Collection Citation: Valeria Fernandez Vallone, Katarzyna Ludwik, Lyn Healy, Nathalie Lefort, Tamer Onder, Lisa Pavinato, Fatma Visal OKUR, Harald Stachelscheid 2025. Good practices in hPSC handling. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk287wl5r/v1
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: April 30, 2025
Last Modified: May 23, 2025
Collection Integer ID: 198125
Keywords: reproducibility, hPSC, pluripotent stem cells, hiPSC, hESC, standards, culture, E8 medium, passaging, cryopreservation, maintenance, survival factors, ROCK inhibitor, CEPT, CloneR2, single cells, reference images, matrix, manual picking, colony, mTeSR, E8 preparation, thawing, healthy hpsc morphology under various culture condition, standardization of stem cell culture technique, human pluripotent stem cell, stem cell culture technique, pluripotent stem cell, illustrating healthy hpsc morphology, stem cell, cell quality assessment, critical visual benchmarks for cell quality assessment, enhanced cell viability, extensive experience from established hpsc core facility, established hpsc core facility, cell viability, hpsc, cell viability during critical manipulation, foundational resource for laboratory, including rock inhibitor, cell
Funders Acknowledgements:
COST CorEuStem
Grant ID: CA20140
Abstract
This protocol collection provides a comprehensive and practical guide for the maintenance, passaging, freezing, and thawing of human pluripotent stem cells (hPSCs). Designed to support reproducibility and transferability, especially among new users, the protocols are informed by extensive experience from established hPSC core facilities.
In addition to detailed step-by-step procedures, the collection includes high-quality reference images illustrating healthy hPSC morphology under various culture conditions, as well as visual examples of spontaneous differentiation—serving as critical visual benchmarks for cell quality assessment. The protocols also cover the in-house preparation of Essential 8 (E8) medium and the use of key survival-supporting compounds, including ROCK inhibitors (ROCKi), CEPT cocktail, and CloneR2, ensuring enhanced cell viability during critical manipulations.
By consolidating expert-validated practices and making them accessible through a user-friendly format, this collection aims to serve as a foundational resource for laboratories working with hPSCs and to facilitate the standardization of stem cell culture techniques across different research environments.
Guidelines
Always work aseptically in a Class II biosafety cabinet.
Ensure that you keep the safety cabinet clean and tidy.
For incubation, place culture vessels on trays to contain any accidental spillage of media and reduce the risk of contamination.
To avoid cross contamination between cell lines:
Only one cell line at a time should be handled in a biosafety cabinet when feeding or passaging the cells.
Bottles or aliquots of media should be prepared for separately for each cell line .
Aliquots should be labelled with the name of the line and the date prepared and stored and handled appropriately.
This protocol collection describes the culture of hPSC under normoxia conditions (5%CO2) due to availability in most cell culture laboratories. However, to reduce genomic instability during culture we recommend to incubate hPSC under hypoxia conditions: 5%CO2 and 5% O2.
Troubleshooting
Safety warnings
The estimated time for an individual protocol does not account for the time required to complete supporting protocols.
Before start
Before starting this protocol, ensure that you have all of the materials required to complete the protocol with ease and efficiency.
