NCI-PBCF-HTB38 (HT-29)

Colon Adenocarcinoma

(ATCC®HTB-38™)
February 27, 2012; Version 1.6
Table of Contents
1. BACKGROUND INFORMATION ON HT-29 CELL LINE.............................................................. 3

2. GENERAL INFORMATION FOR THE THAWING, PROPAGATING AND CRYOPRESERVING

OF NCI-PBCF- HTB38 (HT-29) ............................................................................................................. 3

3. REAGENTS .................................................................................................................................... 5

A. PREPARATION OF COMPLETE GROWTH MEDIUM (MCCOY’S 5A + 10 % (V/V) FBS) ............................ 5

4. THAWING AND PROPAGATION OF CELLS ............................................................................... 6

A. THAWING CELLS ............................................................................................................................ 6

B. PROPAGATING CELLS .................................................................................................................... 7

C. SUBCULTURING CELLS................................................................................................................... 8

5. HARVESTING OF CELLS FOR CRYOPRESERVATION............................................................. 9

6. CRYOPRESERVATION OF CELLS ............................................................................................ 11

A. CRYOPRESERVATION USING A RATE-CONTROLLED PROGRAMMABLE FREEZER ................................ 11

i. Using the Cryomed ............................................................................................................... 11

B. CRYOPRESERVATION USING “MR. FROSTY” .................................................................................. 12

7. STORAGE .................................................................................................................................... 13

APPENDIX 1: PHOTOMICROGRAPHS OF NCI-PBCF-HTB38 (HT-29) .......................................... 14

APPENDIX 2: GROWTH PROFILE FOR NCI-PBCF-HTB38 CELLS ............................................... 16

APPENDIX 3: CYTOGENETIC ANALYSIS OF NCI-PBCF-HTB38 (HT-29) CELLS ........................ 17

APPENDIX 4: GLOSSARY OF TERMS ............................................................................................. 20

APPENDIX 5: REFERENCES ............................................................................................................ 21

APPENDIX 6: REAGENT LOT TRACEABILITY AND CELL EXPANSION TABLES ...................... 24

APPENDIX 7: CALCULATION OF POPULATION DOUBLING LEVEL (PDL) ................................ 26

APPENDIX 8: SAFETY PRECAUTIONS ........................................................................................... 26

 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

Protocol for Thawing, Propagating and Cryopreserving of

NCI-PBCF-HTB38 (HT-29, ATCC®HTB-38™) cells

colorectal carcinoma

1. Background Information on HT-29 cell line

Designations: HT-29
Biosafety Level: 1
Shipped: frozen (in dry ice)
Growth Properties: adherent (see appendix 1)
Organism: Homo sapiens
Organ colon
Source:
Disease colorectal adenocarcinoma

For more information visit the ATCC webpage:

http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?A
TCCNum=HTB-38&Template=cellBiology

2. General Information for the thawing, propagating and

cryopreserving of NCI-PBCF- HTB38 (HT-29)

 The cryoprotectant (DMSO) should be removed by centrifugation.


5 2
The seeding density to use with a vial of HT-29 cells is about 5 x 10 cells/cm or
Culture Initiation
the content of 1 vial into a T-25 flask containing 10 mL complete growth medium
(McCoy’s 5a + 10 % (v/v) FBS).

 The complete growth medium used to expand HT-29 cells is McCoy’s 5a + 10 %

(v/v) FBS.
 Complete growth medium (McCoy’s 5a + 10 % (v/v) FBS) should be pre-warmed
Complete growth o o
before use by placing into a water bath set at 35 C to 37 C for 15 min to 30 min.
medium
 After 30 min, the complete growth medium (McCoy’s 5a + 10 % (v/v) FBS)
should be moved to room temperature until used. Complete growth medium
o o
(McCoy’s 5a + 10 % (v/v) FBS) should be stored at 2 C to 8 C when not in use.


o o
The growth temperature for HT-29 is 37 C ± 1 C
Cell Growth
 A 5 % + 1 % CO2 in air atmosphere is recommended.

Growth Properties  Population doubling time (PDT) is approximately 23 h (Figure 4).

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 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

Special Growth  Subculture HT-29 cells at 80 % to 90 % confluence or when cell density is about
5 2
Requirements 5 x 10 viable cells/cm .

 0.25 % (w/v) trypsin-0.53 mM EDTA (ATCC cat no. 30-2101).

 Subculturing reagents should be pre-warmed before use by placing into a water
o o
bath set at 35 C to 37 C for 15 min to 30 min.
Subculture Medium
 After 30 min, the subculturing medium should be moved to room temperature
o o
until used. Subculturing reagents should be stored at 2 C to 8 C when not in
use.

