increase TH. The P-value for the difference in the total
amount of cells in between sample 150% (10% DMSO and 2.08mg/mL RmAFP#1) and sample
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125% (10% DMSO and 1.74mg/mL RmAFP#1) (p = 0.289), as well as sample 75% (10% DMSO
and 1.04mg/mL RmAFP#1) and sample 50% (10% DMSO and 0.69 RmAFP#1) (p = 0.811), were
non-significant. This could indicate that these samples represent the maximal beneficial
concentration (sample 150% and 125%) and the lowest beneficial concentration (75% and 50%). If
this is the case, it would not be expected that adding any more, or any less RmAFP#1, would lead to
an increase, or a decrease respectively, in the total amount of intact cells. This is seen when sample
150%, 125%, 75% and 50% are compared with the control (P < 0.001).
Stated differently, there might be both an upper and lower threshold for the beneficial concentration
of RmAFP#1. This might be because the medium is saturated with AFP, in the sense that there is
not enough space for AFP to bind to the surface of ice crystals, when the concentration is around
1.735-2.082 mg/mL (125%-150% of TH). The lower limit could be due to the opposite; there is not
enough RmAFP#1 in the media to occupy enough of the ice crystal surface, this would decrease the
hindrance of recrystallization. It cannot be excluded, but is estimated unlikely, that the results
observed are due merely to the increase in osmolarity caused by RmAFP#1. AFPs are known to
work in a non-colligative manner and the concentration of RmAFP#1 is considered too small to
cause the drastic effect seen.
The T-test for the total amount of intact cells in between the control and sample 75%, as well as
sample 50%, are significant (P < 0.001), indicating that the 75% and 50% samples, which has a
higher total amount of intact cells, are different from the control. This could be due to a lesser
degree of cell disruption by recrystallization in these samples, even though the amount is small
(<1.04 mg/mL RmAFP#1). This, however, is not consistent with the amount of intact dead cells in
sample 100%. The amount of dead cells in this sample is on the same level as in the control, but a
greater amount would have been expected, if RmAFP#1 solely decreases the amount of cell
disruption. The same is the case with sample 150% and sample 125%. The amount of dead cells in
these samples is higher than in the control, but less than in the samples 75% and 50%. This could be
explained by that the cell has a maximal stress threshold, as suggested earlier; by adding RmAFP#1
the stress put on the cells by recrystallization is lowered. When the amount is 0 mg/mL RmAFP#1
the stress on the cells is above the threshold, and they will disintegrate. If the amount of RmAFP#1
is equal to those in sample 75% and 50%, the stress was lowered enough to ensure that the cells
were intact, but it is not enough to avoid that the cells will die due to the collective stress of IIF and
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recrystallization. If RmAFP#1 is added in the same amount as in sample 150% and 125%, the stress
on the cell is lowered enough to avoid disintegration and cell death due to stress (see Figure 10).
Amount of living cells
The number of living cells increases in all samples with RmAFP#1, compared to the control (P <
0.011). This increase ranges from 2 to 6.5 fold, with the highest increase in the sample 125 %
RmAFP#1 although sample 150 % RmAFP#1 has close to the same amount (P = 0.289). As stated
earlier, these numbers can be seen as survival rate, since the initial amount of cells in all samples
was the same. This observation is consistent with the hypothesis of the project, wherein it is stated
that RmAFP#1 could increase the cell survival rate by reducing the stress caused by
recrystallization, and possibly uneven nucleation, imposed on the cell and its membrane.
Cell survival rate
Based on the percentage of survived cells the RmAFP#1 has some effect on the survival rate. The
survival rate of samples with the concentrations 150%, 125% and 100% RmAFP#1 is slightly
higher than the control (see Figure 22 in the Results under Maximal RmAFP#1). When the
concentration of RmAFP#1 is below 100% of the maximal TH concentration the cell survival
percentage decreases. This would explain why sample 75% and 50% has a lower survival rate since
recrystallization could still happen. Some cells are killed but because some of the recrystallization is
hindered, the dead cells do not disintegrate. Furthermore, this explanation would is consistent with
the assumption that the cell survival percentage in the control could represent the amount of cells
with a mature membrane at the time of cryopreservation. If the membrane of these cells is more
resilient than that of the larger part of cells, they could survive the recrystallization without being
affected by it, but the other cells with a less resilient membrane might be disrupted in the
recrystallization. This would lead to a smaller total amount of cells (dead and alive), and a larger
cell survival percentage. Due to this, the survival rate might be misleading due to the loss of cells.
To show the real picture, a new diagram was made, where the data is normalized by dividing the
amount of living cells in each sample, with the total amount of cells in sample 150% and 125% (See
Figure 28). This diagram shows that adding RmAFP#1 in a range from 0.69 to 2.08 mg/mL increase
the cell survival rate 1.5-9 fold.
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Figure 28: The percentage of survived cells when the amount of living cells in each sample is divided by the amount of cells in
the sample with the highest total cell amount (sample 150%). This is done since the samples have been added the same
amount of cells from start so the difference in cell amount, is only due to a change in the amount of intact cells.
Illustration of P-values
Since the T-test was made on an extensive amount of samples, the following diagram presents the
values in a more comprehensible way than a table can do. The diagram is presented to give the
reader a possibility to make up their own mind about the significance of our results. The red line
shows the cut-off point for significance of the P-value since this is the value chosen for .
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DMSO variation experiment constant RmAFP#1

