May 23, 2025

Public workspaceFreezing of hPSC

DOI
dx.doi.org/10.17504/protocols.io.rm7vzx278gx1/v1
  • 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
Harald Stachelscheid
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzx278gx1/v1
Protocol Citation: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid 2025. Freezing of hPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzx278gx1/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: 91274
Keywords: cryopreservation, freezing, hPSC, iPSC, Bambanker
Funders Acknowledgements:
COST Action CorEuStem
Grant ID: CA20140
Abstract
This protocol describes cryopreservation of hPSC as clumps or single cells.
Guidelines
hPSC culture quality is highly relevant for a successful freezing/thawing procedure.
We recommend freezing cultures at ~70-80% confluency. High quality cultures show spontaneous differentiation below 5-10% of cultured surface.






Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
  • 1/5/10 mL serological pipettes
  • 15/50 mL conical tubes
  • 10/200/1000µL tips and micropipettes (optional)
  • 1 or 1.5 mL cryo-vials
  • Cell scraper
  • Cell counting equipment
  • Aspirator pump with disposable pipette
  • Centrifuge
  • Microscope, if available Stereo Microscope
  • Freezing container (Mr. Frosty) filled with 100% 2-propanol (pre-chilled), alternatively equipment for automated controlled freezing.
  • Class II Biosafety Cabinet


MEDIA AND REAGENTS
ReagentBambankerâ„¢ 1x120mLNippon GeneticsCatalog #BB01
ReagentSTEM-CELLBANKER - GMP GradeamsbioCatalog #11890
ReagentCryostar CS10Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2874

Alternatively, fetal bovine serum 90% / DMSO 10% solution. However, this freezing medium is less defined and not xeno-free.
Protocol materials
ReagentBAMBANKERBioCat GmbHCatalog #BB03-NP
ReagentBambankerâ„¢ 1x120mLNippon GeneticsCatalog #BB01
ReagentSTEM-CELLBANKER - GMP GradeamsbioCatalog #11890
ReagentCryostar CS10Merck MilliporeSigma (Sigma-Aldrich)Catalog #C2874
Before start
Prepare labelled or barcoded cryo-vials.
Preparation
Preparation
25m
25m
Visually inspect hPSC culture using a microscope. Culture ready for freezing should be of good quality (see Guidelines section).
10m
Refer to Table 1 to prepare adequate amount of dissociation reagent and freezing medium according to the culture vessel format and amount of vessels/wells to be frozen.
ABCD
Culture vesselDissociation reagent [mL]Cryopreservation media [mL]Re-suspension media [mL]
24 well0.250.250.5
12 well0.50.51
6 well112
T25336
T7561010
10 cm dish31010
Table 1. Recommended volume according to vessel format

Note
Freezing as single cells additionally requires the use of re-suspension media (culture media supplemented with survival factors as described in protocol: Survival factors for hPSC growth) to wash out enzymatic dissociation reagent.

Note
Dissociation reagent will depend on the freezing method choice: as clumps or as single cells see STEP CASE.

Freezing medium options are described in material and methods, we recommend the use of ReagentBAMBANKERBioCat GmbHCatalog #BB03-NP

15m
Choose freezing method:
Step case

hPSC freezing as clumps
10 steps

Clump freezing
Clump freezing
1d 0h 16m
1d 0h 16m
Aspirate and discard medium from the vessel.
1m
Rinse once with required volume of non-enzymatic dissociation reagent, refer to Table 1 for recommended volumes.
Note
A variety of non-enzymatic dissociation reagents can be used. For options refer to protocol:
Non-enzymatic passaging of hPSC

2m
Add required volume of non-enzymatic dissociation reagent to the culture vessel, refer to Table 1 for recommended volumes.
1m
Incubate for Duration00:03:00 - 00:05:00 at TemperatureRoom temperature .
Note
Monitor under microscope. When hPSC colonies look losen, stop the incubation.

5m
Gently aspirate non-enzymatic dissociation reagent gently without disturbing the cells and discard.
1m
Add required volume of freezing medium to the culture vessel, refer to Table 1 for recommended volumes.
1m
Gently tap the plate Duration00:00:05 - 00:00:10 to dislodge the cells from the plastic surface.
Note
Gently scraping the vessel surface aids in clump harvesting when plate tapping alone is insufficient.

1m
Use a 2 mL pipette to transfer hPSC suspension (ideally Amount1 mL ) into pre-labeled cryo-vials.
Note
If multiple wells or vessels from same culture are going to be frozen, hPSC suspensions have to be pooled and homogenize in a 15 or 50 mL tube before freezing. Thus, every cryo-vial contains equal material.

1m
Immediately place cryo-vials into the freezing container and place at Temperature-80 °C DurationOvernight
Note
When available, controlled freezing using automated devices e.g. Viafreeze (Cytiva) is recommended.

3m
Transfer hPSC containing cryo-vials to a liquid N2 tank after Duration24:00:00
1d