 The attached HT-29 cells are subcultured using 0.25 % (w/v) trypsin-0.53 mM
EDTA (ATCC cat no. 30-2101).
 The enzymatic action of the trypsin-EDTA is stopped by adding complete growth
Subculture Method
medium to the detached cells.

4 4
A split ratio of 1:10 to 1:12 or a seeding density between 4 x 10 and 5 x 10
2
viable cells/cm is used when subculturing HT-29 cells.


6
Viable The target number of viable cells/mL/cryovial is: 3 x 10 (acceptable range: 3.0 x
6 6
Cells/mL/Cryovial 10 cells/mL to 4.0 x 10 cells/mL).

Cryopreservation  The cryopreservation medium for HT-29 cells is complete growth medium
Medium (McCoy’s 5a + 10 % (v/v) FBS) containing 5 % (v/v) DMSO (ATCC cat no. 4-X).

General Procedure to be applied throughout the SOP

 Use of good aseptic technique is critical. Any materials that are contaminated,
Aseptic Technique as well as any materials with which they may have come into contact, must be
disposed of immediately.

 Record the manufacturer, catalog number, lot number, date received, date
Traceability of
expired and any other pertinent information for all materials and reagents used.
material/reagents Record information in the Reagent Lot Traceability Table 4 (Appendix 6).

 Record the subculture and growth expansion activities, such as passage number,
% confluence, % viability, cell morphology (see Figures 1, 2,2- Appendix 1) and
Expansion of cell line
population doubling levels (PDLs), in the table for Cell Expansion (Table 5,
Appendix 6). Calculate PDLs using the equation in Appendix 7.

Medium volumes  Medium volumes are based on the flask size as outlined in Table 1.

Glossary of Terms  Refer to Glossary of Terms used throughout the document (see Appendix 4).

 Refer to Safety Precautions pertaining to the thawing, propagating and

Safety Precaution cryopreserving of HT-29 (see Appendix 8).

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 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

Table 1: Medium Volumes

Flask Size Medium Volume Range

2
12.5 cm (T-12.5) 3 mL to 6 mL
2
25 cm (T-25) 5 mL to 13 mL
2
75 cm (T-75) 10 mL to 38 mL
2
150 cm (T-150) 30 mL to 75 mL
2
175 cm (T-175) 35 mL to 88 mL
2
225 cm (T-225) 45 mL to 113 mL

3. Reagents

Follow Product Information Sheet storage and/or thawing instructions. Below is a list of
reagents for the propagation, subcultivation and cryopreservation of HT-29 cells.

Table 2: Reagents for Expansion, Subculturing and Cryopreservation

of HT-29 cells

Complete growth Subculturing reagents Cryopreservation

medium reagents medium reagents
McCoy’s 5a Medium Trypsin-EDTA (0.25 % (w/v)
McCoy’s 5a Medium Modified
Modified Trypsin/0.53 mM EDTA )
(ATCC cat no. 30-2007)
(ATCC cat no. 30-2007) (ATCC cat no. 30-2101)
Dulbecco’s Phosphate Buffered Saline
10 % (v/v) Fetal Bovine (DPBS); modified without calcium
10 % (v/v) FBS
Serum (FBS) chloride and without magnesium
(ATCC cat no. 30-2020)
(ATCC cat no. 30-2020) chloride
(ATCC cat no. 30-2200)
5 % (v/v) Dimethyl Sulfoxide
(DMSO)
(ATCC cat no. 4-X)

a. Preparation of complete growth medium (McCoy’s 5a + 10 %

(v/v) FBS)

The complete growth medium is prepared by aseptically combining:

1. 56 mL FBS (ATCC cat no. 30-2020) to a 500 mL bottle of basal medium – McCoy’s
5a (ATCC cat no. 30-2007).
2. Mix gently, by swirling.

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 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

4. Thawing and Propagation of Cells

Reagents and Material:

 Complete growth medium (McCoy’s 5a + 10 % (v/v) FBS)

 Water bath

 T-25 cm2 polystyrene flask

 15 mL polypropylene conical centrifuge tubes

 Plastic pipettes (1 mL,10 mL, 25 mL)

a. Thawing cells

Method:

1. Place complete growth medium (McCoy’s 5a + 10 % (v/v) FBS) in a water bath set at
35 oC to 37 oC. .

2. Label T-25 flask to be used with the (a) name of cell line, (b) passage number, (c)
date, (d) initials of technician.

3. Wearing a full face shield, retrieve a vial of frozen cells from the vapor phase of the
liquid nitrogen freezer.

4. Thaw the vial by gentle agitation in a water bath set at 35 oC to 37 oC. To reduce the
possibility of contamination, keep the O-ring and cap out of the water.

