May 23, 2025
Manual picking and passaging of hPSC colonies
- Nathalie Lefort1,
- Valeria Fernandez Vallone2,
- Lyn Healy3,
- Katarzyna Ludwik2,
- Tamer Onder4,
- Lisa Pavinato5,6,
- Fatma Visal OKUR7,
- Harald Stachelscheid2
- 1Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.;
- 2Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
- 3The Francis Crick Institute;
- 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: Nathalie Lefort, Valeria Fernandez Vallone, Lyn Healy, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal OKUR, Harald Stachelscheid 2025. Manual picking and passaging of hPSC colonies. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g75rmqlwz/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 26, 2025
Last Modified: May 23, 2025
Protocol Integer ID: 125207
Keywords: hpsc colonies this protocol, hpsc colony, picking of single colony, manual clump, selection, single colony, picking, many colony, protocol, positive selection, expansion of many colony
Funders Acknowledgements:
COST CorEuStem
Grant ID: CA20140
Abstract
This protocol describes picking and expansion of many colonies at once (manual clump passaging) and the picking of single colony (positive selection).
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.
Recommended split ratio for established cultures is around 5 colonies per 10 cm2 surface area (i.e. 5 colonies per well of a 6-well plate (or 35 mm dish) or 10 colonies per 60 mm dish.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
- 1/5/10 mL serological pipettes
- 15/50 mL conical tubes
- 200/1000µL tips and micropipettes
- Cell culture treated plastic vessels of choice e.g. 24, 12 or 6-well plates, 10cm dishes)
- Syringes (1mL) and needles (23 to 25G) or stem cell knife (see examples in pictures below)
1 mL syringe and needles (23G and 25G)
1 mL syringe and needles (23G and 25G)
Stem Cell Knife
- Aspirator pump with disposable pipette
- Class II Biosafety Cabinet
- Microscope, if available Stereo Microscope
Different models of Stereo Microscopes
MEDIA AND REAGENTS
hPSC culture conditions and survival factors choice depend on hPSC line and individual lab practices. For options refer to protocols:
Troubleshooting
Safety warnings
Manual clump passaging is recommended for colonies grown on vitronectin* or on feeder cells since their surface adhesion is less strong than when cultured on matrigel, geltrex or laminin.
Prepare vitronectin-coated vessels according to how many clones are to be picked, refer to protocol Coating of tissue Culture Vessels for hPSC
Manual clump passaging12 steps
Picking and passaging of many colonies at once
Pre-warm the required volume medium (refer to Table 1) atRoom temperature for 00:30:00 in 50 mL sterile Falcon tubes.
| A | B | C | D | |
| Culture Vessel | Surface Area [cm2]* | Total volume[ml] | ||
| 6 well plate | 1 well | ~9,6 | 2 | |
| 35 mm dish | 1 dish | ~9.6 | 2 | |
| 60 mm dish | 1 dish | ~20 | 4 | |
| 12 well plate | 1 well | ~4 | 1 | |
| 24 well plate | 1 well | ~2 | 0,5 | |
| 24 well plate | 24 wells | 12 |
Table 1. volume of medium to be prepared according to culture format
30m
Remove the culture vessel containing the cells from the incubator.
Visually inspect the quality of the hiPSC culture under a stereomicroscope. For assesment of culture quality see Reference pictures of hiPSC cultured in defined conditions. If needed remove the differentiated regions as described in protocol: Maintenance of hPSC
15m
Aspirate the medium containing floating and/or differentiated cells.
1m
Add the appropriate volume of hPSC medium, refer to Table 1 for recommended volumes.
2m
Using a syringe with a needle or a stem cell knife cut about 5 colonies/10cm2 surface area into small pieces (i.e. 5 colonies per well of a 6-well plate (or 35 mm dish) or 10 colonies per 60 mm dish).
Figure 1. Schematic of the mechanical cutting of a colony from IMAGINi004-A iPSC line
15m
Use a 200µL pipette with volume adjusted to 100µL to gently dislodge the pieces of colonies with the liquid stream from the pipette.
5m
Aspirate the residual coating solution from the destination culture vessel.
1m
Transfer the medium containing the pieces of colonies to the destination culture vessel using 1000µL pipette with volume adjusted to 1000µL.
2m
If needed adjust the volume of medium in the destination dish to the total volume. Refer to Table 1 for recommended volumes.
2m
If necessary to repeat, add additional media to the source culture vessel and repeat from step 7 to 14 as many times as necessary, depending on the number of new dishes required.
Incubate the vessels at 37 °C and at 5 % volume CO2.
Protocol references
- Ellerström C, Strehl R, Moya K, Andersson K, Bergh C, Lundin K, et al. Derivation of a xeno-free human embryonic stem cell line. Stem Cells. 2006;24(10):2170–2176. https://doi.org/10.1634/stemcells.2006-0130
- Stephenson E, Jacquet L, Miere C, Wood V, Kadeva N, Cornwell G, et al. Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment. Nat Protoc. 2012;7(7):1366–1381. https://doi.org/10.1038/nprot.2012.080
- De Sousa PA, Downie JM, Tye BJ, Bruce K, Dand P, Dhanjal S, et al. Development and production of Good Manufacturing Practice grade human embryonic stem cell lines as source material for clinical application. Stem Cell Res. 2016;17(2):379–390. https://doi.org/10.1016/j.scr.2016.08.011.