Randy Mckanobb
Methods of Biotechnology
Cryopreservation of
Mammalian
Cells in Culture
Freezing/Thawing
Techniques
Cell Count, and Viability of CHO Cells
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Randy Mckanobb
Title: Recovery of Frozen Mammalian Cells
Purpose: To be able Freeze CHO Cells, and to determine the recovery rate upon thawing.
Materials
DMEM (Dulbecco Modified Eagle Medium)
Maker: Gibco
Lot# 1507992
Cat# 10569-010
Exp: 02-12-15
Non-Essential Amino Acid
Maker: Gibco
Lot# 577926
Cat# 11140
Exp: 2010-03
Hot Water Bath
Maker: Precision
Equipment ID: WB-009
Model# 184
Serial# 9309-109
Conical Tubes
Maker: BD Falcon
Ref# 352196-9363993-0421963
Style 17X20mm
Trypan Blue Stain (0.4%)
Maker: Gibco
Ref# 15250-061
Lot# 1555623
Exp: 03-2017
Micro Pipet
Maker: Gibco
Ref# N-84-13447
Fetal Bovine Serum
Maker: Gibco
Lot# 1403414
Cat# 16000-044
Exp: 2012-03
Pipet-Aid
Maker: Drummer Scientific Co.
Serial # P89895
Micro Pipet Tips
Maker: Maxymum Recovery
Lot# 07100924
Pen Strep
Maker: Gibco
Cat# 15140-122
Lot# 559271
Exp: 2010-03
Trypsin-EDTA 0.05%
Maker Gibco
Lot# 75335
Ref#25300-054
Exp: 2012-02
Cell Counter
Maker: VWR
Lot# 0895
Para Film
Maker: Pehiney Plastic Packaging
Cat# PM-992
PBS
Maker:Gibco
Lot# 1553237
Ref# 14040-141
Exp: 2017-03
Microscope
Maker: Leica Microsystems
Type: 090-135001
SterileGuard Hood
Maker: The Baker Company
Class2 Type A/B3
Papertowels
Maker Appeal
White Multifold
Hemacytometer
Maker: Housser Scientific
Lot# 426233
Serological Pipets
Maker: Sarstedt
Lot# 3192E-9292E
Cat#861254001
Exp: 2016-07; 2012-10
NUNC Cryotube
Bio Freeze-Polypropylene
Maker: Costar
Lot# 8601
Droppette Disposable Transfer
Pipets
Maker Simport
Ref# P200-72
Lot# D22136726
Freezing Media Contains DMSO
Cat# 12648-010
Lot# 1559017
Exp: 04-2015
CHO 1-15 500
Description: Ovary
Total cells/ mL: 1.4x106
Expected Viability: 89.9% - 97.1%
Dilute Ampule Content: T-15 Flask
Volume Ampule: 1mL
Date Frozen:3-10-10
Lot# 59111689
T-25 Flask
Maker: Srtedt
Lot# 0099035
Cat# 2015-04
Exp: NA
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Methods:
Media Preparation: (SOP)
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Disinfect the work area with 70% ethanol.
Disinfect the biological hood with 70% ethanol.
Turn the UV (Ultra Violet) light on in the biological hood for at least 15 minutes.
Heat inactivate the Sera by placing the Serum bottle on a 56 degree Celsius water bath for 30 minutes.
Mark the bottle of Serum with date, name and the Triangle sign, signifying “heat inactivated.”
Turn off the UV light on the biological hood.
Have all reagents and equipment necessary for the media preparation inside the biological hood.
Aseptically add the following ingredients to an autoclaved bottle.
a. 78.9 mL of Media
b. 20.0 mL of Sera
c. 0.1 mL of Pen Strep
d. 1.0 mL of Non-Essential Amino Acids
9.
10.
11.
12.
13.
Mix all ingredients; write name, date, and the term “Complete Media.”
Take 1mL aliquot from the bottle containing the complete media.
Place the aliquot in a centrifuge tube and keep inside of the 5% CO2 incubator for at least 24 hours.
After 24 hours check the aliquot for the presence or absence of contamination.
The bottle containing the complete media should be stored in the refrigerator.
Thawing Mammalian Cells (CHO)
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9.
Make sure all media is pre warmed and all reagents and equipment is ready.
Thaw the vial by hand, then using a 5mL pipet added 2.0 mL of pre-warmed media into a centrifuge tube.
(Deviation)
Transfer 0.5 mL of media back and forth from the vial to the centrifuge tube, until all the frozen media are
thawed, and cell are removed from vial.
Centrifuge at 1500 RPM for 5 Minutes.
Remove supernatant, leave 0.1 mL.
Jaysonize, flick, flick, flick.
Re-suspend the cell pellet in 1 mL fresh media (Per T-25 Flask), transfer to a 25 cm2culture flask.
Add 4mL of complete media to the tissue culture flask a total of 5mL.
Incubate at 37 Degrees Celsius.
