TITLE: Coating of tissue Culture Vessels for hPSC 
AUTHORS: Lisa Pavinato, Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
This protocol describes procedures to coat culture vessels for the maintenance of hPSC using different extracellular matrices.

[STEPS]
SECTION: Aliquoting and storage of Vitronectin
2. Upon receipt, aliquot Vitronectin and store vials according to manufacturer instructions (Table 2).


 

Do not store aliquots in frost-free freezer. Do not repeatedly freeze and thaw.
SECTION: Coating
3. Thaw a vial of Vitronectin at Room temperature for 2 min. 
SECTION: Matrix selection
1. 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.
 
 
 

SECTION: Coating
8. If not used immediately, coated plates can be stored at 2 °C to 8 °C  up to seven days.
 

For stored plates, prior to plating the cells, allow plates to equilibrate at Room temperature for about 20 min and     
 


 
SECTION: Aliquoting and storage of Matrigel/Geltrex
2. Upon receipt, store Matrigel/Geltrex according to the manufacturer indication indicated in the "Materials" section.
SECTION: Coating procedure
5. Remove an aliquot of Geltrex from-20 °C storage and thaw on wet ice for about  30 min.  
 

SECTION: Coating procedure
6. Mix the content of one vial of  125 µL  Matrigel/Geltrex with 12.5 mL of cold DMEM/F-12 in a 15 mL falcon tube. Gently pipette up and down to resuspend the diluted matrix. Keep working on ice. 
SECTION: Coating procedure
7. Dispense diluted Matrigel/Geltrex on the vessel of choice, being careful that the surface is completely covered. If necessary, rock or tap the plate to facilitate this process. Refer to Table 1 for recommended volumes.
SECTION: Coating procedure
8. Incubate the coated tissue-culture vessels for 30 min at37 °C or for 60 min at Room temperature.
 

SECTION: Coating procedure
10. If plates were already coated and stored at 4 °C , prior to plating the cells, allow plates to equilibrate at Room temperature for about 20 min and     
 

SECTION: Coating procedure
9. After incubation, aspirate the Matrigel/Geltrex 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.
 

SECTION: Coating
4. In a 15-ml tube, dilute the Vitronectin in 6 mL DPBS, gently pipetting up and down to properly resuspend it.
 

SECTION: Coating
6. Incubate the coated tissue-culture vessels for 30 min to 60 min at Room temperature. 
SECTION: Coating
7. 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.
 

SECTION: Coating
5. 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.

SECTION: Aliquoting and storage of Matrigel/Geltrex
1.1. Before starting
To prevent gelling of Matrigel/Geltrex, pre-chill serological pipettes, tubes and plates to be stored at -20 °C for at least 20 min. Keep working on ice for all the procedures.
SECTION: Aliquoting and storage of Matrigel/Geltrex
4. Mix Matrigel/Geltrex by slowly inverting the original tube and prepare aliquots according to your needs. 
SECTION: Aliquoting and storage of Matrigel/Geltrex
3. Thaw Matrigel/Geltrex stock on wet ice for 180 min.
 
 

SECTION: Aliquoting and storage of Laminin
3. Prepare aliquots according to your needs: the format we suggest in Table 3 for the aliquoting is enough to coat a full 6-well vessel when diluted.

 

SECTION: Coating procedure
4. Follow the appropriate coating procedures depending on the chosen Laminin.
SECTION: Coating procedure
6. Mix 60 µL of Laminin 521 with 60 mL PBS containing Ca2+Mg2+ in a 15 mL tube (1:100 dilution). Final working concentration = 5 μg/mL.
8. Incubate the coated cell culture vessel at least 120 min at 37 °C. 
7. Dispense diluted Laminin 521 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 recommended volumes according to vessel format.
9. After incubation, aspirate the Laminin 521 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.
 

10. If not used immediately, coated plates can be stored at 4 °C   sealed with Parafilm up to seven days; however, a significant reduction in performance is expected.
SECTION: Coating procedure
5. Thaw a vial of Laminin 521 at Room temperature for 2 min. 
SECTION: Coating procedure
5. Mix 57.6 µL of iMatrix-511 with 12 mL PBS without Ca2+Mg2+ in a 50 ml tube. Final working concentration = 2.4 μg/mL.
SECTION: Coating procedure
6. Dispense diluted iMatrix-511 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 recommended volumes according to vessel format.
SECTION: Coating procedure
7. Incubate the coated cell culture vessel 60 min at 37 °C. 
 

SECTION: Coating procedure
8. After incubation, aspirate the iMatrix-511 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.
 

SECTION: Aliquoting and storage of Matrigel/Geltrex
4.1. Prepare aliquots of 125 µL keeping always the tubes on ice. Store at -20 °C.
 

SECTION: Aliquoting and storage of Laminin
2. Upon receipt, store Laminin according to the manufacturer's instructions.