Note: Thawing should be rapid (approximately 2 min to 3 min, just long enough
for most of the ice to melt).

5. Remove vial from the water bath and process immediately.

6. Remove excess water from the vial by wiping with sterile gauze saturated with 70 %
ethanol.

7. Transfer the vial to a BSL-2 laminar-flow hood.

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SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

b. Propagating cells
Method:

1. Add 9 mL of complete growth medium (McCoy’s 5a + 10 % (v/v) FBS) to a 15-mL

conical centrifuge tube.

2. Again wipe the outer surface of the vial with sterile gauze wetted with 70 % ethanol.

3. Using sterile gauze, carefully remove the cap from the vial.

4. With a 1 mL pipette transfer slowly the completely thawed content of the vial (1 mL
cell suspension) to the 15-mL conical centrifuge tube containing 9 mL complete
growth medium (McCoy’s 5a + 10 % (v/v) FBS). Gently resuspend cells by pipetting
up and down.

5. Centrifuge at 125 xg, at room temperature, for 8 min to 10 min.

6. Carefully aspirate (discard) the medium, leaving the pellet undisturbed.

7. Using a 10 mL pipette, add 10 mL of complete growth medium (McCoy’s 5a + 10 %

(v/v) FBS).

8. Resuspend pellet by gentle pipetting up and down.

9. Using a 1 mL pipette, remove 1 mL of cell suspension for cell count and viability. Cell
counts are performed using either an automated counter (such as Innovatis Cedex
System; Beckman-Coulter ViCell system) or a hemocytometer.

10. Record total cell count and viability. When an automated system is used, attach
copies of the printed results to the record.

11. Plate cells in pre-labeled T-25 cm2 flask at about 1.6 x 105 cells/cm2.

12. Transfer flask to a 37 °C ± 1 °C in 5 % CO2 incubator if using flasks with vented caps
(for non-vented caps stream 5 % CO2 in the headspace of flask).

13. Observe culture daily by eye and under an inverted microscope to ensure culture is
free of contamination and culture has not reached confluence. Monitor, visually, the
pH of the medium daily. If the medium goes from red through orange to yellow,
change the medium.

14. Note: In most cases, cultures at a high cell density exhaust the medium faster
than those at low cell density as is evident from the change in pH. A drop in
pH is usually accompanied by an increase in cell density, which is an indicator
to subculture the cells. Cells may stop growing when the pH is between pH 7
to pH 6 and loose viability between pH 6.5 and pH 6.

15. If fluid renewal is needed, aseptically aspirate the complete growth medium from the
flask and discard. Add an equivalent volume of fresh complete growth medium to
the flask. Alternatively, perform a fluid addition by adding fresh complete growth

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SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

medium to the flask without removing the existing medium. Record fluid change or
fluid addition on the Cell Line Expansion Table (see Table 5 in Appendix 6).

16. If subculturing of cells is needed, continue to ‘Subculturing cells’.

Note: Subculture when cells are 80-90 % confluent (see photomicrographs in

Appendix 1).

c. Subculturing cells
Reagents and Material:

 0.25 % (w/v) Trypsin-0.53 mM EDTA

 DPBS
 Complete growth medium (McCoy’s 5a (ATCC cat no. 30-2007) + 10 % (v/v) FBS
(ATCC cat no. 30-2020)
 Plastic pipettes (1 mL, 10 mL, 25 mL)
 T-75 cm2, T-225 cm2 polystyrene flasks

Method:

1. Aseptically remove medium from the flask

2. Add appropriate volumes of sterile Ca2+- and Mg2+-free DPBS to the side of the flask
opposite the cells so as to avoid dislodging the cells (see Table 3).

3. Rinse the cells with DPBS (using a gently rocking motion) and discard.

4. Add appropriate volume of 0.25 % (w/v) Trypsin-0.53 mM EDTA solution to the flask
(see Table 3).

5. Incubate the flask at 37 oC ± 1 °C until the cells round up. Observe cells under an
inverted microscope every 5 min. When the flask is tilted, the attached cells should
slide down the surface. This usually occurs after 5 min to 10 min of incubation.

Note: Do not leave trypsin-EDTA on the cells any longer than necessary as
clumping may result.

6. Neutralize the trypsin-EDTA/cell suspension by adding an equal volume of complete

growth medium (McCoy’s 5a + 10 % (v/v) FBS) to each flask. Disperse the cells by
pipetting, gently, over the surface of the monolayer. Pipette the cell suspension up
and down with the tip of the pipette resting on the bottom corner or edge until a
single cell suspension is obtained. Care should be taken to avoid the creation of
foam.

7. Using a 1 mL pipette, remove 1 mL of cell suspension for total cell count and

viability.