This experiment was designed to test whether or not the amount of DMSO could be lowered if the
maximal beneficial concentration of RmAFP#1 (2.08 mg/mL, found in the previous experiment)
was added to the cryopreservation media. The heating rate during the thawing was lowered to
10oC/minute to favour recrystallization, thus ensuring that the effect of RmAFP#1 was visible. The
control had 10% DMSO and no RmAFP#1. All the samples had 2.08 mg/mL RmAFP#1, but varied
in amount of DMSO (10%-0%).
The total amount of intact cells increased when adding RmAFP#1 to the cryopreservation medium
(see Figure 23 in the Results under Varying DMSO constant RmAFP#1), although the control
(10% DMSO) and Exp. Sample 1 (10% DMSO and 2.08 mg/mL RmAFP#1) did not as much differ
from each other as in the previous experiment. The P-value for the difference in the total amount of
intact cells is insignificant (P = 0.078). But since the amount of living cells comprises the largest
part of the total amount of intact cells in all the samples, and the difference between the control and
sample 1 is significant (P=0.046), the difference in the total amount of intact cells between the
control and exp. Sample 1 might not be due to chance.
The higher P-values in this experiment might be due to the fact that the heating rate during the
thawing was 10oC/minute, and not 17oC/minute, as in the former experiment. This could lead to an
increase in the intracellular recrystallization, which could lead to a higher variation in the amount of
cells, both dead and alive, since intracellular recrystallization would affect different types of cells
(as in dead, strong or weak cells), in different ways. The difference between the observations in this
experiment and the previous indicates that the higher the thawing rate the more important is the
effect of RmAFP#1. This means that adding RmAFP#1 could make the thawing procedure less
critical and, thus, much easier to handle.
Cell survival rate
As with the previous experiment the amount of RmAFP#1 did not have a direct impact on the
percentage of survived cells, when the percentage was calculated based on the amount of cells in
each sample. However, the percentage of survived cells in the samples with a very low DMSO
(sample 4, 2.5% DMSO) concentration, and with no DMSO (sample 5), was higher than expected
(see Figure 24 in the Results under Varying DMSO constant RmAFP#1), and 0-2.5% DMSO and
2.08 mg/mL RmAFP#1 leads to a higher survival rate (approx. 10%), than 10% DMSO and 0%
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RmAFP#1 (P < 0.003). This might be due to that intracellular nucleation is homogenous (see the Ice
crystallization and nucleation section), and happens later than the extracellular heterogeneous
nucleation (for the control), thus the cells have a lower efflux time. When RmAFP#1 is added the
heterogeneous nucleation is postponed, since RmAFP#1 hinders nucleation by nucleators. This
coupled with the intracellular homogenous nucleation happening at a later time, increases the efflux
time. The increase in efflux time could lead to a higher intracellular osmolarity and thus a decrease
in IIF. Furthermore, the cytotoxicity of DMSO is decreased with the decreasing amount, leading to
a lower amount of dead cells. This could be the reason why the samples with a higher amount of
DMSO than sample 4 and 5 (sample 2 and 3) have a lower survival rate (P < 0.019).
Another explanation could be, that in the samples with a low DMSO concentration only the
strongest cells survive the cryopreservation because these can survive IIF, and the internal stress is
enough to disintegrate those who cannot. This would lead to a greater survival rate when the
percentage is calculated based on the total amount of cells within the sample. But if this was the
case, it would be expected that the survival rate would match that of the control, since this is
subjected to extracellular ice formation.
When the percentage of survived cells within the different samples are normalized according to the
sample with the highest amount of intact cells, the survival percentage for the samples with a low
DMSO concentration decreases (see Figure 29), although the samples with a low amount, or no,
DMSO is still greater than the control (P < 0.003).
Figure 29: Normalized percentage of survived cells for the DMSO variation experiment. The percentage of survived cells are
calculated by dividing amount of living cells in each sample by the amount of cells in the sample with the highest total cell
amount (sample 1). This is done since the samples have been added the same amount of cells from start so the difference in
cell amount, is only due to a change in the amount of intact cells.
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This suggests that the cells can survive without the DMSO thus implicating that recrystallization is
as an important factor as the IIF during cryopreservation of cells. This hypothesis is somewhat
supported by the fact that the Exp. Sample 1 has the highest percentage of survived cells, since this
sample has 10% DMSO and 150% of the RmAFP#1 concentration where the maximal TH is
reached, and thus should be protected against both recrystallization and IIF. If IIF was the most
important factor in the survival of cells during cryopreservation, it would be assumed that there
would be a difference between the control (10% DMSO) and Exp. Sample 2 (7.5% DMSO, 2.08
mg/mL RmAFP#1). Furthermore, it would be expected that the difference in between sample 1 and
the sample 2 was smaller. A reason for the big difference in between Exp. Sample 2 and 1 could be
that the beneficial concentration of DMSO is reached in Exp. Sample 1, but not in exp. Sample 2.
When exp. Sample 2 and the control is compared it is apparent that the survival rate of these two
samples are close to equal (see Figure 24 in the Results under Varying DMSO constant
RmAFP#1), as well as the amount of living cells (see Figure 23 in the Results under Varying
DMSO constant RmAFP#1). This indicates that 2.5% DMSO can be substituted by 2.08 mg/mL
RmAFP#1 and result in the same survival rate.
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DMSO and RmAFP#1 variation experiment

