May 23, 2025

Public workspaceMaintenance of hPSC

DOI
dx.doi.org/10.17504/protocols.io.bp2l6xmjrlqe/v1
  • 1The Francis Crick Institute;
  • 2Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
  • 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.bp2l6xmjrlqe/v1
Protocol Citation: Lyn Healy, Valeria Fernandez Vallone, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid 2025. Maintenance of hPSC. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6xmjrlqe/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 15, 2023
Last Modified: May 23, 2025
Protocol Integer ID: 90972
Keywords: iPSC, hPSC, confluence, maintenance, spontaneous differentiation
Funders Acknowledgements:
COST CorEuStem
Grant ID: CA20140
Disclaimer
The reported protocols are based on the authors experience, and may partially differ from the original protocols provided by the companies.
Abstract
This protocol describes maintenance of established hPSC lines in expansion media on matrix coated culture vessels.
Guidelines
Depending on the growth characteristics of specific hPSC line, cells must be passaged every 5-7 days in order to maintain log phase of growth and avoid induction of differentiation.
In general hPSC lines are maintained in a 6 well format, however information on other tissue culture vessel formats are also included in this protocol as a guidance.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
  • 1/5/10 mL serological pipettes
  • 15/50 mL conical tubes
  • Cell culture treated plastic vessels of choice e.g. 24, 12 or 6-well plates, T25, T75 flasks, 10cm dishes
  • 10/200/1000µL tips and micropipettes (optional)
  • Aspirator pump with disposable pipette
  • Centrifuge
  • Microscope, if available Stereo Microscope
  • Disposable scraping tools (pipette tips, glass rods, small size cell scrapper, syringe and needle)
  • Incubator at 37oC and 5% CO2  or under hypoxic conditions, 5% O2/ 5% CO2
  • Class II Biosafety Cabinet


MEDIA AND REAGENTS

Vitronectin

ReagentVitronectin (VTN-N) Recombinant Human Protein, TruncatedThermo FisherCatalog #A14700
ReagentVitronectin XF™STEMCELL Technologies Inc.Catalog ##07180

Laminin

ReagentMACSmatrix Laminin 511Miltenyi BiotecCatalog #130-136-454
ReagentrhLaminin-521Thermo FisherCatalog #A29248 ReagentCellAdhere™ Laminin-521 100 µg STEMCELL Technologies Inc.Catalog #77003

Basement Membrane Products

ReagentGeltrex™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1413301
ReagentMatrigel hESC-qualified (Corning Cat# 354277)CorningCatalog #354277

Cell Culture Media

ReagentEssential 8™ Medium Gibco - Thermo Fisher ScientificCatalog #A1517001
ReagentEssential 8 Flex complete medium (E8)Gibco - Thermo Fisher ScientificCatalog #A2858501
ReagentStemFlex MediumThermo Fisher ScientificCatalog #A3349401
ReagentmTeSR™1 500 mL Kit STEMCELL Technologies Inc.Catalog #85850
ReagentmTeSR plus media kitSTEMCELL Technologies Inc.Catalog #100-0276
ReagenteTeSRSTEMCELL Technologies Inc.Catalog #100-1215
ReagentStemMACS™ iPS-Brew XF human: Basal medium and supplementMiltenyi BiotecCatalog #130-104-368

Homemade Cell Culture Media: Essential 8 (E8) Medium (see protocol E8 media production)


Selection of culture conditions
Selection of culture conditions
15m
15m
The following points should be considered when choosing hPSC culture conditions:

  • A number of media will work with an array of different matrices.
  • The rate of growth of cell lines will differ between different matrix/media combinations and cell lines may exhibit a transient change in morphology until adapted to the new culture conditions.
  • When transitioning cell lines between different media/matrix combinations follow the manufacturers guidance. Usual recommendations: do not switch the media and matrix at the same time. Make a gradual change of media including proportions until full transition.
  • hPSC often require a couple of passages to adapt to the new culture conditions.
  • Following transition to new culture conditions, perform quality control testing as appropriate for the intended use of the cells.
  • For commercial media, prepare, store and use as directed by the manufacturer. For media made in house, use and store under the same conditions established for the validation of the media.

ABCDEFG
Media/MatrixTruncated VitronectinVitronectin XFMatrigelGeltrexLaminin 521Laminin 511
Essential E8XXXXXXX
Essential E8 FlexXXXXXXXX
mTeSR1XXXXXX
mTeSR PlusXXXX
StemMACS iPSC-Brew XFXXXXXXXX
Stem FlexXXXXXX
Table 2. Media and matrix possible combinations
XX = recommended by manufacturer and tested by CoreEuStem member
X = tested and recommended by CoreEuStem member
Homemade Cell Culture Media: Essential 8 (E8) Medium (see protocol E8 media production)

Note
The matrix media list is not exhaustive. It reflects the media and matrices that are used by the members of CorEuStem for their maintenance protocols.

15m
Inspect hPSC cultures daily using a microsope to monitor morphology, the presence of spontaneous differentiated cells, and confluence. Based on these observations, determine if the cultures require further action (e.g. removal of differentiated cells, passaging). Use reference images in the protocol: Reference pictures of hiPSC cultured in defined conditions to guide your assessment of the cultures. Cell lines may vary in their growth rate and morphology in different media/matrix combinations.
15m
Step case

Culture >70% confluent
1 step

Culture is >70% confluent and < 10% of the colonies in the culture appear differentiated upon visual inspection.
If the cells require passaging, refer to the protocol Non-enzymatic passaging of hPSC or Single cell passaging of hPSC. Otherwise performe a complete media change (refer to Culture <70% confluent tab).

Note
Assessing confluency is inherently subjective. Whenever possible, use imaging systems capable of calculating the surface area of the culture vessel occupied by cells to ensure a consistent and objective evaluation of cell culture confluency.
If such imaging tools are not available, utilize a collection of images captured during the culture process at various stages of confluency to train staff in recognizing and estimating different confluency percentages.
Keep in mind that the growth rate required to achieve confluency is influenced by both the specific cell line and the cell culture system used.

Protocol references
  • Ludwig T, Bergendahl V, Levenstein M, et al. Feeder-independent culture of human embryonic stem cells. Nat Methods. 2006;3(8):637–646. https://doi.org/10.1038/nmeth902

  • Chen G, Gulbranson D, Hou Z, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424–429. https://doi.org/10.1038/nmeth.1593


  • Braam SR, Zeinstra L, Litjens S, et al. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells. 2008;26(9):2257–2265. https://doi.org/10.1634/stemcells.2008-0291

  • Rodin S, Antonsson L, Niaudet C, et al. Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment. Nat Commun. 2014;5:3195. https://doi.org/10.1038/ncomms4195

  • Nakagawa, M., Taniguchi, Y., Senda, S. et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep 4, 3594 (2014). https://doi.org/10.1038/srep03594