8. Record total cell count and viability.

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 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

9. Add appropriate volume of fresh complete growth medium (McCoy’s 5a + 10 % (v/v)

FBS) and transfer cell suspension (for volume see Table 1 on page 4) into new pre-
labeled flasks at a seeding density of about 5 x 104 viable cells/cm2 or a split ratio of
1:10 to 1:12.

10. Label all new flasks with the (a) name of cell line, (b) passage number, (c) date, (d)
initials of technician.

Table 3: Volume of Rinse Buffer and Trypsin

Flask Flask DPBS Rinse Trypsin-EDTA

Buffer
Type Size
2
T-flask 12.5 cm (T-12.5) 1 mL to 3 mL 1 mL to 2 mL
2
25 cm (T-25) 1 mL to 5 mL 1 mL to 3 mL
2
75 cm (T-75) 4 mL to 15 mL 2 mL to 8 mL
2
150 cm (T-150) 8 mL to 30 mL 4 mL to 15 mL
2
175 cm (T-175) 9 mL to 35 mL 5 mL to 20 mL
2
225 cm (T-225) 10 mL to 45 mL 5 mL to 25 mL

5. Harvesting of Cells for Cryopreservation

Reagents and Material:

 0.25 % (w/v) Trypsin-0.53 mM EDTA

 DPBS
 Complete growth medium (McCoy’s 5a (ATCC cat no. 30-2007) + 10 % (v/v) FBS
(ATCC cat no. 30-2020))
 50 mL or 250 mL conical centrifuge tube
 Plastic pipettes (1 mL, 10 mL, 25 mL)

 Sterile DMSO

 1 mL to 1.8 mL cryovials

 Ice bucket with ice

Method:

1. Label cryovials to include information on the (a) name of cell line, (b) passage

number (c) date.

2. Prepare cryopreservation medium by adding DMSO to cold complete growth medium

(McCoy’s 5a + 10 % (v/v) FBS) at a final concentration of 5 % (v/v) DMSO. Place
cryopreservation medium on ice until ready to use.

3. Aseptically remove medium from the flask(s).

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SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

4. Add appropriate volumes of sterile Ca2+- and Mg2+-free DPBS to the side of the flask
so as to avoid dislodging the cells (see Table 3).

5. Rinse the cells with DPBS (using a gentle rocking motion) and discard.

6. Add appropriate volume of 0.25 % (w/v) Trypsin-0.53 mM EDTA solution to the flask
(see Table 3).

7. Incubate the flask at 37 oC ± 1 °C until the cells round up. Observe cells under an
inverted microscope every 5 min. When the flask is tilted, the attached cells should
slide down the surface. This usually occurs after 5 min to 10 min of incubation.

Note: Do not leave trypsin-EDTA on the cells any longer than necessary as
clumping may result.

8. Neutralize the trypsin-EDTA/cell suspension by adding an equal volume of complete

growth medium (McCoy’s 5a + 10 % (v/v) FBS) to each flask. Disperse the cells by
pipetting, gentle, over the surface of the monolayer. Pipette the cell suspension up
and down with the tip of the pipette resting on the bottom corner or edge until a
single cell suspension is obtained. Care should be taken to avoid the creation of
foam.

9. Using a 1 mL pipette, remove 1 mL of cell suspension for total cell count and

viability.

10. Record total cell count and viability.

11. Spin cells at approximately 125 xg for 5 min to 10 min at room temperature.

Carefully aspirate and discard the medium, leaving the pellet undisturbed

12. Calculate volume of cryopreservation medium based on the count performed at step
9 and resuspend pellet in cold cryopreservation medium at a viable cell density of 3 x
106 viable cells/mL (acceptable range: 3.0 x 106 viable cells/mL to 4.0 x 106 viable
cells/mL by gentle pipetting up and down..

13. Dispense 1 mL of cell suspension, using a 5 mL or 10 mL pipette, into each 1.0 mL

cryovial.

14. Place filled cryovials at 2 oC to 8 oC until ready to cryopreserve. A minimum

equilibration time of 10 min but no longer than 45 min is necessary to allow DMSO to
penetrate the cells.

Note: DMSO is toxic to the cells. Long exposure in DMSO may affect viability.

