Primary Human Epidermal Keratinocytes, adult (C-005-5C)
Reagents (do not warm in water bath):
50mL Epilife medium (M-EPI-500CA)
500uLHuman Keratinocytes Growth Supplement (S-005-1)
Prepare a 37C water bath for the thawing of cryopreserved primary cells.
Sterilize tissue culture hood with 75% alcohol.
Mix the two reagents (do not warm in water bath) in laminar flow culture hood and pour 7mL into a 10cm plate (Corning). Label with date, cell type, passage number, and your name.
Thaw the primary cells in 37C water bath with gentle agitation (around 1 to 2 minutes). Do not thaw thoroughly, a movable sliver of ice should still be seen in the cryogenic tube. Wipe cryogenic tube with 75% alcohol.
Transfer thawed primary cells into plate in laminar flow culture hood.
Use 500uL of fresh medium to wash cryogenic tube by pipetting up and down. Transfer medium into plate.
Store cell in a humidified 37C incubator with 5% CO^2, 95% air.
Do not disturb cell for at least 24 hours before changing cell media the next day by aspirating media then pouring in 7.5mL of fresh media.
They should have a cobblestone appearance, as shown in the picture below.
Reagents (do not warm in water bath)
Maintenance medium (Epilife + HKGS)
Change media every other day until 85% confluence.
Trypsin/EDTA solution 0.025%
Maintenance media (Epilife medium + HKGS)
Trypsin Neutralizer Solution,TNS (R-002-100)
Thaw TNS in 37C water bath, and take out when thawed to cool off.
Sterilize tissue culture hood with 75% alcohol.
Move culture plate to laminar flow culture hood, aspirate medium.
Wash cells with 1mL trypsin/EDTA solution, remove solution after ensuring that entire surface was covered.
Add 1mL trypsin/EDTA and incubate at room temperature for approximately 8 to 10 minutes (check every 4 minutes) until the cells are completely round.
Check under microscope and lightly rap/tap under and on the sides of the the plate to dislodge cells.
When cells are detached, tilt the plate a little and add 3mL TNS into plate. “Wash” cells by gently pipetting solution over the plate several times.
Transfer solution into a sterile 15mL conical tube.
Add 3mL of fresh TNS into plate while tilting the plate. Repeat “wash” action, and transfer solution to the 15mL conical tube. A total of 7mL of solution should be in the 15mL conical tube.
Centrifuge cells at 180g (or 1268rpm for a 100mm radius motor) for 7 minutes.
Observe pellet, then remove supernatent gently from tube.
Resuspend cells gently in maintenance medium by pipetting up and down for a homogenous mixture.
Determine concentration of cells in suspension.
Seed new culture plate with at least 2.5x10^3cells/cm^2.
Incubate in humidified incubator at 37C, with 5% CO^2 and 95% air.
CryoStor CS10 Cell Crypreservation Media (C2874)
Maintenance medium (Epilife medium + HKGS)
Steps (if you would like to cryoperserve a tube and continue on with culturing)
Ensure 95% confluency of cells.
Follow “Subculture procedure” steps 1 to 9.
Transfer 4mL of solution to conical tube 2 and centrifuge both tubes at 180g (1268rpm for a 100mm radius motor).
Aspirate solution from both tubes.
Add 1mL to 2 mL cold CryoStor solution to conical tube 1 (previously with 3mL trypsin/EDTA/TNS solution) and resuspend pellet by gentle pipetting. Transfer solution to cryogenic tube and store in -80C overnight before transferring cryogenic tubes to liquid nitrogen.
Resuspend conical tube 2 (previously with 4mL trypsin/EDTA/TNS solution) with maintenance medium and add back to plate with fresh maintenance medium. Incubate plate in humidified incubator at 37C with 5% CO^2 and 95% air.
I guess that’s it for HEKa cell culture. If you have any questions relating to my protocol or otherwise, feel free to DM me 💖