This experiment was designed to test whether DMSO could be substituted by RmAFP#1 in different
amounts. RmAFP#1 was added according to the amount of DMSO removed meaning that when
2.5% DMSO is removed 75% of 2.08 mg/mL RmAFP#1 is added. The total amount of intact cells
decreased with the concentration of DMSO, but the amount of living cells did not show the same
decrease. The cell survival rate was highest in sample 1, next to this was sample 2 and samples 0, 3,
4 and 5 were close to the same (see Figure 25 and Figure 26 in Results under Variation of DMSO
and RmAFP#1).
The total amount of intact cells varied in between the different samples, although the P-value
indicated that the difference in between sample 0 and samples 3 (5% DMSO and 1.04mg/mL
RmAFP#1), 4 (2.5% DMSO 1.56mg/mL RmAFP#1) and 5 (2.08mg/mL RmAFP#1) was
insignificant (P > 0.105). This could be due to that the heating rate was not consistent for all the
samples (will be discussed further below). The same insignificance is suggested by the P-value
when sample 1 (10% DMSO) and samples 2 (7.5% DMSO and 0.52mg/mL RmAFP#1), 3 and 4 are
compared (P > 0.134). If the insignificance is not due to an uneven heating rate then the total
amount of cells in the different samples, might be the same, as in that the difference in between
them are due to chance. This could be a result from the stress put on the cells. As hypothesized
earlier different stages in cell development leads to a stronger or weaker cell. If there are different
kinds of matured cells, and these are distributed randomly, some at the surface of an expanding ice
crystal some in a smaller ice crystal which is sacrificed, the resulting total amount of intact cells
would be highly due to chance (see Figure 27).
Amount of living cells
When the significance of the difference in the amount of living cells in between the samples are
tested, they are mostly insignificant (P > 0.125). The only difference which is significant is the
difference in between sample 1 and sample 3 (P = 0.012). This could indicate that RmAFP#1
hinders enough stress on the cells to avoid them dying due to the removal of DMSO.
Sample 0 vs. sample 1
The data for sample 0 and sample 1 should be the same, since these have the same amount of
DMSO and no RmAFP#1, and were treated the same way during the thawing period. This is,
however, not the case. The difference in amount of cells and percentage of survived cells might be
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due to an uneven heating rate, but the difference in-between the two samples are discussed later. If
the heating rate is the reason why the samples differ, the results indicate that the previous
hypothesis based on the results from the Results under Maximal RmAFP#1 section might be true.
The proposed hypothesis, suggests that the RmAFP#1 hinders the recrystallization in the media,
thus lowering the stress put on the cell membrane, and increases the amount of intact cells. This
would fit with the survival difference between sample 0 and 1, because sample 0 might have been
exposed to a quicker heating rate and thus the amount of time at which recrystallization, both intraand extracellular, happens, is smaller.
Cell survival rates
When the survival rates are compared it is apparent that sample 1 (10% DMSO) and sample 2
(7.5% DMSO + 0.52 mg/mL RmAFP#1) had the highest survival rate of all the samples (P < 0.027).
It should be noted that the difference in the survival rate of sample 1 and 2 is insignificant (P =
0.170), as well as the total amount of cells (P = 0.735), and the difference then might be due to
chance. This indicates that 0.052 mg/mL RmAFP#1 might be enough to ensure the same total
amount of cells and survival rate, when 2.5% DMSO is removed, supporting the hypothesis that
RmAFP#1 can partially substitute DMSO.
Since the survival rate of sample 3 (5% DMSO and 1.04 mg/mL RmAFP#1), as well as sample 4
and 5, are lower than sample 1 and 2, 5% might be a lower limit for the concentration needed to
achieve a beneficial effect of DMSO.
When the data is normalized based on highest total amount of cells (sample 5), the samples that resulted
in most cells as well as the highest percentage of survived cells, is sample 1 (10% DMSO), sample 2
(7.5% DMSO and 0.52 mg/mL RmAFP#1) and sample 5 (0% DMSO + 2.08 mg/mL RmAFP#1) (P <
0.027). This supports the survival of the fittest theory, indicating that the pressure caused by IIF and
external ice recrystallization is damaging the cells, in such a way that either RmAFP#1 or DMSO are
needed to obtain a high survival rate compared to the total cell amount.
If the data is modified and the sample with the highest amount of total cells (sample 5 and the control, P
= 0.927) is used as a reference point for the total amount of cells expected in each sample (see Figure
30), it is apparent that DMSO is not necessarily needed to ensure cell survival.
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Figure 30: Normalized percentage of survived cells for the DMSO+RmAFP#1 variation experiment. The percentage of
survived cells when the amount of living cells in each sample is divided by the amount of cells in the sample with the highest
total cell amount (sample 5 and control). This is done since the samples have been added the same amount of cells from start
so the difference in cell amount, is only due to a change in the amount of intact cells.
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Conclusion
The conclusion will be made on the basis of the Maximal RmAFP#1 experiment and on the DMSO
variation experiment, not the DMSO and RmAFP#1 variation experiment since there is a great deal
of uncertainty regarding the consistency of the heating rate in between the samples. The uncertainty
regarding the heating rate is discussed previously and since the method was more reliable in the first
two experiments, the data from these should be reliable.
When adding RmAFP#1 to a medium with 10% DMSO the total amount of intact cells increases
depending on the concentration of RmAFP#1. The heating rate is ~17oC/min and the range of
RmAFP#1 is 0.69-2.08 mg/mL. This heating rate and RmAFP#1 concentration range results in a
2.5-7.5 fold increase of intact cells, compared to a cryopreservation medium with 10% DMSO.
Furthermore, there is a 1.5-7 fold increase in amount of intact living cells when using the same
range of RmAFP#1 concentration. Based on this there is a lower and upper limit for the
concentration of RmAFP#1. The lower limit is in between 0.69 mg/mL and 1.04 mg/mL, whereas
the upper limit is in between 1.74 mg/mL and 2.08 mg/mL. It is proposed that this increase is due to
that RmAFP#1 hinder recrystallization, thus the amount of stress on the cells is decreased leading to
a lesser disintegration of the cells. An unexpected low amount of dead cells in a sample with 1.39
mg/mL RmAFP#1 and 10% DMSO did lead to the assumption that the decrease in stress might also
lead to a lesser degree of apoptosis, but further investigation is needed.
Adding 2.08 mg/mL RmAFP#1 to a cryopreservation medium with 7.5% DMSO leads to the same
total amount of intact cells, and amount of living cells, as a cryopreservation medium with 10%
DMSO. This suggests that RmAFP#1 can partially substitute DMSO in a cryopreservation medium,
and the medium will result in the same amount of cells as the standard cryopreservation medium.
Cell survival rate
The cell survival rate calculated as the amount of living cells divided total amount of intact cells
within the same sample is misleading. This is due to the loss of intact cells during the
cryopreservation. When RmAFP#1 is added the amount of intact cells increase and when the
survival rate is calculated the survival rate is not much higher than that for a cryopreservation
medium which has 10% DMSO and no RmAFP#1. The cell survival rate should be calculated by
dividing the amount of intact living cells, with the total amount of intact cells in the sample with the
highest amount of cells. It could also be approximated on the basis of the amount of intact living
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cells in the medium if the amount of cells added from the start is the same, as it is in these
experiments. When the cell survival rate is revised according to the previous conclusion, certain
ranges of RmAFP#1 and DMSO increase the survival rate.
When the heating rate is ~17oC/min adding RmAFP#1 in a range of 0.69-2.08, to a cryopreservation
medium with 10% DMSO, results in a 2-9 fold increase in the survival rate, compared to a
cryopreservation medium which has 10% DMSO and no RmAFP#1.
When the heating rate is ~10oC/min adding 2.08 mg/mL RmAFP#1 to a 10% DMSO
cryopreservation medium results in a 2 fold increase in survival rate. Furthermore, the survival rate
of the sample having 10% DMSO, the sample with 7.5% DMSO and 2.08 mg/mL RmAFP#1 and
the sample having 2.08 mg/mL RmAFP#1 and no DMSO, does not differ greatly. This indicates
that 2.08 mg/mL RmAFP#1 can partially or entirely substitute DMSO when the heating rate is
~10oC/min.
Overall conclusion
The current data suggests that it is beneficial adding RmAFP#1 to a cryopreservation media since
the data indicates that the cell survival increases (1.5-9 fold) up until a threshold point in between
1.74 and 2.08 mg/mL RmAFP#1. This suggests that recrystallization during the thawing is as an
important factor as IIF.
As stated in the Cryopreservation theory part, the benefits of adding AFP to cryopreservation media
have been tested before (Friis, 2010). These experiments have not shown that adding AFP to the media
would be beneficial for the survival rate of cells. The greatest part of these experiments has been done
with Fish AFPs and AFGPs which, for the most part, make bipyramidal hexagonal ice crystals during
the TH temperature range and hexagonal spicules when the temperature reaches below TH. These ice
crystals could be unfavourable for the cells, since the pointy ends could penetrate the membrane and
destroy the cell. If this is the case, it could explain why experiments with Fish AFPs have not shown the
same benefits of adding AFPs to a cryopreservation medium, as our experiment. Another explanation
could be the amount added. As shown in our experiment number 1, the concentration of AFP does make
a difference on the amount of cells that survive. Some experiments have been done with insect AFPs,
including the RmAFP#1 used in this project. These experiments have not all concluded that the insect
AFPs are beneficial, but it is noted that the concentration of the AFP is not as high as the known
concentration that leads to the highest TH.
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Perspective
An improvement of the current protocol could be taking pictures and making cell counts before the
cryopreservation to ensure that the cells are in the best shape. This might also reveal the amount of
less mature cells, which would be an advantage when the cryopreservation data is interpreted.
Another improvement which might be able to prove or disprove the hypothesis would be adding a
control with neither RmAFP#1 nor DMSO. This cells which survives the cryopreservation treatment
in this sample would be those with a very mature and well developed membrane. If no cells are in
this sample, or a high degree less than those in the DMSO cryopreservation medium, the hypothesis
stating that there are a difference in the integrity of the membrane and the strongest survive the
recrystallization in the DMSO cryopreservation medium, will not be true.
The hypothesis stated in the report that the beneficial effect of RmAFP#1 is due to a decrease in the
extracellular recrystallization is not entirely proven by the data in this project. Further studies in the
mechanism are needed. The hypothesis could be proven by investigating the membrane stability
during cryopreservation. This could be done by tagging the RmAFP#1 with a fluorescent tag, such
as Green Fluorescent Protein (GFP) and having a cell tagged with a different fluorescent tag. The
cryopreservation could then be observed using a cooling stage, or another kind of instrument
capable of freezing the samples under a microscope. By doing this the integrity of the membrane
could be observed during the entire cryopreservation. As an addition it could be an option to dye/tag
the essential cell organelles, if the cell had very few essential cell organelles inside it. Then one
might be able to see whether the RmAFP#1 affects the intracellular stress leading to apoptosis. If it
has then the hypothesis is right; adding RmAFP#1leads to a less stressful environment and as a
result the degree of apoptosis and disintegration is decreased.
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calcium through a divalent cation receptor in renal distal epithelial A6 cells. Pflugers Arch. 445, 4050.
Fletcher, G. L., Hew, C. L. and Davies, P. L. (2001). Antifreeze proteins of teleost fishes. Annu.
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Appendices
Appendix #1: Calculations protein purification
Based on the gel filtration chromatography (data not shown) for the different samples some of the
bound
protein
samples
were
excluded
from
the
1st
BCA
(see
Figure
31
and
Figure 31: Standard curve for 1st BCA, based on samples (data not shown) made from 1mg/mL standard protein. The
equation from this was used for concentration determination.
Table 2: BCA for the selected NiNTA samples.
Figure 31: Standard curve for 1st BCA, based on samples (data not shown) made from 1mg/mL standard protein. The
equation from this was used for concentration determination.
Table 2: BCA for the selected NiNTA samples.
Sample name
Absorbance
Average
Standard
Concentration Volume Amount
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ABS
Deviation
(mg/mL)
in (mL)
of
undiluted
50 (1:5 dilution)
1.200
50* (1:5 dilution)
1.170
25 (1:10 dilution)
0.689
25* (1:10 dilution)
0.695
10 (1:25 dilution)
0.357
10* (1:25 dilution)
0.355
5 (1:40 dilution)
0.254
5* (1:40 dilution)
0.221
RmAFP#1
1.185
0.015
5.885
9.000
52.97
0.692
0.003
6.725
9.000
60.52
0.356
0.001
8.214
9.000
73.93
0.238
0.017
8.291
9.000
74.62
Based on this BCA the concentration of proteins in the sample is calculated to be 61.51 mg, with a
significantly high standard deviation of 9.17. The lowest concentration obtained by using 2 of the
samples with different concentrations (50 and 25) was used as a reference concentration in future
determination experiments.
Since the strain used (HR012) is known to produce about 10 mg RmAFP#1 per litre culture, and the
volume of the HR012 culture was 12L the outcome of the protein production should be 120 mg.
Because of the poor outcome a SDS PAGE was run on the unbound protein samples to determine
whether or not the RmAFP#1 was lost during the first column rinse (DATA NOT SHOWN).
When the dialysis and freeze drying had been carried out, another BCA was done to calculate the exact
protein concentration in the newly freeze dried samples (DATA NOT SHOWN).
Table 3: AFP P. 1 BCA
Sample
Absorbanc
Average
Standard
Concentratio
Amount of protein
Deviation
n (mg/mL)
in solution (mg/mL)
50
0.772
0.746
0.020
0.673
3.299
50
0.742
----
----
----
----
50
0.724
----
----
----
----
25
0.393
0.383
0.026
0.608
2.977
25
0.347
----
----
----
----
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25
0.409
----
----
----
----
10
0.213
0.223
0.010
0.564
2.761
10
0.219
----
----
----
----
10
0.237
----
----
----
----
Based on this the concentration in the sample is 2.89 mg/mL (SD = 0.221) making the 10g
calculated concentration 5g and the 5g calculated concentration 2.5g, which fits with the results
seen on the gel.
Appendix #2: Solutions and standard procedures

