May 23, 2025

Public workspaceSurvival factors for hPSC growth

DOI
https://dx.doi.org/10.17504/protocols.io.3byl4wmdrvo5/v1
Survival factors for hPSC growth
  • Tamer Onder1,
  • Valeria Fernandez Vallone2,
  • Lyn Healy3,
  • Nathalie Lefort4,
  • Katarzyna Ludwik2,
  • Lisa Pavinato5,6,
  • Fatma Visal OKUR7,
  • Harald Stachelscheid2
  • 1Koc University;
  • 2Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
  • 3The Francis Crick Institute;
  • 4Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.;
  • 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: https://dx.doi.org/10.17504/protocols.io.3byl4wmdrvo5/v1
Protocol Citation: Tamer Onder, Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Lisa Pavinato, Fatma Visal OKUR, Harald Stachelscheid 2025. Survival factors for hPSC growth. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wmdrvo5/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: March 11, 2025
Last Modified: May 23, 2025
Protocol Integer ID: 124189
Keywords: survival, apoptosis, ROCK inhibitor , CEPT, CloneR2, hPSC, iPSC, supplementation of hpsc culture media, survival factors for hpsc growth, hpsc culture media, hpsc growth, supplementation, survival factor, survival
Funders Acknowledgements:
COST Action CorEuStem
Grant ID: CA20140
Abstract
This protocol describes the preparation and supplementation of hPSC culture media with survival factors.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
  • 1/5/10 mL pipettes
  • 15/50 mL conical tubes
  • 10/200/1000µL tips and micropipettes
  • 0.5 or 1.5 mL tubes
  • Centrifuge
  • Vortex
  • Class II Biosafety Cabinet

MEDIA AND REAGENTS

ReagentY-27632SelleckchemCatalog #S1049
ReagentY-27632 dihydrochlorideMedChemExpressCatalog #HY-10583
ReagentY-27632 (Dihydrochloride) 5 x 10 mg STEMCELL Technologies Inc.Catalog #72307
Reagent• Chroman I MedChemExpressCatalog #HY-15392
ReagentEmricasan (IDN-6556)SelleckchemCatalog #S7775
ReagentISRIB (trans-isomer)MedChemExpressCatalog #HY-12495
ReagentTrans-ISRIBTocrisCatalog #5284
ReagentPolyamine Supplement (1000×)Merck MilliporeSigma (Sigma-Aldrich)Catalog #P8483
ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D2650
ReagentCloneR2STEMCELL Technologies Inc.Catalog ## 100-0691

hPSC culture conditions and survival factors choice depend on hPSC line and individual lab practices.
For options refer to protocols:
Maintenance of hPSC
Coating of tissue Culture Vessels for hPSC
Troubleshooting
Select survival factor according to the desired culture condition or lab-workflow from the dropdown below.

Note
Presence of survival factors in the culture medium can have strong effects on hPSC morphology. Refer to Reference pictures of hPSC cultured in defined conditions for examples of typical morphologies.


Step case

Y-27632 ROCK inhibitor
7 steps

Stock solution preparation and aliquoting
40m
Spin down the vial containing ROCK inhibitor Y-27632 (ROCKi) powder prior to opening.
2m
Stock solution preparation and aliquoting
40m
Reconstitute in required volume of sterile water in a 15 mL Falcon tube. Refer to Table 1 for quantity and corresponding volume.

ABC
Amount of ROCKiVolume of waterFinal concentration
1mg625ul5 mM (500x)
5mg3.125ml5 mM (500x)
10mg6.25ml5 mM (500x)
50mg31.25ml5 mM (500x)
Table 1. Guidance to volumes needed to dissolve different amounts of ROCKi.

Note
Alternatively, use DMSO as solvent.
ROCKi solubility in both water and DMSO is maximum Concentration100 millimolar (mM)


5m
Prepare Amount200 µL aliquots in 0.5 mL eppendorf tubes.

Note
If ROCKi is provided as ready-to-use solution e.g. Concentration10 millimolar (mM) , prepare Amount50 µL - 100 µL aliquots and store as described below.

15m
Store aliquots atTemperature-20 °C or atTemperature-80 °C for long-term storage (up to two years).
2m
Preparation of culture medium supplemented with ROCKi
5m
Thaw required number of ROCKi aliquots according to culture media volume needed.

Note
After thawing, ROCKi aliquots can be stored at Temperature4 °C for up to two weeks.
Do not repeatedly freeze-thaw this reagent.

2m
Supplement culture media to the final concentration of Concentration10 micromolar (µM) ROCKi (Table 2).
ABC
Volume Medium [mL]Volume ROCKi 5mM [µL]Volume ROCKi 10mM [µL]
5 10 5
12 24 12
50 100 50
500 1000 500
Table 2. ROCKi volume per culture media volume supplemented accorsing to ROCKi stock concentration.

3m
Always supplement fresh the culture media with ROCKi prior to use.
Proceed with cell culture, refer to protocols:
Maintenance of hPSC
Single cell passaging of hPSC
Reference pictures of hPSC cultured in defined conditions

Protocol references
  • Tristan CA, Hong H, Jethmalani Y, et al. Efficient and safe single-cell cloning of human pluripotent stem cells using the CEPT cocktail. Nat Protoc. 2023;18:58–80. https://doi.org/10.1038/s41596-022-00753-z
  • Chen Y, Tristan CA, Chen L, et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat Methods. 2021;18:528–541. https://doi.org/10.1038/s41592-021-01126-2
  • Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007;25(6):681–686. https://doi.org/10.1038/nbt1310
  • Kalamegam G, Fong CY, Bongso A. Effect of ROCK inhibitor Y-27632 on normal and variant human embryonic stem cells (hESCs) in vitro: its benefits in hESC expansion protocols. Stem Cell Rev Rep. 2010;6(1):86–95. https://doi.org/10.1007/s12015-009-9107-8