Sep 06, 2022
Thawing, Passaging and Freezing of hPSCs on MEFs
- 1University of California, Berkeley;
- 2Albert Einstein College of Medicine
External link: https://doi.org/10.7554/eLife.79208
Collection Citation: Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner 2022. Thawing, Passaging and Freezing of hPSCs on MEFs. protocols.io https://dx.doi.org/10.17504/protocols.io.b4msqu6e
Manuscript citation:
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 03, 2022
Last Modified: December 13, 2024
Collection Integer ID: 57746
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This collection contains protocols which describe the standard procedure of culturing human pluripotent stem cells (hPSCs) on inactivated mouse embryonic fibroblasts (MEFs).
Collection overview
Thawing of hPSCs grown on MEFs
Passaging of hPSCs grown on MEFs
Freezing of hPSCs grown on MEFs
A. Freezing of hPSCs as single cell suspension using trypsin
B. Freezing of hPSCs as cell aggregates using collagenase
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, 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. While freezing hPSCs as single cell solution (using Rock Inhibitor) results in better cell recovery, some laboratories prefer freezing of hPSCs as cell clusters. We have used both approaches and do not observe obvious differences.
4. While bulk/collagenase passaging is used for routine maintenance of hPSC cultures,
manual/microdissection passaging is used to enrich for undifferentiated hPSC colonies (“clean-up” of culture based on undifferentiated hPSC colony morphology) or to expand (“pick”) individual colonies (e.g.,for clonal expansion of individual targeted cells in the process of establishing genome edited cell lines). Manual passaging/ microdissection requires (i) the identification and discrimination of undifferentiated and differentiated hPSC colonies and (ii) the capacity to excise the undifferentiated cells and transfer them to a new plate and can be performed using various approaches as established in many hPSC laboratories.
Materials
| A | B | C | |
| Item | Vendor | Catalog # | |
| DMEM/F12 | Thermo Fisher | 11320082 | |
| DPBS w/o Calcium and magnesium | Corning | MT21031CV | |
| Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
| Knockout Serum Replacement | Thermo Fisher | 10828-028 | |
| FB Essence | Avantor | 10803-034 | |
| Newborn Calf Serum | Sigma | N4762 | |
| L-Glutamine | Sigma | G8540 | |
| Penicillin & Streptomycin (100X) | Thermo Fisher | 15140163 | |
| MEM Non-Essential Amino Acids (100X) | Thermo Fisher | 11140050 | |
| Heat Stable Recombinant Human FGF2 | Thermo Fisher | PHG0360 | |
| Collagenase type IV | Thermo Fisher | 17104019 | |
| DMSO | Fisher Scientific | BP231-100 | |
| BSA | Sigma | A4503 | |
| Y-27632 | Chemdea | CD0141 | |
| 2-Mercaptoethanol | Sigma | M3148 | |
| 0.25% Trypsin with EDTA | Thermo Fisher | 25200114 | |
| Styrofoam microtube freezer box | Labnet | R8000 | |
| Nalgene® Mr. Frosty® Cryo 1°C Freezing Containers | Thermo Fisher |