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 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

6. Cryopreservation of Cells

Material:

 Liquid nitrogen freezer

 Cryomed Programmable freezer (Forma Scientific, cat no. 1010) or

 Mr. Frosty (Nalgene, cat no. 5100)

 Isopropanol
 Cryovial rack

a. Cryopreservation using a rate-controlled programmable freezer

Method:

A slow and reproducible cooling rate is very important to ensure good recovery of
cultures. A decrease of–1 °C per min to -80 °C followed by rapid freeze at about 15 °C to
30 °C per min drop to -150 °C usually will usually work for most animal cell cultures. The
best way to control the cooling process is to use a programmable, rate-controlled,
electronic freezer unit. Refer to the manufacturer’s handbook for detailed procedure.

i. Using the Cryomed

Starting the Cryopreservation Process

1. Check that the liquid nitrogen valve that supplies the Cryomed is open.

2. Check the gauge to ensure that there is enough liquid nitrogen in the open tank to
complete the freeze.

3. Install the thermocouple probe so that the tip is immersed midway into the control
fluid

Note: Be sure that the thermocouple is centered in the vial and the vial is
placed centered in the rack. The probe should be changed after three uses or
if it turns yellow to ensure accurate readings by the controller during the
freezing process. Old medium may have different freezing characteristics.

4. Close and latch Cryomed door.

5. Turn on microcomputer, computer and monitor.

6. Double click the “Cryomed” icon. The machine may need to be pre-programmed for
specific cell type and medium.

7. From the top of the screen, select MENU  RUN FUNCTIONS  START RUN.

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SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

8. Fill out the box which appears on the screen. Cell line ID; TYPE OF SAMPLE;
MEDIA; NUMBER OF SAMPLES.

9. Hit the ESCAPE key and the Cryomed will cool to 4C.

10. Once Cryomed chamber has cooled to 4 C, load cryovials onto racks and close the
door.

11. When the Cryomed’s chamber temperature and the sample temperature have
reached approximately 4C; press the space bar to initiate the rate controlled
cryopreservation process.

Completing the Cryopreservation Process

1. When samples have reached -80C, an alarm will sound. To silence this, select
ALARM from the options at the top of the screen.
2. Select MENU RUN FUNCTIONS→ STOP. Hit the ESCAPE key to return to the
main menu and select EXIT.
3. Immediately transfer vials to liquid nitrogen freezer.
4. Shut down the microcomputer and then turn off the monitor.

b. Cryopreservation using “Mr. Frosty”

1. One day before freezing cells, add 250 mL isopropanol to the bottom of the container
and place at 2 oC to 8 oC.

2. On the day of the freeze, prepare cells for cryopreservation as described above.

3. Insert cryovials with the cell suspension in appropriate slots in the container.

4. Transfer the container to a -70 °C to -90 °C freezer and store overnight.

5. Next day, transfer cryovials to the vapor phase of liquid nitrogen freezer.

Note: Each container has 18 slots which can accommodate 18 cryovials in one
freeze.

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Important information when using the rate-controlled programmable freezer or a

manual method (Mr. Frosty) for cryopreservation of mammalian cells.

 Regardless which cooling method is used, it is important that the transfer to the final
storage location (between -130 °C and -196 °C) be done quickly and efficiently. If the
transfer cannot be done immediately, the vials can be placed on dry ice for a short
time. This will avoid damage to cultures by inadvertent temporary warming during the
transfer process. Warming during this transfer process is a major cause of variation
in culture viability upon thawing.

 Always keep the storage temperature below -130 °C for optimum survival. Cells may
survive storage at higher temperatures but viability will usually decrease over time.
The ideal storage container is a liquid nitrogen freezer where the cultures are stored
in the vapor phase above the liquid nitrogen.

Note: ATCC does not have experience in the cryopreservation of the HT-29 cells
by any other method than the Cryomed programmable freezer.

7. Storage

 Store cryopreserved cells in the vapor phase of liquid nitrogen freezer (below -130
°C) for optimum long-term survival.

Note: Experiments on long-term storage of animal cell lines at different

temperature levels indicate that a -70 °C storage temperature is not adequate
except for very short period of time. A -90 °C storage may be adequate for longer
periods depending upon the cell line preserved. The efficiency of recovery,
however, is not as great as when the cells are stored in vapor phase of the liquid
nitrogen freezer.

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APPENDIX 1: PHOTOMICROGRAPHS OF NCI-PBCF-HTB38 (HT-29)

Figure 1: Photomicrograph of HT-29 cells after one day, post-freeze recovery.

Cells were plated at 1.6 x 105 viable cells/cm2.

Figure 2: Photomicrograph of HT-29 cells after two days, post-freeze

recovery. Cells were plated at 1.6 x 105 viable cells/cm2.

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40X 500 µm 40X 500 µm 40X 500 µm

100X 200 µm 100X 200 µm 100X 200 µm

Figure 3: Photomicrographs of HT-29 cells at various time points after seeding

at a cell density of 5 x 104 viable cells/cm2.