500 mL LB media
The mixture is made in a 1L capped flask to avoid spilling in the autoclave.
1. 5.0 g Bacto Tryptone
2. 2.5 g Bacto Yeast Extract
3. MilliQ added until the waterline reaches the 300 mL mark.
4. 0.5 mL 1M NaOH
5. MilliQ added until the waterline reaches the 500 mL mark
The lid is loosely placed on top of the flask and secured with autoclave tape. Then the flask is put in
the autoclave. When the flask is autoclaved, and has cooled down below 50oC 0.5 mL Ampicillin is
added. The flask is shaken and put in the refrigerator.
-
If the media is used for plates the procedure is the same except for 2 things:
1. 10 g Agar is added before the autoclave step
2. The temperature of the medium is in between 40 and 50oC when the ampicillin is
added and straight after the gentle shaking it is slowly poured in to petri dishes.
500 mL NaCl-Ringer solution (x1)

1. 13.15 g NaCl
2. 0.42 g NaHCO33. 0.37 KCl
The solution is autoclaved and then 0.29 g CaCl x 2H2O is added. The solution is put in the
refrigerator, and was not prepared more than a week before the experiments to avoid contamination.
10 mL 4M TRIS solution
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4.84 g TRIS and 10mL MilliQ water is mixed in a flask, then autoclaved. In cases where the
amount of RmAFP#1 solution was smaller the 4M solution was too high, autoclaved MilliQ was
added instead so that the final concentration of TRIS in the cryopreservation medium was 10mM.
1mL 0.5M Glucose solution
90.09 mg D-Glucose was mixed with 1 mL MilliQ and then autoclaved. The solution was not made
more than a week before use.
240 mL Protease inhibitor solution
12 mL Protease inhibitor (stock) and 228mL R-buffer is added to a small flask with a lid.
2L R-buffer
The solution was made in two different flasks, see the table for the exact amounts.
Flask #
Imidazole (g)
NaCl (g)
TRIS (g)
0.688
2.922
3.944
0.681
2.929
3.946
Each flask is filled with 1L MilliQ and the pH is adjusted to 8 (8.01 and 8.03) with 1M HCl. Then
the solution is autoclaved.
1L I-buffer
The solution was made in one flask, see the table for the exact amounts.
Flask #
Imidazole (g)
NaCl (g)
TRIS (g)
13.64
2.923
3.945
The flask is filled with 1L MilliQ and the pH is adjusted to 8(8.02) with 1M HCl. Then the solution
is autoclaved.
Stock solutions
Chemical
Concentration
Company
ID #
DMSO
99.7%
Sigma Aldrich
D2650
PenStrep
10,000 Units Penicillin
Sigma Aldrich
P4333
10 mg Streptomycin/mL
Page 68 of 93
Trypane blue
Roskilde University
0.4
mg/mL
(made
from
powder
~40% Sigma Aldrich
T6146
concentration, sterile filtrated)

IPTG (dioxane free)
0.2M concentration (made from powder 100% Research

concentration)
Protease
70571
Organics
inhibitor 2%NaN3, 0.1 Benzamid, 0.2M EDTA
Lab solution
N/A
(x20)
Appendix #3: Detailed lab protocol

Some of the pages from the hand written lab protocol have been excluded since the protein was not
used later on. The SDS-PAGE procedure are leaved out since it is standard procedure, it can be seen
in the hand written protocol.
Protein purification
March 2nd 2015: LB media and plate production + Streaking of plates.
10L LB media and 500mL LB media with agar was made according to the previous recipe. The
table below has the exact value in each media.
Flask #
Tryptone (g)
Bacto Yeast (g)
NaCl (g)
Agar (g)
Approx volume post autoclave
10.01
5.01
10.01
-----------
960 mL
10.03
5.00
10.03
-----------
960 mL
10.00
5.01
10.01
-----------
980 mL
10.05
5.01
10.00
-----------
1000 mL
10.05
5.00
10.02
-----------
950 mL
10.02
5.00
10.00
-----------
980 mL
10.01
5.00
10.01
-----------
920 mL
10.02
5.01
10.01
-----------
960 mL
10.04
5.00
10.02
-----------
980 mL
10
10.00
5.01
10.00
-----------
1000 mL
Agar
5.01
2.50
5.00
10.00
N/A
When the plates were cooled down three of them were chosen for streaking of the HR012 strain.
When the plates had been streaked they were put in a plastic bag and left on a dark patch in the lab.
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March 4th 2015: Over-night culture making.

