Home › Genetics / Genomics › Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium
Genetics / Genomics JoVE (Open Access) Citable · DOI

Feeder-Free Adaptation, Culture and Passaging of Human IPS Cells using Complete KnockOut Serum Replacement Feeder-Free Medium

DOI: 10.3791/2236-v
What you'll learn
  • ✓ Adapt human iPSCs from feeder-dependent to feeder-free culture
  • ✓ Coat culture dishes with Geltrex for optimal cell attachment
  • ✓ Passage and maintain iPSCs in KSR-FF medium long-term
Protocol

The following protocol provides instruction for adapting human induced Pluripotent Stem (iPS) Cells to feeder-free culture using complete KnockOut Serum Replacement Feeder-Free medium (KSR-FF). Once adapted, instructions for continual maintenance are also provided.

Difficulty
intermediate
Total time
~3–5 days (initial adaptation); ~30 min per passage thereafter
Model organism
Human induced pluripotent stem cells (iPSCs)
Biosafety
BSL-1

Steps

1
Coat culture dishes with Geltrex substrate

Prepare culture plates by applying Geltrex-coated matrix to ensure feeder-free attachment and growth of human iPSCs.

▶ 01:33
2
Adapt human iPSCs to feeder-free conditions

Transition iPSCs from feeder-dependent culture to KSR-FF medium through gradual medium exchange and monitoring of cell morphology and pluripotency.

▶ 02:59
3
Passage iPSCs in feeder-free medium

Dissociate and plate adapted iPSCs at appropriate densities in KSR-FF medium to maintain long-term culture and prevent differentiation.

▶ 05:20
4
Evaluate representative culture results

Assess morphology, viability, and pluripotency markers of successfully adapted and passaged feeder-free iPSCs.

▶ 07:36
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