May 23, 2025
Coating of tissue Culture Vessels for hPSC
- Lisa Pavinato1,2,
- Valeria Fernandez Vallone3,
- Lyn Healy4,
- Nathalie Lefort5,
- Katarzyna Ludwik3,
- Tamer Onder6,
- Fatma Visal OKUR7,
- Harald Stachelscheid3
- 1Institute of Oncology Research (IOR), Bellinzona Institutes of Science (BIOS+), Bellinzona, Switzerland;
- 2Faculty of Biomedical Sciences, Università della Svizzera Italiana, Lugano, Switzerland.;
- 3Core Unit pluripotent Stem Cells and Organoids - Berlin Institute of Health @ Charite, Berlin, Germany;
- 4The Francis Crick Institute;
- 5Université de Paris, Imagine Institute, iPSC Core Facility, INSERM UMR U1163, F-75015 Paris, France.;
- 6Koc University;
- 7Hacettepe University, Center for Stem Cell Research and Development (PEDISTEM) and Hacettepe University Faculty of Medicine, Department of Pediatrics
- CorEuStem
Protocol Citation: Lisa Pavinato, Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Fatma Visal OKUR, Harald Stachelscheid 2025. Coating of tissue Culture Vessels for hPSC . protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9pz24g3e/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 20, 2023
Last Modified: May 23, 2025
Protocol Integer ID: 91194
Keywords: Coating, matrigel, vitronectin, geltrex, laminin, hPSC, matrix, iPSC, tissue culture vessel, culture vessels for the maintenance, culture vessel, hpsc, hpsc this protocol, tissue, using different extracellular matrice, different extracellular matrice
Funders Acknowledgements:
COST Action 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 procedures to coat culture vessels for the maintenance of hPSC using different extracellular matrices.
Materials
LABORATORY EQUIPMENT AND CONSUMABLES
Use sterile material
- 1/5/10 mL pipettes
- 1.5/15/50 mL conical tubes
- 10/200/1000µL tips and pipettes (optional)
- Cell culture treated plastic vessels of choice e.g. 24, 12 or 6-well plates, T25, T75 flasks, 10cm dishes
- Aspirator pump with disposable pipette
- Class II Biosafety Cabinet
- Wet ice
MEDIA AND REAGENTS
Vitronectin options tested by authors
- Vitronectin (VTN-N) Recombinant Human Protein, TruncatedThermo FisherCatalog #A14700 (500 μg/ml)
- Vitronectin XF™STEMCELL Technologies Inc.Catalog ##07180 (250 μg/ml)
- Recombinant Human Vitronectin Protein, CFR&D SystemsCatalog #2308-VN
Geltrex options tested by authors
- Geltrex LDEV Free hESC Quality 5 mlThermo Fisher ScientificCatalog #A1413302
- Geltrex™ hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane MatrixThermo FisherCatalog #A1569601
Matrigel options tested by authors
- Growth Factor Reduced (GFR) Matrigel® phenol red-freeCorningCatalog #356231
- Matrigel hESC-Qualified Matrix, LDEV-freeCorningCatalog #354277
Laminin options tested by authors
- BIOLAMININ 521 LN (LN521)BiolaminaCatalog #LN521
- iMatrix-511 Recombinant Laminin E8 Fragments (Laminin matrix)amsbioCatalog #AMS.892 011
Diluent
- DPBS (no Ca, no Mg)ThermofisherCatalog #14190144 or other vendors
- Gibco™ DPBS (10X), calcium, magnesiumFisher ScientificCatalog #14080055
- DMEM/F12Thermo Fisher ScientificCatalog #11320033 or Advanced DMEM/F-12Thermo FisherCatalog #12634028
- CellAdhere™ Dilution BufferSTEMCELL Technologies Inc.Catalog #07183 can be used instead of DPBS in combination with Vitronectin XF™STEMCELL Technologies Inc.Catalog ##07180 (see "Guidelines and Warnings").
| A | B | C | D | E | |
| Product | Company | Catalogue number | Stock concentration | Storage upon receipt | |
| Vitronectin (VTN-N) Recombinant Human Protein, Truncated | ThermoFisher | A14700 | 500 μg/mL | -80°C | |
| Vitronectin XF | StemCell Technologies | 07180 | 250 µg/ml | -20°C to -80°C | |
| Recombinant Human Vitronectin Protein, CF | R&D Systems | 2308-VN | 50 µg | -20°C to -80°C | |
| Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix | ThermoFisher | A1413302 | 12 to 18 mg/ml | -20°C to -80°C | |
| Geltrex hESC-Qualified, Ready-To-Use, Reduced Growth Factor Basement Membrane Matrix | ThermoFisher | A1569601 | 0.12 to 0.18 mg/mL | 2°C to 8°C protected from light | |
| Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-free, LDEV-free | Corning | 356231 | batch dependent | -20°C to -80°C | |
| Corning Matrigel hESC-Qualified Matrix, LDEV-free | Corning | 354277 | batch dependent | -20°C to -80°C | |
| Laminin 521 | Biolamina | LN521-05 | 100 µg/ml | -80°C | |
| iMatrix-511 | amsbio | AMS.892011 | 500 μg/ml | 2°C to 15°C |
Table 4. List of tested coatings, their initial concentration and the temperature of storage upon reception.