The plates were checked for suitable and solitary colonies. 6 1000 mL baffled flasks were added
500 mL LB media and were marked with a number to keep track of them. Each flask was
inoculated with one colony, two from each plate, using a 10L pipette tip. 930; the flasks are put in a
37oC shaking water bath (130RPM). Some extra LB media was made (measurements not shown).
March 5th 2015: Batch culture making and centrifugation.
The over-night cultures are split in four by pouring 250mL of the over-night culture in four 2000mL
baffled flasks. 750mL new LB media is poured in each flask and the baffled flasks are put in the
refrigerator for 30 minutes. Then the baffled flasks are put in a 30 oC shaking water bath for 30
minutes, then 1mL 0.2M IPTG solution is put in each flask. The cultures are left for 3 hours.
After 3 hours approx. 250 mL is divided into 4 centrifuge tubes, these are spun down (4,009G) for
10 minutes, the supernatant is removed and the centrifuge tube is reused. In between spins the
centrifuge tubes with pellet is kept on ice. The table below shows the mL in each tube and the
number of time they are spun down.
Tube#
10
11
12
(mL) (mL) (mL) (mL) (mL) (mL) (mL) (mL) (mL) (mL) (mL) (mL)
1
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
250
225
225
250
250
250
250
250
250
250
250
175
175
250
250
250
250
250
250
Total
1000 1000 1000 1000 925
925
975
975
1000 1000 1000 1000
The pellet collected from each culture was put in the freezer over-night.
March 6th 2015: French press
For every 250 mL medium spun down 5 mL protease inhibitor is added to the centrifuge tubes. The
tubes are put on ice and are then put on a shaking table for approx. 2 hours, until the pellet is resuspended. The pellet is French pressed following the lab protocol. Then they are spun down at
20,000G 4oC for 20 minutes, and the supernatant is collected and frozen down.
March 9th 2015: Denaturation
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The frozen AFP is thawed and a shaking water bath is heated to 70oC. The thawed samples are put
in the water bath for 10 minutes, with shaking. Then they are spun down (10,000G for 10 minutes)
using several Ole Dich tubes and the supernatant from each sample are collected. The samples were
put in the freezer.
March 12th 2015: NiNTA + SDS-page
The samples were thawed, and the NiNTA column was connected to the KTA. The I- and Rbuffer was degassed and checked for precipitate.
The super-loop is connected to the machine and 25 mL AFP liquid is put in it. The program
HisAFP pur on 5 mL NiNTA was run, and the unbound protein sample was collected. The
program took 2-3 hours, and 5 runs were made this day. Several parameters were adjusted during
the run time. The NiNTA column was change after the 4th run, but this was changed to after every
3rd run. The bound protein samples are collected and put in the freezer.
March 13th 2015: NiNTA run
The procedure from yesterday was repeated although the R- and I-buffers used were contaminated,
so new ones were made (DATA NOT SHOWN). The KTA and NiNTA were cleaned with a run
of 20% ethanol before leaving it for the weekend.
March 16th 2015: NiNTA run
The procedure from the 13th of March was repeated although the NiNTA and KTA were run with
100% R-buffer before start. Every frozen bound protein sample is thawed and these were dialysed
against a minimum volume of 250 times the volume in the dialysis tube (1000MW cut-off
membrane). The containers with water and dialysis tubes were put on top of magnet stirrers, the
stirring was started and the containers were left in the refrigerator room over-night.
March 17th 2015: Dialysis and freeze-dry
The dialysis water was changed in the morning and the containers were put back in the refrigerator
room. About four hours after the dialysed liquid is collected and put in a freeze-drying flask. The
flask is put in a -80oC freezer for an hour. When the liquid was frozen the flask was connected to
the freeze drier and the freeze drying was started.
March 18th 2015: Analysis of freeze drying.
The liquid had not sublimated due to a fault in the pressure so the freeze drying procedure was
repeated.
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Some of the freeze drying flasks was not done; these were left for the next day. It was tried to
measure the rest on a scale but this was unsuccessful. It was decided to re-suspend the precipitate in
a known amount of autoclaved MilliQ (3.8 mL), and the sample was saved in the refrigerator.
The rest of the freeze dried protein was also re-suspended in a known amount of autoclaved MilliQ
(5.2 mL) and the two flasks with re-suspended protein were joined together. A BCA was made on
the re-suspended protein. Due to some discrepancies in the expected and produced amount of
protein a SDS-PAGE was made on the unbound protein samples (DATA NOT SHOWN).
March 30th 2015: SDS-PAGE of AFP protein samples.
A SDS-PAGE of the AFP P.1 sample (the first collected protein), the AFP. P. 2 and AFP P. 3
(reruns of the unbound protein). The unmodified SDS-PAGE gel can be seen below.
Figure 32: SDS-PAGE from the 30th of March. The gel was run 1
hour since the mA was too low (13). After 30 minutes the buffer was
changed from a Tricine-Glycine SDS PAGE running buffer, to a MES
SDS PAGE Running buffer. The mA was changed from 13 to 46. This
is also why the gel is smudged. The B, C, D, E, F and G samples are
samples we ran for another one in the lab. The AFP. P1., AFP P. 2
and AFP P. 3 are our samples. The numbers in brackets is the amount
which is suspected due to the BCA done. The positive control is a
control sample from the freezer this one has a concentration of 33.3
g/L RmAFP#1, and is used to confirm the concentration estimated
in our samples by the BCA.
Experiment days
April 14th 2015: Preparation for Maximal RmAFP#1 experiment
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Ringer (x2) and (x10) solution, glucose solution and 4M TRIS solution was made ready for the
experiment, as per the solution guide in the previous part. The 10mg/10,000 unit PenStrep solution
the course lab provided was diluted with autoclaved MilliQ.
April 16th 2015: Maximal RmAFP#1 experiment
The A6 cell solution provided by Marianne Lauridsen at Henning F. Bjerregaards lab was diluted so
a concentration of 1 x 106 cells/mL was reached. The specific cell solution data can be seen in the
table below.
Dead cells (cells/mL)
Living cells (cells/mL)
Cell count (cells/mL)
37,944
2.14 x 106
2.16 x 106
6 2mL Eppendorf tubes was filled with 1.7 mL cell solution and spun down at 800 RPM for 5
minutes. The supernatant was removed and they were filled with the solutions according to the table
below (DMSO was added last). All the amounts are in mL.
R-media
name
R0
Ringer
solution
X2 (and
x10)
Glucose
0.5M
H2O
DMSO
Penstrep
Trisbuffer
AFP
solution
(2.887
mg/mL
conc.)
---------
Amount
of R:
0.850
0.017
0.459
0.170
0.034
0.170
1.700
50.000
0.850
0.017
0.244
0.170
0.034
0.170
0.385
1.700
75.000
0.850
0.017
0.052
0.170
0.034
0.170
0.577
1.700
100.000
0.170
0.017
0.500
0.170
0.034
0.170
0.769
1.660
125.000
0.170
0.017
0.307
0.170
0.034
0.170
0.962
1.660
150.000
0.170
0.017
0.115
0.170
0.034
0.170
1.154
1.660
Then the Eppendorf tubes were whirlpool mixed for a few seconds, and divided into 250L
Eppendorf tubes. The small Eppendorf tubes were put in the Mr. Frosty box, a hole was made in the
lid (with a sterilised needle) and they were put in the freezer. The lid was put on top of the Mr.
Frosty a second after it was put in the -80oC freezer to be sure some ice crystals would be present
within the box. After three hours the lids were covered with parafilm.
April 21st 2015: Cell analysis Maximal RmAFP#1 experiment
The samples were taken out of the freezer and Mr. frosty box and put in an Eppendorf stand on a
revolving table in the lab for about 40 minutes. The samples were put in the refrigerator, and taken
Page 73 of 93
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out at a 10 minute interval. The 10 minutes was to reacclimatize the samples before the Trypane
stain was done. When the sample was acclimatized it was whirlpool mixed, and 20L Trypane blue
and 20L cell sample were put in a new Eppendorf tube. This Eppendorf tube was left for 5 minutes
before it was whirl pooled again and 10L was loaded on to the Burker-Turk. Pictures of each
corner were taken and the next samples were taken out. The different sample replicas are looked at
in the order of their number, meaning that replica number 1 from each sample is analyzed first, then
each replica number 2 and so on.
April 29th 2015: Constant RmAFP#1 varying DMSO experiment
The different cryopreservation solutions were made in the morning. The A6 cell solution provided
by Marianne Lauridsen at Henning F. Bjerregaards lab was diluted so a concentration of 1 x 106
cells/mL was reached. The specific cell solution data can be seen in the table below.
740
3.71 x 106
3.77 x 106
R-media
name
R0
R1
R2
R3
R4
Ringer
solution
X2
0.850
0.850
0.850
0.850
0.850
Glucose
0.5M
0.017
0.017
0.017
0.017
0.017
H2O
DMSO
0.459
0.527
0.527
0.527
0.527
0.170
0.128
0.085
0.043
0.000
Penstrep
Tris-buffer
0.034
0.034
0.034
0.034
0.034
0.170
0.170
0.170
0.170
0.170
AFP
solution
(2,887
mg/mL
conc.)
--------0.348
0.348
0.348
0.348
May 6th 2015: Cell analysis Constant RmAFP#1 varying DMSO experiment
The different cryopreservation solutions were made in the morning. The samples were taken out of
the freezer and Mr. frosty box and put in an Eppendorf stand on a revolving table in a ~10oC
Page 74 of 93
Roskilde University
refrigerator for about 40 minutes. The samples were put in the refrigerator, and taken out at a 10
minute interval. The 10 minutes was to reacclimatize the samples before the Trypane stain was
done. When the sample was acclimatized it was whirlpool mixed, and 20L Trypane blue and 20L
cell sample were put in a new Eppendorf tube. This Eppendorf tube was left for 5 minutes before it
was whirl pooled again and 10L was loaded on to the Burker-Turk. Pictures of each corner were
taken and the next samples were taken out. The different sample replicas are looked at in the order
of their number, meaning that replica number 1 from each sample is analyzed first, then each replica
number 2 and so on.
May 12th 2015: DMSO and RmAFP#1 variation experiment
The A6 cell solution provided by Marianne Lauridsen at Henning F. Bjerregaards lab was diluted so
a concentration of 1 x 106 cells/mL was reached. The specific cell solution data can be seen in the
table below.
740
3.71 x 106
3.77 x 106
R-media name
(name based
on % AFP
maximal
concentration)
R0
0.000
25.000
50.000
75.000
100.000
Ringer
solution
X2 (and
x10)
0.850
0.850
0.850
0.850
0.850
0.850
Glucose
0.5M
0.017
0.017
0.017
0.017
0.017
0.017
H2O
DMSO
0.459
0.459
0.425
0.391
0.357
0.323
0.170
0.170
0.128
0.085
0.043
0.000
Penstrep
0.034
0.034
0.034
0.034
0.034
0.034
Tris-buffer
0.170
0.170
0.170
0.170
0.170
0.170
AFP
solution
(2.887
mg/mL
conc.)
--------0.000
0.077
0.153
0.230
0.306
Page 75 of 93
Roskilde University
May 20th 2015: Cell analysis DMSO and RmAFP#1 variation experiment
The samples were taken out of the freezer and Mr. frosty box and put in an Eppendorf stand on a
revolving table in the lab for about 40 minutes. The samples were put in the refrigerator, and taken
out at a 10 minute interval. The 10 minutes was to reacclimatize the samples before the Trypane
stain was done. When the sample was acclimatized it was whirlpool mixed, and 20L Trypane blue
and 20L cell sample were put in a new Eppendorf tube. This Eppendorf tube was left for 5 minutes
before it was whirl pooled again and 10L was loaded on to the Burker-Turk. Pictures of each
corner were taken and the next samples were taken out. The different sample replicas are looked at
in the order of their number, meaning that replica number 1 from each sample is analyzed first, then
each replica number 2 and so on.
Appendix #4: Data from experiments

