Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: February 02, 2022
Last Modified: December 13, 2024
Collection Integer ID: 57732
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
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
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.
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.
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.
We have routinely maintained feeder-free hPSC cultures on VTN, Matrigel and Geltrex-coated cell culture plates without observing obvious differences.
We have routinely passaged feeder-free hPSCs using either Accutase (as single cell suspension) or ReLeSR (as cell aggregates) without observing obvious differences.
Materials
Item
Vendor
Catalog #
DMEM/F12
Thermo
Fisher
11320082
DPBS w/o
Calcium and magnesium (DPBS)
Corning
MT21031CV
mTeSR-plus
STEMCELL
Technologies
100-0276
StemFlex
Thermo
Fisher
A3349401
FB Essence
Avantor
10803-034
Vitronectin
(VTN-N) Recombinant Human Protein, Truncated