Troubleshooting
Safety warnings
Vitronectin coating
The use of Non-tissue culture-treated cultureware STEMCELL Technologies Inc. and CellAdhere™ Dilution Buffer 100 mL
STEMCELL Technologies Inc.Catalog #7183 is recommended with Vitronectin XF™STEMCELL Technologies Inc.Catalog ##07180 from the vendor.
However, we tested the use of normal tissue culture-treated cultureware and DPBS no calcium, no magnesiumInvitrogen - Thermo FisherCatalog #14190136 with Vitronectin XF™STEMCELL Technologies Inc.Catalog ##07180 , and no major differences were noted. In the protocol, we refer to this last combination.
Select extracellular matrix (ECM) according to desired culture conditions. See protocol Maintenance of hPSC for an overview on medium/matrix combinations that have been validated by CorEuStem members.
Refer to Table 1 for final volumes needed according to vessel format.
| A | B | C | |
| Culture vessel | Approximative growth area (cm2) | Volume of diluted matrix per vessel/well (ml) | |
| 96 well | 0.32 | 0.05-0.1 | |
| 48 well | 1.1 | 0.1-0.2 | |
| 24 well | 1.9 | 0.2-0.5 | |
| 12 well | 3.5 | 0.4-1.0 | |
| 6 well | 9.6 | 1.0-2.0 | |
| 100 mm dish | 56.7 | 5.0-8.0 | |
| T25 flask | 25 | 2.5 | |
| T75 flask | 75 | 7.5 | |
| T175 flask | 175 | 10-18 |
Table 1. Suggested volumes of diluted matrices for coatings.
5m
Vitronectin7 steps
Different versions of Vitronectin are reported in "Materials" section.
Aliquoting and storage of Vitronectin
15m
Upon receipt, aliquot Vitronectin and store vials according to manufacturer instructions (Table 2).
| A | B | C | D | |
| A14700 ThermoFisher | 07180 StemCell Technologies | 2308-VN R&D Systems | ||
| Thawing of stock | RT | RT or ON 4°C | Powder | |
| Reconstitution | Ready-to-use 500μg/mL | Ready-to-use 250μg/mL | 50 µg in 200 µl sterile DPBS to obtain 250μg/mL | |
| Aliquot size* | 60µl/vial | 120µl/vial | 120µl/vial | |
| Storage of aliquots | -80°C | -20°C/-80°C | -80°C |
Table 2. Instructions describing reconstitution and aliquoting of vitronectin variants from different manufacturers
*Prepare aliquots according to your needs: the format we indicated for the aliquoting is enough to coat a full 6-well plate when diluted.
RT = room temperature; ON = over night
Do not store aliquots in frost-free freezer. Do not repeatedly freeze and thaw.
15m
Coating
1h 31m
Thaw a vial of Vitronectin at Room temperature for 00:02:00 .
2m
In a 15-ml tube, dilute the Vitronectin in 6 mL DPBS, gently pipetting up and down to properly resuspend it.
Note
The indicated dilutions are for a final concentration of 5 μg/mL . Vitronectin coating has been tested and validated up to a final concentration of 3.33 μg/mL for hPSC maintenance. Optimal concentration depends on specific hPSC lines, the application or research objectives.
2m
Dispense diluted Vitronectin on the preferred dish, being careful that the surface is completely covered. If necessary, rock or tap the plate to facilitate this process. Refer to Table 1 for suggested volumes according to vessel format.
2m
Incubate the coated tissue-culture vessels for 00:30:00 to 01:00:00 at Room temperature .
1h
After incubation, aspirate the Vitronectin solution right before use, by tilting the plate and ensuring not to scrape the bottom of the well. Directly proceed with the plating of cells. Refer to Maintenance of hPSC, and/or to Single cell passaging of hPSC, Non-enzymatic passaging of hPSC. Do not allow the coated surface to dry.
Note
Vitronectin-coated plates cannot be re-used: always seed cells onto fresh Vitronectin-coated plates.
5m
If not used immediately, coated plates can be stored at 2 °C to 8 °C up to seven days.
Note
In our experience, the coating can be stored up to 30 days; however this is not suggested by the manufacturers.
For stored plates, prior to plating the cells, allow plates to equilibrate at Room temperature for about 00:20:00 and
Note
Do not allow the coated surface to dry out. To prevent excessive evaporation of the coating, tightly seal the plates with Parafilm. Additionally, some DPBS may be added after the incubation time is finished.
20m
Protocol references
- Chen G, Gulbranson DR, 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
- Nishishita N, Shikamura M, Takenaka C, et al. Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naïve state. PLoS One. 2012;7(6):e38389. https://doi.org/10.1371/journal.pone.0038389
- Rodin S, Domogatskaya A, Ström S, et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat Biotechnol. 2010;28(6):611–615. https://doi.org/10.1038/nbt.1620
- 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
- Lu HF, Lim SX, Leong MF, et al. Defined culture conditions of human embryonic stem cells. Methods Mol Biol. 2014;1181:1–15. https://doi.org/10.1007/978-1-4939-1047-2_1