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APPENDIX 2: GROWTH PROFILE FOR NCI-PBCF-HTB38 CELLS

9.00E+05

8.00E+05

7.00E+05

6.00E+05
Viable Cells/cm2

5.00E+05

4.00E+05

3.00E+05

2.00E+05

1.00E+05

0.00E+00
0 1 2 3 4 5 6 7
Days in Culture

Figure 4: Growth curve for HT-29 cells; cells were plated at 5 x 104 viable
cells/cm2; population doubling time (PDT) is approximately 23 h.

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APPENDIX 3: CYTOGENETIC ANALYSIS OF NCI-PBCF-HTB38 (HT-29) CELLS

NCI-PBCF-HTB38 Karyotype Results

Metaphase Spread Karyotype

Number of metaphase spreads counted 20

Band level 300-400

Number of metaphase spreads karyotyped 10

Chromosome range 67-72

Sex Female

Comments Aneuploid*

Karyotype:
67-72,XX,-X,add(3)(p21),ins(3)(p11.2;?),add(4)(q25),der(6)t(6;14)(q23;q13),+7,
add(7)(p10),-8x2,dup(9)(q13q22),+10,del(10)(p12),+11,-13,add(13)(p10),i(13)(q10),
-14x2,+15,del(17)(p12),-18,+19,dup(19)(q13.1q13.3),+20,-21x2,-22,+mar1,+mar2,
+mar3,+mar4,+mar5
(ISCN nomenclature written based on a triploid karyotype).
*Human diploid karyotype (2N): 46,XX (female) or 46,XY (male)

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Karyotype Summary:
In the karyotype image, arrows indicate regions of abnormality. It should be noted that the
karyotype description includes the observed abnormalities from all examined metaphase
spreads, but due to heterogeneity, not all of the karyotyped cells will contain every
abnormality.
This is a highly rearranged human cell line of female origin containing 67 to 72
chromosomes per metaphase spread (hypotriploid to hypertriploid). Structural abnormalities
include rearrangements to approximately 45% of the 22 different autosomal chromosomes.
Also, there are five unidentifiable clonal marker chromosomes (markers present in two or
more of the examined cells) [+mar1 through +mar5].
The rearrangements include:
 Addition of unknown material to the short arms (designated by p) of chromosomes 3,
7, 13;
 Addition of unknown material to the long arm (designated by q) of chromosome 4;
 Deletion of material from the short arms of chromosomes 10 and 17;
 A translocation involving chromosomes 6 and 14 [der(6)t(6;14)(q23;q13)];
 Isochromosome 13 [i(13)(p10)];
 Duplications of chromosomal DNA involving chromosomes 9 [dup(9)(q13q22)] and
19 [dup(19)(q13.1q13.3];
 An insertion of unknown material on the short arm of chromosome 3

[ins(3)(p11.2;?)].

Numerical changes are based on a triploid karyotype which would contain three copies of
each chromosome (3N). Therefore, karyotype designations such as -13 and -18, indicates
two copies of structurally normal chromosomes 13 and 18; +7 indicates four copies of
chromosome 7. (ISCN 2009: An International System for Human Cytogenetic Nomenclature
(2009), Editors: Lisa G. Shaffer, Marilyn L. Slovak, Lynda J. Campbell)

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Karyotype Procedure:
 Cell Harvest: Cells were allowed to grow to 80-90% confluence. Mitotic division was
arrested by treating the cells with KaryoMax® colcemid for 20 minutes to 2 hours at 37°C.
Cells were harvested using 0.05% Trypsin-EDTA, treated with 0.075M KCL hypotonic
solution, and then fixed in three changes of a 3:1 ratio of methanol;glacial acetic acid.

 Slide Preparation: Slides were prepared by dropping the cell suspension onto wet glass
slides and allowing them to dry under controlled conditions.

 G-banding: Slides were baked one hour at 90°C, trypsinized using 10X trypsin-EDTA,
and then stained with Leishman’s stain.

 Microscopy: Slides were scanned using a 10X objective and metaphase spreads were
analyzed using a 100X plan apochromat objective on an Olympus BX-41 microscope.
Imaging and karyotyping were performed using Cytovision® software.

 Analysis: Twenty metaphase cells were counted and analyzed, and representative
metaphase cells were karyotyped depending on the complexity of the study.

Summary of Karyotyping Procedure:

G-band karyotyping analysis is performed using GTL banding technique: G bands produced
with trypsin and Leishman. Slides prepared with metaphase spreads are treated with trypsin
and stained with Leishman’s. This method produces a series of light and dark bands that
allow for the positive identification of each chromosome.

HT-29 cell line karyotyping was carried out by Cell Line Genetics, Inc. (Madison, WI 53719)

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APPENDIX 4: GLOSSARY OF TERMS

Confluent monolayer: adherent cell culture in which all cells are in contact with other cells
all around their periphery and no available substrate is left uncovered.

