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Basic Steps and Cytotoxicity Studies. Cell Culture:: Rita Nieto Montesinos, Phd. Ucsm, July 2018

This document discusses basic steps for cell culture including obtaining cells from suppliers, growing and maintaining cell lines, techniques for subculturing and cryopreserving cells, and considerations for the culture environment including media, pH, temperature, and CO2 levels. It also covers cytotoxicity assays for analyzing the effects of drugs or toxic compounds on cell viability and function.

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29 views41 pages

Basic Steps and Cytotoxicity Studies. Cell Culture:: Rita Nieto Montesinos, Phd. Ucsm, July 2018

This document discusses basic steps for cell culture including obtaining cells from suppliers, growing and maintaining cell lines, techniques for subculturing and cryopreserving cells, and considerations for the culture environment including media, pH, temperature, and CO2 levels. It also covers cytotoxicity assays for analyzing the effects of drugs or toxic compounds on cell viability and function.

Uploaded by

Cielo
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Cell culture:

Basic steps and


cytotoxicity studies.
Rita Nieto Montesinos, PhD.
UCSM, July 2018.
Introduction
In silico: Computer simulation
In vitro studies:
 Cells
 Tissues
 Organs
In vivo studies:
 PK
 BD
 PD
Clinical trials: Phase I, II, III and IV, if it is possible…
Cell culture
Removal of cells from a plant, animal or human being and
their subsequent growth in a favorable artificial environment.
Cell culture
Resected tissue
Subcultured

Primary culture Confluence


Subucultured

Secondary culture Confluence


Subcultred

Cell line Confluence


Subcultured

Cell strain
Cloning

Finite culture Continuos culture


Senescence Immortalization
Cells
Suppliers: American Type Culture Collection (ATCC).
 Human
 Animal
 Insect
 Fish
 Stem cell lines
Caco-2 [Caco2] (ATCC® HTB-37™)
Organism Homo sapiens, human
Tissue Colon
Cell Type Epithelial cells
Product Format Frozen
Morphology Epithelial-like
Culture Properties Adherent
Biosafety Level 1
Disease Colorectal adenocarcinoma
Age 72 years
Gender Male
Ethnicity Caucasian

Applications This cell line is a suitable transfection host.

Storage Conditions Liquid nitrogen vapor phase


Caco-2 [Caco2] (ATCC® HTB-37™)
Eagle's Minimum Essential Medium + fetal
Complete Growth Medium
bovine serum to a final concentration of 20%.

Inoculum is 1 X 104 viable cells/cm2.


Subculture cells when they are about 80%
Subculturing confluent, at a cell concentration between 8 x
104 and 1 x 105 cell/cm2.
Medium Renewal: 1 to 2 times per week

Atmosphere: air, 95%; CO2, 5%


Culture Conditions
Temperature: 37°C

Cryopreservation Liquid nitrogen vapor temperature


Cryopreservation of cells
Protective agent: DMSO or glycerol.
T°: < -130°C.
Vapor or liquid nitrogen.
Culture environment
Media

T° Cells pH

CO2
Culture environment - Media
Provides: Nutrients
Growth factors
Growth hormones
Regulates: pH
Osmotic pressure.

Tissue extracts and body fluids → defined media.


Culture environment - Media
Inorganic salts:
CaCl2, Fe(NO3)3.9H2O, MgSO4, KCl, HCO-3,
NaCl and NaH2PO4.
Amino acids:
L-Alanyl, L-Glutamine,
L-Arginine.HCl, L-Cystine.2HCl, etc.
Vitamins:
Choline chloride, folic acid, myo-inositol,
niacinamide, D-pantothenic acid,
Pyridoxal.HCl, Riboflavin and Thiamine.HCl.
Others:
D-glucose, HEPES, Phenol Red.Na and
Pyruvic acid.Na.
Dubelcco’s Modified Eagle’s Medium
(DMEM)
Culture environment - Media
Antibiotics (strep/pen)
NEAA
FBS

Adjust pH (HCl/NaOH)
Stock at 4°C/-20°C.
Thaw and use at 37°C.
Culture environment - pH
Mammalian and human cell lines: pH 7.35 – 7.45.
Some transformed cell lines: pH 7.0 – 7.4.
Some normal fibroblast: pH 7.4 – 7.7.
pH < 6.8 → death

acidic pH neutral pH basic pH

rapid metabolism high T°


contamination chemical corruption
Culture environment – CO2
Medium buffered by CO2 /H2CO3
H2O + CO2 H2CO3