Data from maximal RmAFP#1 experiment
Sample name
C6
TOTAL
C5
TOTAL
C4
TOTAL
C3
TOTAL
C2
TOTAL
C1
Dead
23
24
20
67
7
8
8
10
33
4
9
15
7
35
11
9
6
10
36
13
5
13
6
37
3
3
10
Live
96
51
41
188
43
54
62
57
216
268
178
141
101
688
152
99
85
157
493
56
77
103
110
346
90
12
132
Amount
119
75
61
255
50
62
70
67
249
272
187
156
108
723
163
108
91
167
529
69
82
116
116
383
93
15
142
%dead
19.33
32.00
32.79
26.27
14.00
12.90
11.43
14.93
13.25
1.47
4.81
9.62
6.48
4.84
6.75
8.33
6.59
5.99
6.81
18.84
6.10
11.21
5.17
9.66
3.23
20.00
7.04
%live
80.67
68.00
67.21
73.73
86.00
87.10
88.57
85.07
86.75
98.53
95.19
90.38
93.52
95.16
93.25
91.67
93.41
94.01
93.19
81.16
93.90
88.79
94.83
90.34
96.77
80.00
92.96
Page 76 of 93
Roskilde University
TOTAL
16
Mean amount of survived (%)
234
TOTAL
150.3
TOTAL
150.2
TOTAL
150.1
93.60
7.26
SD sample of total amount (numeral)
TOTAL
150.4
6.40
398.17
SD sample of survived (%)
TOTAL
150.5
250
88.79
Mean of total amount (numeral)
150.6
23
24
22
17
86
39
16
16
35
106
14
13
26
31
84
39
45
64
41
189
55
83
58
58
254
39
62
32
133
TOTAL
125.6
33
87
44
176.81
618
600
455
430
2103
709
541
680
611
2541
628
546
691
611
2476
840
846
938
753
3377
928
853
716
610
3107
730
794
450
1974
94.94
2638.33
1.5
549.73
728
846
753
641
624
477
447
2189
748
557
696
646
2647
642
559
717
642
2560
879
891
1002
794
3566
983
936
774
668
3361
769
856
482
2107
3.59
3.85
4.61
3.80
3.93
5.21
2.87
2.30
5.42
4.00
2.18
2.33
3.63
4.83
3.28
4.44
5.05
6.39
5.16
5.30
5.60
8.87
7.49
8.68
7.56
5.07
7.24
6.64
6.31
96.41
96.15
95.39
96.20
96.07
94.79
97.13
97.70
94.58
96.00
97.82
97.67
96.37
95.17
96.72
95.56
94.95
93.61
94.84
94.70
94.40
91.13
92.51
91.32
92.44
94.93
92.76
93.36
93.69
761
933
797
4.34
9.32
5.52
95.66
90.68
94.48
Page 77 of 93
Roskilde University
TOTAL
125.5
2327
730
768
585
655
2738
592
681
1273
832
719
684
748
2983
821
749
744
716
3030
267
639
747
838
2491
92.67
2665.5
1.41
591.92
164
38
36
59
33
TOTAL
166
125.4
79
55
TOTAL
134
125.3
55
79
72
80
TOTAL
286
125.2
66
69
56
52
TOTAL
243
125.1
31
25
39
63
TOTAL
158
2491
768
804
644
688
2904
671
736
1407
887
798
756
828
3269
887
818
800
768
3273
298
664
786
901
2649
6.58
4.95
4.48
9.16
4.80
5.72
11.77
7.47
9.52
6.20
9.90
9.52
9.66
8.75
7.44
8.44
7.00
6.77
7.42
10.40
3.77
4.96
6.99
5.96
93.42
95.05
95.52
90.84
95.20
94.28
88.23
92.53
90.48
93.80
90.10
90.48
90.34
91.25
92.56
91.56
93.00
93.23
92.58
89.60
96.23
95.04
93.01
94.04
Data from variation of DMSO, constant RmAFP#1 experiment

Sample name
C6
TOTAL
C5
TOTAL
C4
Dead
35.00
15.00
19.00
29.00
22.00
44.00
25.00
51.00
66.00
77.00
63.00
Live
33.00
10.00
15.00
32.00
90.00
54.00
38.00
28.00
59.00
179.00
72.00
47.00
64.00
Amount
68.00
25.00
34.00
61.00
188.00
76.00
82.00
53.00
110.00
321.00
138.00
124.00
127.00
%Live
48.53
40.00
44.12
52.46
47.87
71.05
46.34
52.83
53.64
55.76
52.17
37.90
50.39
Page 78 of 93
Roskilde University
60.00
79.00
262.00
64.00
77.00
82.00
89.00
96.00
84.00
98.00
104.00
TOTAL
354.00
C2
14.00
10.00
13.00
35.00
3.00
30.00
4.00
28.00
TOTAL
103.00
C1
28.00
65.00
25.00
87.00
41.00
56.00
31.00
66.00
TOTAL
274.00
58.02
377.83
10.29
191.58
TOTAL
C3
1.6
TOTAL
1.5
TOTAL
1.4
TOTAL
1.3
TOTAL
1.2
48.00
28.00
22.00
32.00
130.00
67.00
154.00
129.00
388.00
738.00
46.00
65.00
70.00
84.00
265.00
39.00
29.00
31.00
44.00
143.00
11.00
139.00
528.00
141.00
171.00
180.00
202.00
694.00
24.00
48.00
33.00
32.00
137.00
93.00
112.00
97.00
97.00
399.00
23.00
24.00
32.00
43.00
122.00
516.00
307.00
207.00
357.00
56.83
49.62
54.61
52.05
46.67
51.49
51.01
41.67
72.92
90.91
87.50
75.18
69.89
77.68
57.73
68.04
68.67
71.00
52.00
54.00
75.00
181.00
583.00
461.00
336.00
745.00
1387
72.00
61.00
37.00
107.00
277.00
74.00
43.00
86.00
124.00
327.00
46.00
1542
118.00
126.00
107.00
191.00
542.00
113.00
72.00
117.00
168.00
470.00
57.00
32.39
46.15
59.26
57.33
67.40
88.51
66.59
61.61
47.92
89.95
61.02
48.41
34.58
56.02
51.11
65.49
59.72
73.50
73.81
69.57
80.70
Page 79 of 93
Roskilde University
7.00
4.00
11.00
TOTAL
33.00
1.1
38.00
28.00
26.00
20.00
TOTAL
112.00
26.00
21.00
31.00
124.00
71.00
66.00
119.00
59.00
315.00
83.51
525.50
23.90
479.48
2.6
TOTAL
2.5
TOTAL
2.4
TOTAL
2.3
TOTAL
2.2
TOTAL
2.1
TOTAL
31.00
31.00
13.00
17.00
92.00
26.00
37.00
99.00
36.00
198.00
65.00
142.00
28.00
235.00
144.00
61.00
133.00
61.00
399.00
4.00
5.00
9.00
4.00
22.00
3.00
13.00
28.00
15.00
59.00
33.00
25.00
42.00
100.00
109.00
94.00
145.00
79.00
318.00
47.00
24.00
29.00
28.00
128.00
33.00
26.00
76.00
132.00
267.00
100.00
156.00
35.00
291.00
84.00
64.00
108.00
67.00
323.00
25.00
22.00
20.00
21.00
88.00
50.00
22.00
13.00
16.00
101.00
78.00
55.00
42.00
45.00
142.00
59.00
63.00
175.00
168.00
406.00
165.00
298.00
63.00
526.00
228.00
125.00
241.00
128.00
722.00
29.00
27.00
29.00
25.00
81.00
53.00
35.00
41.00
31.00
107.00
78.79
84.00
73.81
124.00
65.14
70.21
82.07
74.68
99.06
60.26
43.64
69.05
62.22
90.14
55.93
41.27
43.43
78.57
65.76
60.61
52.35
55.56
55.32
36.84
51.20
44.81
52.34
44.74
86.21
81.48
68.97
84.00
108.64
94.34
62.86
31.71
51.61
94.39
Page 80 of 93
Roskilde University