Split ratio: the divisor of the dilution ration of a cell culture to subculture (e.g., one flask
divided into four, or 100 mL up to 400 mL, would be split ratio of 1:4).

Subculture (or passage): the transfer or transplantation of cells, with or without dilution,
from one culture vessel to another.

Passage No: the total number of times the cells in the culture have been subcultured or
passaged (with each subculture the passage number increases by 1).

Population doubling level (PDL): the total number of population doublings of a cell line
since its initiation in vitro (with each subculture the population doubling increases in
relationship to the split ratio at which the cells are plated). See Appendix 7.

Population doubling time (doubling time): the time interval, calculated during the
logarithmic phase of growth in which cells double in number.

Seeding density: recommended number of cells per cm2 of substrate when inoculating a
new flask.

Epithelial-like: adherent cells of a polygonal shape with clear, sharp boundaries between
them.

Fibroblast-like: adherent cells of a spindle or stellate shape.

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APPENDIX 5: REFERENCES
1. Culture Of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, 6th
edition, published by Wiley-Liss, N.Y., 2010.
2. Didier ES, et al. Characterization of Encephalitozoon (Septata) intestinailis isolates
cultured from nasal mucosa and bronchoalveolar lavage fluids of two AIDS patients.
J. Eukaryot. Microbiol. 43: 34-43, 1996. PubMed: 8563708

3. Human tumor cells in vitro. New York: Plenum Press; 1975.

4. Chen TR, et al. WiDr is a derivative of another colon adenocarcinoma cell line, HT­
29. Cancer Genet. Cytogenet. 27: 125-134, 1987. PubMed: 3472642

5. Fogh J, et al. Absence of HeLa cell contamination in 169 cell lines derived from
human tumors. J. Natl. Cancer Inst. 58: 209-214, 1977. PubMed: 833871

6. Fogh J, et al. One hundred and twenty-seven cultured human tumor cell lines
producing tumors in nude mice. J. Natl. Cancer Inst. 59: 221-226, 1977. PubMed:
327080

7. Adachi A, et al. Productive, persistent infection of human colorectal cell lines with
human immunodeficiency virus. J. Virol. 61: 209-213, 1987. PubMed: 3640832

8. Fantini J, et al. Human colon epithelial cells productively infected with human
immunodeficiency virus show impaired differentiation and altered secretion. J. Virol.
66: 580-585, 1992. PubMed: 1727501

9. Butzow R, et al. A 60-kD protein mediates the binding of transforming growth factor-
beta to cell surface and extracellular matrix proteoglycans. J. Cell Biol. 122: 721-727,
1993. PubMed: 8335695

10. Trainer DL, et al. Biological characterization and oncogene expression in human
colorectal carcinoma cell lines. Int. J. Cancer 41: 287-296, 1988. PubMed: 3338874

11. Hanski C, et al. Tumorigenicity, mucin production and AM-3 epitope expression in
clones selected from the HT-29 colon carcinoma cell line. Int. J. Cancer 50: 924-929,
1992. PubMed: 1372882

12. Reiter LS, et al. The role of the urokinase receptor in extracellular matrix degradation
by HT29 human colon carcinoma cells. Int. J. Cancer 53: 444-450, 1993. PubMed:
8381394

13. Barnett SW, et al. Characterization of human immunodeficiency virus type 1 strains
recovered from the bowel of infected individuals. Virology 182: 802-809, 1991.
PubMed: 2024498

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14. Shabahang M, et al. 1,25-Dihydroxyvitamin D3 receptor as a marker of human colon

carcinoma cell line differentiation and growth inhibition. Cancer Res. 53: 3712-3718,
1993. PubMed: 8393379

15. Lesuffleur T, et al. Differential expression of the human mucin genes MUC1 to MUC5
in relation to growth and differentiation of different mucus-secreting HT- 29 cell
subpopulations. J. Cell Sci. 106: 771-778, 1993. PubMed: 8308060

16. Pollack MS, et al. HLA-A, B, C and DR alloantigen expression on forty-six cultured
human tumor cell lines. J. Natl. Cancer Inst. 66: 1003-1012, 1981. PubMed: 7017212

17. Fantini J, et al. Infection of colonic epithelial cell lines by type 1 human
immunodeficiency virus is associated with cell surface expression of
galactosylceramide, a potential alternative gp120 receptor. Proc. Natl. Acad. Sci.
USA 90: 2700-2704, 1993. PubMed: 8464878

18. Devedjian JC, et al. Regulation of the alpha 2A-adrenergic receptor in the HT29 cell
line. Effects of insulin and growth factors. J. Biol. Chem. 266: 14359-14366, 1991.
PubMed: 1677644