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Trypsinization of Mammalian Cells (CHO)
10. Remove cell culture media from flask, discard supernatant.
11. Wash attached cells with 3mL of PBS (Phosphate Buffer Saline). Note: Add PBS to the side of the flask
opposite the cells. Rinse the cell sheet by gently rocking the flask for 1 minute. Discard PBS.
12. Add 2 mL f Trypsin solution to the side of the flask opposite the cells sheet. Incubate the flask at 37
degrees Celsius 5% CO2incubator. Monitor the cells dissociation process carefully by examining the flask
under the microscope every 1-2 minutes.
13. When cells are completely detached, stand the flask in the upright position to allow the cells to drain to
the bottom of the flask. Add 2mL of complete media on top of the cells.
14. Dispense the cells by pipetting repeatedly over the surface of the monolayer.
15. Remove all 4 mL of cell and trypsin and complete media solution from the flask and transfer the solution
to a centrifuge tube.
16. Centrifuge the cells for 5 minutes at 1500 PRM.
17. Remove all but 0.5mL of the supernatant and discard, be careful not to disturb the pellet of cells.
18. Jasonize- flick, flick, flick (Deviation)
19. Add 2 mL of complete media to the cells pellet. Dispense the cells by pipetting repeatedly up and down.
20. Add 1mL of cells to a T-25 flask the other 1mL to another flask i.e., 1:2 slit. The remaining 0.1 mL of cells
should be used for cell count and viability determination.
21. Add 4 mL of complete media to each of the two T-25 Flasks for a total volume of 5 mL. Make a back-up
flask.
22. Observe the flask containing cells under a microscope. Incubate them at 37 degrees Celsius 5% CO2
incubator.
Cell Count and Viability Determination of Mammalian Cells (CHO) - Part-1
1.
2.
Making appropriate dilutions of the cells suspension with growth media or PBS just prior to counting. The
optima concentration of cells for counting is 5-10 x 105 cells/mL (50-100 cells per large square) after
dilution in the counting solution. If dilution is necessary, be sure to include the dilution factor in your
calculation at the last step.
Using a Pasteur pipette with figure control, let the cell suspension flow under the coverslip until the grid
area is just full and not overflowing into the overflow well. See Figure A. If the chamber is loaded too
heavily, clean it and begin again; do not attempt to remove excess liquid. Allow cells to settle.
Figure A – Hemacytometer
1.
Figure B - Grid System (Magnified view)
Count all of the contained in each of the 4 large squares (1-4 in Figure B). Some cells will be touching the
outside borders. Count only those cells touching two of the outside borders (for example, upper and left
border). A minimum of 200 cells should counted. Determine the average number of cells per large square
after counting in each square. This is the number of cells per 104 mL.
In order to determine the cells/mL, use the formula below.
Cells/mL = Average number of cells per large square x 104/mL x dilution factor
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Part-2
2.
3.
To 1 part of the trypan blue saline solution, add 1 part of the cell suspension (1:1 dilution).
Load cells into hemacytometer and count the number of unstained (viable) mammalian cells and stained
(dead) cells separately. For greater accuracy, count more than a combined total of 200 cells.
Viable cells/mL =
Average number of viable cells in large square (X 104/mL X 2* X) dilution factor
*(for 1:1 dilution)
% viable cells =
number of viable cells
x 100
number of viable cells + number of dead cells
Cryopreservation/Freezing (SOP)
1.
2.
Count cells and determine viability. Refer to previous module “Cell Count and Viability” for SOP.
Ideal density is 1-2 million cells per 1 mL of freezing media.
If high cell density: aliquot required volume of cells into tube. And freeze media w/DMSO, mix quickly
(exothermic reaction) and dispense into vials.
If low cell density: aliquot required volume of cells into tube, centrifuge, remove supernatant, and resuspend pellet in freezing media with DMSO. Mix and dispense into vials.
3.
4.
Put vials in pre-cooled ethanol bath in -20 degrees Celsius freezer for 30-60 minutes. Note: Cells lose
viability when kept in 10% DMSO at room temperature or at -20 degrees Celsius.
Transfer vials to liquid nitrogen storage tank.
Percentage of Cells after Recovery of Thawing Cells - (CHO)
Number of cells Frozen
6.7x106
6.1x106
Viability of frozen cells
99%
78%
Number of cells thawed
2.2x106
2.03x106
Viability of thawed cells
18%
22%
Percent of recovery of thawed cells
32.8%
33.2%
# of thawed cells
x100
# of cells frozen
Results
Reached 100% confluent
Reached 100% confluent
Discussion: I figured out there are several different way to be unsuccessful in this process of freezing
and thawing of the mammalian cell line, the whole process has to be done aseptically, with a full
understanding of the SOP of, Trysinization of attached cells, Thawing, Freezing, and Cell Count and
Viability. You need to be able to obtain a proper procedure to have a technique that works well for
successful results.
Conclusion: Everything was done aseptically according to the SOP, all deviations were recorded during
the process. After freezing and thawing my CHO cells I was able to achieve full confluence, and was able
to freeze CHO cells for the summer classes.
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