76.50
330.67
22.77
239.77
3.6
13.00
14.00
9.00
17.00
53.00
72.00
88.00
39.00
52.00
251.00
113.00
86.00
63.00
81.00
343.00
33.00
39.00
62.00
42.00
176.00
10.00
16.00
3.00
6.00
35.00
16.00
2.00
19.00
13.00
50.00
74.21
298.00
15.81
232.32
38.00
29.00
30.00
31.00
TOTAL
3.5
TOTAL
3.4
TOTAL
3.3
TOTAL
3.2
TOTAL
3.1
TOTAL
4.6
40.00
17.00
15.00
15.00
24.00
20.00
25.00
21.00
90.00
69.00
52.00
36.00
43.00
200.00
108.00
85.00
74.00
113.00
380.00
46.00
86.00
92.00
56.00
280.00
23.00
40.00
16.00
19.00
98.00
19.00
21.00
21.00
17.00
78.00
37.00
34.00
34.00
38.00
106.00
141.00
140.00
75.00
95.00
310.00
221.00
171.00
137.00
194.00
723.00
79.00
125.00
154.00
98.00
456.00
33.00
56.00
19.00
25.00
100.00
35.00
23.00
40.00
30.00
93.00
78.00
46.00
45.00
46.00
64.86
58.82
73.53
55.26
84.91
48.94
37.14
48.00
45.26
64.52
48.87
49.71
54.01
58.25
52.56
58.23
68.80
59.74
57.14
61.40
69.70
71.43
84.21
76.00
98.00
54.29
91.30
52.50
56.67
83.87
48.72
63.04
66.67
67.39
Page 81 of 93
TOTAL
4.5
TOTAL
4.4
TOTAL
4.3
TOTAL
4.2
TOTAL
4.1
Roskilde University
87.00
11.00
9.00
17.00
15.00
52.00
55.00
36.00
28.00
54.00
173.00
36.00
30.00
39.00
36.00
141.00
128.00
26.00
28.00
46.00
103.00
203.00
108.00
85.00
74.00
113.00
380.00
86.00
75.00
110.00
87.00
358.00
8
11
3
7
29.00
31
24
27
54
136.00
11
6
6
3
TOTAL
26.00
126.00
74.15
306.50
8.40
91.54
5.6
9.00
24.00
26.00
20.00
TOTAL
79.00
5.5
13.00
35.00
12.00
11.00
TOTAL
71.00
5.4
71.00
40.00
39.00
47.00
29
42
15
40
27.00
29.00
41.00
36.00
133.00
36.00
21.00
40.00
23.00
120.00
71.00
77.00
67.00
91.00
215.00
37.00
37.00
63.00
118.00
255.00
163.00
121.00
102.00
167.00
553.00
122.00
105.00
149.00
123.00
499.00
39.00
35.00
30.00
61.00
165.00
40.00
48.00
21.00
43.00
152.00
36.00
53.00
67.00
56.00
212.00
49.00
56.00
52.00
34.00
191.00
142.00
117.00
106.00
138.00
59.53
70.27
75.68
73.02
87.29
79.61
66.26
70.25
72.55
67.66
68.72
70.49
71.43
73.83
70.73
71.74
79.49
68.57
90.00
88.52
82.42
72.50
87.50
71.43
93.02
82.89
75.00
54.72
61.19
64.29
62.74
73.47
37.50
76.92
67.65
62.83
50.00
65.81
63.21
65.94
Page 82 of 93
TOTAL
5.3
TOTAL
5.2
TOTAL
5.1
Roskilde University
197.00
36.00
48.00
55.00
139.00
12.00
12.00
11.00
7.00
42.00
21.00
8.00
7.00
15.00
51.00
TOTAL
306.00
72.00
98.00
77.00
247.00
51.00
33.00
36.00
73.00
193.00
161.00
107.00
29.00
124.00
421.00
503.00
108.00
146.00
132.00
386.00
63.00
45.00
47.00
80.00
235.00
182.00
115.00
36.00
139.00
472.00
60.83
66.67
67.12
58.33
63.99
80.95
73.33
76.60
91.25
82.13
88.46
93.04
80.56
89.21
89.19
70.29
279.33
11.10
118.34
Data from DMSO + RmAFP#1 variation experiment

Sample name
Dead
Live
Amount
%Live
0.1
69.00
20.00
89.00
22.47
97.00
21.00
118.00
17.80
118.00
20.00
138.00
14.49
96.00
19.00
115.00
16.52
TOTAL
95.00
20.00
115.00
17.39
0.2
17.00
11.00
28.00
39.29
27.00
37.00
64.00
57.81
18.00
20.00
38.00
52.63
21.00
19.00
40.00
47.50
TOTAL
20.75
21.75
42.50
51.18
0.3
66.00
37.00
103.00
35.92
77.00
24.00
101.00
23.76
Page 83 of 93
Roskilde University
63.00
20.00
83.00
24.10
60.00
17.00
77.00
22.08
TOTAL
66.50
24.50
91.00
26.92
0.4
64.00
23.00
87.00
26.44
82.00
23.00
105.00
21.90
96.00
25.00
121.00
20.66
98.00
21.00
119.00
17.65
TOTAL
85.00
23.00
108.00
21.30
0.5
13.00
16.00
29.00
55.17
18.00
25.00
43.00
58.14
23.00
67.00
90.00
74.44
22.00
95.00
117.00
81.20
19.00
37.91
170.50
21.02
50.75
69.75
72.76
26.47
Dead
Live
Amount
32.00
14.00
46.00
30.43
52.00
9.00
61.00
14.75
23.00
15.00
38.00
39.47
37.00
18.00
55.00
32.73
TOTAL
36.00
14.00
50.00
28.00
5.5
39.00
46.00
85.00
54.12
42.00
46.00
88.00
52.27
221.00
91.00
25.00
246.00
10.16
22.88