19. Santoro IM, Groden J. Alternative splicing of the APC gene and its association with
terminal differentiation. Cancer Res. 57: 488-494, 1997. PubMed: 9012479

20. Bermudez LE, et al. Exposure to low oxygen tension and increased osmolarity
enhance the ability of Mycobacterium avium to enter intestinal epithelial (HT-29)
cells. Infect. Immun. 65: 3768-3773, 1997. PubMed: 9284150

21. Tsao H, et al. Novel mutations in the p16/CDKN2A binding region of the Cyclin­
dependent Kinase-4 gene. Cancer Res. 58: 109-113, 1998. PubMed: 9426066

22. Qian XC, Brent TP. Methylation hot spots in the 5' flanking region denote silencing of
the O6-methylguanine-DNA methyltransferase gene. Cancer Res. 57: 3672-3677,
1997. PubMed: 9288770

23. Morin PJ, et al. Apoptosis and APC in colorectal tumorigenesis. Proc. Natl. Acad.
Sci. USA 93: 7950-7954, 1996. PubMed: 8755583

24. White LJ, et al. Attachment and entry of recombinant norwalk virus capsids to

cultured human and animal cell lines. J. Virol. 70: 6589-6597, 1996. PubMed:

8794293

25. Kolanus W, et al. alphaLbeta2 integrin/LFA-1 binding to ICAM-1 induced by

cytohesin-1 a cytoplasmic regulatory molecule. Cell 86: 233-242, 1996. PubMed:
8706128

26. Wang R, et al. Cellular adherence elicits ligand-independent activation of the Met
cell-surface receptor. Proc. Natl. Acad. Sci. USA 93: 8425-8430, 1996. PubMed:
8710887

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27. Young SW, et al. Gadolinium(III) texaphyrin: a tumor selective radiation sensitizer

that is detectable by MRI. Proc. Natl. Acad. Sci. USA 93: 6610-6615, 1996. PubMed:

8692865

28. Groh V, et al. Cell stress-regulated human major histocompatibility complex class I

gene expressed in gastrointestinal epithelium. Proc. Natl. Acad. Sci. USA 93: 12445­
12450, 1996. PubMed: 8901601

29. Takahashi K, et al. Keratan sulfate modification of CD44 modulates adhesion to

hyaluronate. J. Biol. Chem. 271: 9490-9496, 1996. PubMed: 8621620

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APPENDIX 6: REAGENT LOT TRACEABILITY AND CELL EXPANSION TABLES

Table 4: Reagent Lot Traceability

Reagent Vendor Catalog # Lot # Expiration

Date

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 Table 5: Cell Expansion

Observation under
FROM FLUID CHANGE microscope CELL COUNT TO

By / Date Flask qty; Pass # % Add/ Volume Viable Total % Split Flask Pass #
size Confluence Replace (in mL) cells/mL viable Viability Ratio qty; PDL#
cells size
Add /
Replace

Add /
Replace

Add /
Replace

Add /
Replace

Add /
Replace

Add /
Replace

Add /
Replace

Add /
Replace

Add /
Replace
 SOP: Thawing, Propagation and Cryopreservation of NCI-PBCF-HTB38 (HT-29)

APPENDIX 7: CALCULATION OF POPULATION DOUBLING LEVEL (PDL)

Calculate the PDL of the current passage using the following equation:

PDL = X + 3.322 (log Y – log I)

Where: X = initial PDL

I = cell inoculum (number of cells plated in the flask)

Y = final cell yield (number of cells at the end of the growth period)

APPENDIX 8: SAFETY PRECAUTIONS

 Use at least approved Biological Safety Level 2 (BSL-2) facilities and procedures.
 Wear appropriate Personal Protective Equipment (PPE), such as isolation gown, lab
coat with sleeve protectors, face shield and gloves.
 Use safety precautions when working with liquid nitrogen, nitrogen vapor and

cryogenically cooled fixtures..

 Use liquid nitrogen freezers and liquid nitrogen tanks only in areas with adequate

ventilation. Liquid nitrogen reduces the concentration of oxygen and can cause

suffocation.

 Wear latex gloves over insulating gloves to prevent liquid nitrogen from soaking in and
being held next to the skin. Liquid nitrogen is extremely cold and will cause burns and
frostbite. Metal inventory racks, tank components, and liquid nitrogen transfer hoses
exposed to liquid nitrogen or nitrogen vapor quickly cool to cryogenic temperatures and
can cause burns and frostbite.
 Wear a full face mask when thawing and retrieving vials from liquid nitrogen freezer.
Danger to the technician derives mainly from the possibility that liquid nitrogen can
penetrate the cryovial during storage. On warming, rapid evaporation of the nitrogen
within the confines of such cryovial can cause an aerosol or explosion of the cryovial and
contents.

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