Reagents needed
Complete growth media - DMEM + 10% FBS + 1X P/S T-25 flask
- Equilibrate the media to 37o
C
- Remove the frozen cells from the liquid nitrogen storage wearing the appropriate protective equipment
- Place the frozen cells in a 37o
C water bath to thaw
- Aseptically transfer the thawed cells into the cell culture hood by spraying down with 70% ethanol
- Transfer the thawed cells to a 15 mL conical tube and dilute the cells stored in DMSO 10-fold with complete media.
- Spin down gently at 300g for 5 minutes and aspirate all media from the culture - Resuspend the cells with 5 mL of pre-warmed complete media and seed into the
T-25 flask - Incubator at 37o
C, 5% CO2 and change the media after overnight incubation
- Passage the cells when they reach 80-90% confluence
A.1.1.2 CHO-K1 adherent cells passaging protocol
Cells need to be passaged every 2-4 days or when they reach up to 90% confluence. This protocol is for expanding cells from a T-25 into a T-75 flask. The reagent volumes can be scaled up for larger flasks.
Reagents needed
Complete growth media - DMEM + 10% FBS + 1X P/S PBS
Trypsin-EDTA
- Aspirate all media from the culture - Wash cells 2X with pre-warmed PBS
- Add 1 mL of pre-warmed Trypsin-EDTA into the T-25 flask
- Return cells to incubator for 2-10 minutes to allow the adherent cells to detach - Tap the bottom of the flask to allow cells to completely detach
- Add 4mls of pre-warmed complete media to the cells. The FBS in the complete media quenches the trypsin
- Re-seed the cells using the desired dilution into a T-75 flask (~12mL per T-75 flask).
o Eg: To re-seed at a 1:12 dilution, add 1mL of the cells into 12 mL of media in the T-75 flask
o To re-seed at a desired cell concentration, count the cells using the hemacytometer and dilute to the desired concentration
A.1.1.3 Hemacytometer cell counting and determination of cell viability
Reagents needed 0.2% trypan blue
Hemacytometer and cover slip Microscope
Hand counter
- Add 10µl of 0.2% trypan blue to 10µl of the cell suspension.
- Load one side of the hemacytometer with 10µl of the 2X diluted cell suspension and place on the microscope.
- Count the number of viable cells in 5 out of 9 squares of the hemacytometer. Viable cells are impermeable to the trypan blue dye and remain bright, while non- viable cells take up the dye and are blue.
- Divide the total number of cells counted by 5 to get the average number of cells per square.
- Multiply the average count per square by 104
to get the number of cells per ml of the diluted cell suspension.
- Determine the concentration of cells before dilution by multiplying by 2 since the cells were diluted into trypan blue.
o To determine cell viability Eg for suspension cells, count the total number of cells, and total number of viable cells.
- Clean the cover slip and hemacytometer with 70% ethanol
A.1.1.4 Preparation of 0.2% trypan blue for determining cell viability Reagents needed
Trypan blue powder Distilled water
- Dissolve 0.1 g of trypan blue in 50 mL of distilled water - Spin down at 3000g for 5 minutes to remove large precipitates.
- Clarify by filtering with a 0.45 µm or 0.22 µm filter to remove particulates
A.1.1.5 Cryopreservation of CHO-K1 cells Reagents needed
Complete growth media - DMEM + 10% FBS + 1X P/S Freezing media - DMEM + 10% FBS + 1X P/S + 10% DMSO PBS
Trypsin-EDTA 0.2% trypan blue
Hemacytometer and cover slip Microscope
Hand counter Cryovials
Ethanol freezing container
- Equilibrate all media to 37o
C
- Aspirate all media from the culture and trypsinize as for passaging - After cells are detached, dilute 10-fold with complete media
- Count the cells using trypan blue staining. Cell viability to be greater than 90% - Spin the cells down at 300g for 5 minutes and aspirate off the growth medium - Resuspend the cells in freezing medium to a concentration of 2 x 106
cells/mL - Aliquot the cells into cryovials and transfer the cells immediately into the -80o
C freezer in an ethanol slow freezing container
- Freeze the cells overnight at -80o
C and then transfer to the liquid nitrogen storage Adapted from
A.1.1.6 Transfection of CHO-K1 cells for transient antibody expression Materials needed
Low passage umber (<10 passages) CHO-K1 Cells
150 µg Midi-prepped single vector plasmid DNA containing the heavy and light chains or
75 µg midi-prepped light chain plasmid DNA and 75 µg midi-prepped heavy chain plasmid DNA
Opti-MEM (Life Technologies)
Lipofectamine 2000 (Life Technologies)
Complete growth media: DMEM + 10% FBS + 1X Penicillin/Streptomyin
Antibody growth media: DMEM + 10% Low IgG FBS + 1X Penicillin/Streptomyin 1 M Tris pH 8.0, sterile
Day 1 - Cell preparation:
- Trypsinize CHO-K1 cells - Count using the hemocytometer - Plate 5 x 106
cells per T-150 flask (3 flasks total) in 24 mL of complete growth media
- Grow overnight in jacketed incubator at 37C and 5% CO2
- Transfect when the cells reach ~95% confluency Day 2 - Transfection:
- Mix 12 mL Opti-MEM with 75 µg of the heavy chain and 75 µg of the light chain plasmid
o For single plasmid vectors, dilute 150 µg of the vector in 12.5 mL of Opti- MEM
- Mix 12 mL Opti-MEM with 375 µL Lipofectamine 2000 - Allow reagents to sit at RT 5 min
- Mix the diluted DNA and Lipofectamine (24 mL total) - Allow to sit at RT for 20 minutes
- While reagents are sitting, remove media from each flask of cells and replace with 16 mL antibody growth media
- Add 8 mL of the transfection mix (DNA + Lipofectamine) to each flask - Mix gently and incubate overnight at 37o
C, 5% CO2
Day 3 - Media change:
- Remove the old media from the cells
- Spin down the old media at 5000g for 5 minutes to pellet the cells - Save the clarified culture supernatant for further antibody purification - Add fresh antibody growth media to the cells and return to the incubator - To increase yields, the cells from the old flasks can be reseeded into two new
- Replace the media on the cells with fresh antibody growth media every other day for a total of three media changes. Saved the media for further purification.
A.1.1.7 Guidelines for scaling up or down transfection of DNA into different tissue culture vessels
Table A.1: Required volumes for transfection reagents
Scale up/down the concentration of cells plated depending on the culture vessel so that the cells will be approximately 95% confluent at the time of transfection.
Culture vessel Low IgG media Opti- MEM (2 tubes) DNA in µg, (L+H), Dilution volume (µL) Lipofectamine- 2000, dilution volume (µL) Complex added 24-Well 0.4 mL 50 µL 0.6 µg in 50 µL 1.5 µL in 50 µL 100 µL 12-Well 0.8 mL 100 µL 1.2 µg in 100 µL 3 µL in 100 µL 200 µL 6-Well 1.5 mL 250 µL 3 µg in 250 µL 7.5 µL in 250 µL 500 µL T-150 16 mL 4 mL 50 µg in 4 mL 125 in 4 mL 8 mL Adapted from
- Lipofectamine® reagent protocols.
- Optimized transfection protocols from Ellen Wagner
A.1.2 Bacteria growth and maintenance