May 23, 2025
Single cell passaging of hPSC
- Valeria Fernandez Vallone1,
- Lyn Healy2,
- Nathalie Lefort3,
- Katarzyna Ludwik1,
- Tamer Onder4,
- Lisa Pavinato5,6,
- Fatma Visal 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 OKUR, Harald Stachelscheid 2025. Single cell passaging of hPSC . protocols.io https://dx.doi.org/10.17504/protocols.io.261gedjwdv47/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: 91254
Keywords: enzymatic, single cells, hPSC, iPSC, passaging, split, hpsc this protocol, using enzymatic dissociation reagent, enzymatic dissociation reagent, single cell, hpsc, cell
Funders Acknowledgements:
COST Action CorEuStem
Grant ID: CA20140
Abstract
This protocol describes single cell passaging of hPSC using enzymatic dissociation reagents.
Guidelines
The protocol is adaptable to different enzyme-free reagents described in Materials section and various vessel formats.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
- 1/5/10 mL 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)
- Cell scraper
- Cell counting equipment
- Aspirator pump with disposable pipette
- Centrifuge
- Microscope, if available Stereo Microscope
- Incubator at 37oC and 5% CO2 or under hypoxic conditions, 5% O2/ 5% CO2
- Class II Biosafety Cabinet
MEDIA AND REAGENTS
StemPro™ Accutase™ Cell Dissociation ReagentGibco - Thermo Fisher ScientificCatalog #A1110501
TrypLE™ Express EnzymeThermo Fisher ScientificCatalog #12604013
Gentle Cell Dissociation Reagent 100 mL
STEMCELL Technologies Inc.Catalog #7174
Trypan Blue Solution 0.4% Sterile-filtered Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8154
hPSC culture conditions and survival factors choice depend on hPSC line and individual lab practices. For options refer to protocols:
Troubleshooting
Preparation of destination vessel
30m
Prepare coated tissue culture vessels and culture media according to hPSC line requirements and desired format (Table 1).
| A | B | E | F | |
| Culture vessel | Dissociation reagent (mL) | hPSC media + survival factor (harvest: mL/per well) | hPSC media + survival factor (final: mL/per well | |
| 96 well | 0.05 | 0.15 | 0.2 | |
| 24 well | 0.25 | 1 | 0.5 | |
| 12 well | 0.5 | 1.5 | 1 | |
| 6 well | 1 | 3 | 2 | |
| T25 | 2 | 6 | 7 |
Table 1. Recommended volumes according to vessel format
Equilibrate prepared destination vessel at 37 °C until usage.
30m
hPSC single-cell passaging
1h 3m
Prepare required volume of the reagents and media according to the Table 1. Equilibrate the media to Room temperature .
Note
Culture media aliquot to be used can be warmed up at 37°C for 00:15:00 . However, to preserve recombinant proteins activity Room temperature equilibration is recommended.
20m
Aspirate and discard medium from the selected source vessel.
1m
Add enzymatic dissociation reagent, refer to Table 1 for recommended working volumes.
1m
Incubate the vessel at 37 °C for 00:05:00 - 00:10:00 .
Note
Incubation time depends on reagent used, coating and hPSC line attachment properties. Therefore, monitoring under microscope every 2-3 min until hPSC detachment observation is recommended.
If necessary, gently tap culture vessel from the side to detach hPSC.
10m
Add hPSC media supplemented with survival factors to the wells containing dissociation reagent, refer to Table 1 for recommended harvesting volumes.
1m
Pipette up and down several times to dislodge the cells from the culture surface.
Note
Pipette selection for this step depends on total hPSC suspension volume. The smaller the tip of the pipette the better cells will be singularized.
3m
Transfer hPSC suspension to a 15 or 50 mL conical tube.
Note
Several wells or vessels can be combined for centrifugation step.
2m
Centrifuge at 300 x g for 00:05:00 .
5m
Aspirate and discard supernatant without disrupting the cell pellet.
1m
Resuspend cell pellet in hPSC media supplemented with survival factors. Resuspension volume will depend on pellet size in a range of 1-10 mL.
2m
Perform hPSC counting using viability staining such as Trypan Blue 0.4%.
Note
Counting can be performed manually using e.g. Neubauer Chamber or automated cell counting devices.
10m
Adjust hPSC suspension volume according to desired number of cells for seeding, refer to Table 1 for recommended final media volumes.
Note
As example, for hPSC single cell passaging for hPSC maintenance in 6 well-plates, we recommend a seeding density between 200,000-400,000 cells per well. Seeding densities have to be tested and adjusted per hPSC line for optimal passaging.
2m
Aspirate coating solution from the destination vessel.
1m
Pipette cell suspension into the coated recipient wells according to desired hPSC seeding density, refer to Table 1 for recommended final media volumes.
3m
Gently move destination vessels with hPSC in suspension in cross shape to distribute them evenly.
1m
Incubate the vessels at 37 °C and at 5 % volume CO2.
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