May 23, 2025
Thawing of cryopreserved hPSC
- Valeria Fernandez Vallone1,
- Lyn Healy2,
- Nathalie Lefort3,
- Katarzyna Ludwik1,
- Tamer Onder4,
- Lisa Pavinato5,6,
- Fatma Visal FVO OKUR7,
- Harald Stachelscheid1
- 1Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
- 2The Francis Crick Institute;
- 3Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.;
- 4Koc University;
- 5Institute of Oncology Research (IOR), Bellinzona Institutes of Science (BIOS+), Bellinzona, Switzerland;
- 6Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland.;
- 7Hacettepe University, Center for Stem Cell Research and Development (PEDISTEM) and Hacettepe University Faculty of Medicine, Department of Pediatrics
- CorEuStem
Protocol Citation: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid 2025. Thawing of cryopreserved hPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd282lmk/v1
License: This is an open access protocol 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 protocol and it's working
Created: November 21, 2023
Last Modified: May 23, 2025
Protocol Integer ID: 91286
Keywords: hPSC, thawing, cryopreservation, iPSC
Funders Acknowledgements:
COST Action CorEuStem
Grant ID: CA20140
Abstract
This protocol describes thawing of hPSC.
Guidelines
Thawing procedure is described for one cryovial. The protocol description is general and applies to multiple hPSC lines frozen as clumps or single cells.
It is recommended to thaw hPSC in same culture conditions that were used before freezing.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
- 2/5/10 mL pipettes
- 15/50 mL conical tubes
- 10/200/1000µL tips and pipettes (optional)
- Cell culture treated plastic vessels of choice e.g. 24, 12 or 6-well plates
- Cell counting equipment
- Aspirator pump with disposable pipette
- Centrifuge
- Microscope
- Water/bead warming bath
- Class II Biosafety Cabinet
- Incubator at 37oC and 5% CO2 or under hypoxic conditions, 5% O2/ 5% CO2
MEDIA AND REAGENTS
hPSC culture conditions and survival factors choice depend on hPSC line and individual lab practices. For options refer to protocols:
Safety warnings
hPSC culture media supplementation with survival factors is required for hPSC survival upon thawing.
Preparation of destination vessel
Preparation of destination vessel
35m
35m
Prepare coated tissue culture vessels according to hPSC line requierements and desired format. For coating procedure refer to protocol Coating of tissue Culture Vessels for hPSC
Note
It is recommended to thaw hPSC on the same matrix that was used before freezing.
5m
Equilibrate prepared destination vessel at 37 °C until usage.
30m
hPSC thawing
hPSC thawing
43m
43m
Prepare required volume of the reagents and media supplemented with survival factors according to Table 1. Additionally prepare 10 mL of media for Step 8. Equilibrate the media to Room temperature or 37 °C .
| A | F | |
| Culture vessel | hPSC media + survival factor (final: mL/per well | |
| 24 well | 0.5 | |
| 12 well | 1 | |
| 6 well | 2 | |
| T25 | 7 |
Table 1. Recommended volumes according to vessel format
Note
hPSC culture media: it is recommended to thaw hPSC in same media used before freezing.
For options refer to protocol Maintenance of hPSC. For thawing media has to be supplemented with Survival factors. For options refer to protocol Survival factors for hPSC growth.
10m
Remove cryovial containing frozen hPSC from liquid N2.
5m
Sanitize the tube with 70 % volume ethanol.
2m
Thaw hPSC suspension at 37 °C using water or bead bath for about 00:02:00 -00:04:00 leaving small amount of un-thawed media.
Note
If using water bath, carefully swirl vial in the water, avoid immersing the vial above the level of the cap.
6m
Using 2 mL pipette transfer slowly hPSC suspension from the cryovial to a 15 mL conical tube.
3m
Dropwise add 10 mL of culture media supplemented with survival factors to hPSC suspension while gently swirling the tube, refer to Table 1 for recommended volumes.
1m
Centrifuge hPSC suspension at 300 x g, 00:05:00
5m
Aspirate and remove the supernatant without disrupting the cell pellet.
1m
Using a 5 mL pipette gently add 2 mL of hPSC culture medium supplemented with survival factors to the pellet.
1m
Gently resuspend the hPSC pellet by pipetting up and down.
Note
After homogeneous re-suspension and if hPSC were frozen as single cells, cell counting and viability assessment using e.g. Trypan Blue 0.4%. solution can be performed.
2m
Aspirate coating solution from the destination vessel.
30s
Seed hPSC at desired density according to well size/surface.
5m
Rock the plate side to side, back and forth to spread the cells across the well.
1m
Incubate the vessels at 37 °C and at 5 % volume CO2.
30s
After 24:00:00 , perform full media change with respective hPSC maintenance media without survival factors. For further hPSC culture refer to protocol Maintenance of hPSC.
Note
hPSC display different morphology when cultured in medium containing survival factors. This morphology will change after media is replaced with medium without survival factors.
For detailed morphology, refer to protocol Reference pictures of hPSC cultured in defined conditions