Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.
Total time
~30 min thawing per vial; ~2–4 hours plating and recovery; ~2–4 hours freezing per batch
Model organism
Human embryonic stem cells (hES)
Steps
1
Thaw human embryonic stem cells rapidly
Remove hES cell vial from liquid nitrogen and rapidly thaw at 37°C to minimize ice crystal formation and osmotic stress. Work quickly to transfer cells to recovery medium.
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2
Plate thawed cells on feeder layer
Transfer thawed hES cells onto pre-prepared mouse embryonic feeder cell monolayers. Allow cells to attach and expand in culture with appropriate medium changes.
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3
Slowly freeze hES cells for storage
Suspend hES cells in cryoprotectant solution and freeze slowly (controlled-rate freezer or −80°C overnight) before transfer to liquid nitrogen for long-term preservation.
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