SD sample of total amount
(numeral)
Sample name
5.6
%Live
Page 84 of 93
Roskilde University
27.00
118.00
TOTAL
98.25
36.00
134.25
26.82
5.4
44.00
75.00
119.00
63.03
56.00
19.00
75.00
25.33
TOTAL
50.00
47.00
97.00
48.45
5.3
25.00
8.00
33.00
24.24
24.00
18.00
42.00
42.86
21.00
16.00
37.00
43.24
21.00
20.00
41.00
48.78
TOTAL
22.75
15.50
38.25
40.52
5.2
71.00
14.00
85.00
16.47
67.00
22.00
89.00
24.72
53.00
34.00
87.00
39.08
51.00
96.00
147.00
65.31
TOTAL
60.50
41.50
102.00
40.69
5.1
68.00
70.00
138.00
50.72
25.00
20.00
45.00
44.44
24.00
13.00
37.00
35.14
49.00
44.00
93.00
47.31
41.50
39.95
182.17
9.27
36.75
78.25
46.96
Live
Amount
TOTAL
(numeral)
Sample name
4.6
31.77
Dead
%Live
81.00
42.00 123.00
34.15
55.00
27.00 82.00
32.93
Page 85 of 93
Roskilde University
22.00
23.00 45.00
51.11
52.00
31.00 83.00
37.35
52.50
30.75 83.25
36.94
164.00
62.00 226.00
27.43
87.00
73.00 160.00
45.63
83.00
33.00 116.00
28.45
78.00
63.00 141.00
44.68
TOTAL
103.00
57.75 160.75
35.93
4.4
185.00
12.00 197.00
6.09
38.00
12.00 50.00
24.00
15.00
21.00 36.00
58.33
35.00
32.00 67.00
47.76
TOTAL
68.25
19.25 87.50
22.00
4.3
23.00
28.00 51.00
54.90
26.00
15.00 41.00
36.59
19.00
21.00 40.00
52.50
28.00
12.00 40.00
30.00
24.00
19.00 43.00
44.19
TOTAL
4.5
TOTAL
4.2
TOTAL
4.1
7.00
22.00
10.00
5.00
22.00
7.00
13.00
15.00
13.00
29.00
75.86
15.00
33.33
29.00
24.14
28.00
53.57
12.25 25.25
48.51
21.00
20.00
33.00
36.00
41.00
48.78
Page 86 of 93
TOTAL
(numeral)
3.6
Roskilde University
69.00
52.17
50.00
64.00
44.00
45.45
27.00 51.00
52.94
18.00
32.00
24.00
20.00
24.00
40.08
150.25
10.06
44.07
106.00
14.00
120.00
11.67
42.00
24.00
66.00
36.36
67.00
18.00
85.00
21.18
38.00
33.00
71.00
46.48
63.25
22.25
85.50
26.02
74.00
28.00
102.00
27.45
54.00
47.00
101.00
46.53
32.00
14.00
46.00
30.43
TOTAL
53.33
29.67
83.00
35.74
3.4
128.00
36.00
164.00
21.95
55.00
9.00
64.00
14.06
39.00
12.00
51.00
23.53
119.00
7.00
126.00
5.56
TOTAL
85.25
16.00
101.25
15.80
3.3
75.00
20.00
95.00
21.05
34.00
35.00
69.00
50.72
39.00
24.00
63.00
38.10
12.00
25.00
37.00
67.57
TOTAL
3.5
Page 87 of 93
Roskilde University
TOTAL
40.00
26.00
66.00
39.39
3.2
20.00
32.00
52.00
61.54
18.00
11.00
29.00
37.93
6.00
8.00
14.00
57.14
14.00
27.00
41.00
65.85
TOTAL
14.50
19.50
34.00
57.35
3.1
20.00
13.00
33.00
39.39
27.00
16.00
43.00
37.21
10.00
27.00
37.00
72.97
16.00
25.00
41.00
60.98
18.25
37.82
136.08
14.32
20.25
38.50
52.60
10.00
7.00
17.00
41.18
5.00
22.00
27.00
81.48
17.00
64.00
81.00
79.01
19.00
22.00
41.00
53.66
TOTAL
12.75
28.75
41.50
69.28
2.5
17.00
31.00
48.00
64.58
8.00
36.00
44.00
81.82
12.00
34.00
46.00
73.91
9.00
19.00
28.00
67.86
TOTAL
11.50
30.00
41.50
72.29
2.4
44.00
28.00
72.00
38.89
TOTAL
(numeral)
2.6
24.72
Page 88 of 93
Roskilde University
27.00
27.00
54.00
50.00
44.00
30.00
74.00
40.54
55.00
25.00
80.00
31.25
TOTAL
42.50
27.50
70.00
39.29
2.3
23.00
41.00
64.00
64.06
25.00
44.00
69.00
63.77
42.00
61.00
103.00
59.22
56.00
74.00
130.00
56.92
TOTAL
36.50
55.00
91.50
60.11
2.2
24.00
7.00
31.00
22.58
21.00
8.00
29.00
27.59
16.00
13.00
29.00
44.83
24.00
6.00
30.00
20.00
TOTAL
21.25
8.50
29.75
28.57
2.1
26.00
14.00
40.00
35.00
22.00
18.00
40.00
45.00
27.00
22.00
49.00
44.90
40.00
10.00
50.00
20.00
28.75
50.88
106.33
17.04
16.00
44.75
35.75
TOTAL
(numeral)
1.6
20.99
21.00
24.00 45.00
53.33
10.00
51.00 61.00
83.61
10.00
33.00 43.00
76.74
Page 89 of 93
Roskilde University
19.00
22.00 41.00
53.66
15.00
32.50 47.50
68.42
5.00
31.00 36.00
86.11
9.00
36.00 45.00
80.00
18.00
34.00 52.00
65.38
16.00
19.00 35.00
54.29
TOTAL
12.00
30.00 42.00
71.43
1.4
54.00
30.00 84.00
35.71
65.00
59.00 124.00
47.58
65.00
30.00 95.00
31.58
55.00
26.00 81.00
32.10
TOTAL
59.75
36.25 96.00
37.76
1.3
26.00
28.00 54.00
51.85
58.00
48.00 106.00
45.28
22.00
27.00 49.00
55.10
35.00
21.00 56.00
37.50
TOTAL
35.25
31.00 66.25
46.79
1.2
20.00
14.00 34.00
41.18
5.00
23.00 28.00
82.14
11.00
30.00 41.00
73.17
18.00
15.00 33.00
45.45
TOTAL
13.50
20.50 34.00
60.29
1.1
15.00
39.00 54.00
72.22
15.00
16.00
21.00 36.00
26.00
58.33
TOTAL
1.5
Page 90 of 93
TOTAL
(numeral)
Roskilde University
42.00
61.90
26.00
36.00 62.00
58.06
18.00
30.50 48.50
62.89
57.93
111.42
11.93
20.47
Page 91 of 93

Antifreeze Proteins Enhance Survival of Cells in Cryopreservation - Substituting DMSO With RmAFP#1 in Cryopreservation of Cells 1.2

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Antifreeze Proteins Enhance Survival of Cells in Cryopreservation - Substituting DMSO With RmAFP#1 in Cryopreservation of Cells 1.2

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What is cryopreservation and why is it important?

What is cryopreservation and why is it important?

What are the main causes of freezing damage to cells?

What are the main causes of freezing damage to cells?

Antifreeze Proteins Enhance Survival of

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Theoretic summary and elaboration on the hypothesis .................................................................. 26

Cell viability determination ........................................................................................................... 34

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Cryopreservation: experimental outline for RmAFP#1 and DMSO experiments.......................... 36

DMSO variation experiment constant RmAFP#1 .......................................................................... 51

DMSO and RmAFP#1 variation experiment ................................................................................. 54

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Appendix #2: Solutions and standard procedures .......................................................................... 67

Appendix #4: Data from experiments ............................................................................................ 76

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Definitions for selected words

Antifreeze proteins or Ice structuring proteins

Nomenclature list and abbreviations

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Bacterial contamination testing

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Cryopreserving living cells or tissues is essential in a variety of areas, such as in assisted

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Figure 2: The structure of Dimethyl Sulfoxide (DMSO). The

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Ice crystallization and nucleation

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