→ exogenous CO2
(incubator)
Culture environment – T°
Mammalian and human cell lines: 36 – 37°C.
→ incubator
Culture environment - Media

Adherent culture (monolayers) Suspension culture


Trypsination
Dissociation of adherent cells from the flasks using trypsin
(proteolytic enzyme which breaks down proteins).
Trypsination
Protocol:
 Remove medium
 Wash with PBS
 Add trypsine
 Incubate at 37°C for 6’
 Add medium (FBS)
 Centrifuge at 1000 g for 6’
 Remove supernatant
 Add 1 mL medium (Blue Trypan)
Safety
Hazards:
 Human or animal cells and tissues.
 Toxic, corrosive, or mutagenic reagents.

Risks:
 Accidental punctures (syringe needles or contaminated
sharps) onto skin and mucous membranes.
 Ingestion through mouth pipetting.
 Inhalation exposures to infectious aerosols.
Biosafety
Objective: Reduce or eliminate exposure of laboratory
workers and the outside environment to potentially harmful
biological agents.

→ Standard microbiological practices and


techniques.
BSL (1 – 4): Asepsis.
Safety equipment – primary barriers
Pipettes and pipettors

Aspiration systems
Safety equipment – primary barriers
Flow laminar hoods Waste containers
Safety equipment - disinfectants
EtOH 70% Detergents
Personal protective equipment (PPE)
Cell culture basics
https://www.youtube.com/watch?v=kbhQ29vRqs4
Passing cells: Trypsination
https://www.youtube.com/watch?v=7hEUmLz1Ew0
Contamination
Chemical: Biological:
 Water • Bacteria
 Serum • Molds
 Plasticizers • Yeasts
 Detergents • Viruses
• Mycoplasma

Cross contamination by
other cell lines.
Cell culture applications:
 Physiology and biochemistry of cells (metabolic and aging
studies).
 Manufacture of biological compounds (vaccines and
therapeutic proteins).
 Drug screening and development.
 Effects of drugs and toxic compounds on the cells.
 Mutagenesis and carcinogenesis.
Cytotoxicity
The quality of inducing interference in structures and/or
processes essential for cell function, proliferation or survival.

Cells response:
 Lose membrane integrity (cell lysis).
 Stop growing and dividing (viability decrease).
 Activate a genetic program of controlled cell death
(apoptosis).
Cytotoxicity assays
Trypan blue:
Cytotoxicity assays – Trypan blue
Usage:While passaging (after trypsination).

Protocol:
 Before culturing, take 50 µL of cells suspension.
 Mix with 50 µL of trypan blue.
 Count all the viable cells (not stained) in three squares of the
hemocytometer chamber.
Cytotoxicity assays – Trypan blue
Cytotoxicity assays – Trypan blue

Advantages: Disadvantages:

 Routine material  Not selective


 Cheap  Healthy cells?
 Rapid  Are cells losing their
functions?

→ Supplementary assays !
Cytotoxicity assays
Cytotoxicity assays
MTT: Colorimetric

Viable cell
Cytotoxicity assays – MTT
Usage: Effects of drugs or toxic compounds.

Protocol (1):
 Remove medium and wash with PBS.
 Add samples.
 Incubate at 37°C, 5% CO2 for 4h.
 Remove samples and wash with PBS.
Cytotoxicity assays – MTT
Usage: Effects of drugs or toxic compounds.

Protocol (2):
 Add 100 µL of MTT in PBS (4 mg/mL).
 Incubate at 37°C, 5% CO2 for 2h.
 Remove MTT from cells and wash with PBS.
 Add DMSO to dissolve formazan and read at 570 nm.
Cytotoxicity assays – MTT
Usage: Effects of drugs or toxic compounds.

Protocol (3):
Cytotoxicity assays – MTT

Advantages: Disadvantages:

 Routine material  At the confluence.


 Cheap  Colorimetric, not highly
 Easy sensitive.

→ For initial drug screening!


Cytotoxicity assay – MTT

https://www.youtube.com/watch?v=vn6enA6lSKs
References
• Gibco
• Dojindo
• Biorelevant media resistant co-culture model mimicking
permeability of human intestine.
Antoine D1, PellequerY1, Tempesta C1, Lorscheidt S2, Kettel
B2, Tamaddon L1, Jannin V3, Demarne F3, Lamprecht A4,
Béduneau A5. Int J Pharm. 2015 Mar 15;481(1-2):27-36.
doi: 10.1016/j.ijpharm.2015.01.028. Epub 2015 Jan 16.

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