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Genetics / Genomics JoVE (Open Access) Citable · DOI

Cryopreserving and Recovering of Human iPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

DOI: 10.3791/2237-v
What you'll learn
  • ✓ Cryopreserve human iPS cells using KnockOut SR medium without feeder layers
  • ✓ Thaw and recover cryopreserved iPS cells with high viability
  • ✓ Culture recovered iPS cells in feeder-free or feeder-based conditions
Protocol

This protocol describes the detailed procedure for cryopreserving human iPS cells in KnockOut SR cryopreservation medium and recovering these cells in complete KnockOut SR Feeder Free (KSR-FF) medium or feeder-based KnockOut SR medium.

Difficulty
intermediate
Total time
~30 min (cryopreservation); ~1-2 hrs (thawing and initial recovery); cell viability assessment within 24 hrs
Model organism
Human induced pluripotent stem cells (iPSCs)
Biosafety
BSL-1

Steps

1
Prepare and execute cryopreservation of iPSCs

Suspend human iPS cells in KnockOut SR cryopreservation medium at appropriate density. Transfer to cryovials and freeze using controlled-rate or passive cooling protocols prior to storage in liquid nitrogen.

▶ 01:31
2
Thaw cryopreserved iPSCs and assess viability

Remove cryovials from liquid nitrogen storage, rapidly thaw at 37°C water bath, and transfer cells to recovery medium. Perform initial viability assessment and culture in either feeder-free KSR-FF or feeder-based KnockOut SR medium.

▶ 03:48
3
Evaluate recovery and cell morphology outcomes

Examine recovered iPS cell cultures for colony morphology, pluripotency markers, and post-thaw viability. Document representative results demonstrating successful cryopreservation and recovery.

▶ 05:54
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