Feeder-free culturing of hPSCs

This collection describes the standard procedure of feeder-free culturing of human pluripotent stem cells (hPSCs) using mTeSR-plus or StemFlex

Collection overview

Coating plates

A. VTN

B. Matrigel

C. Geltrex

Adapting hPSCs cultured on MEFs to feeder-free system

Thawing of feeder-free hPSCs

Passaging of feeder-free hPSCs

A. Accutase

B. ReLeSR

Freezing of feeder-free hPSCs

A. Accutase

B. ReLeSR

General Notes

1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. Until otherwise indicated, feeder-free hPSCs are routinely grown in a humidified cell culture incubator under “low” oxygen conditions. We have successfully maintained hPSCs using either 3% O2 (3% O2, 5% CO2) or 5% O2 (5% O2, 5% CO2) conditions.
3. We have routinely maintained feeder-free cells in either mTeSR-plus or StemFlex. However, these two media are not interchangeable. Pick one and stick to it.
4. We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex-coated cell culture plates without observing obvious differences.
5. We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences .