TITLE: Non-enzymatic passaging of hPSC
AUTHORS: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
This protocol describes the procedure to passage hPSC culture using enzyme-free reagents. 

[BEFORE_START]
If using EDTA as enzyme-free reagent, prepare EDTA 0.5 millimolar (mM)  as follows:
Dilute 1 to 1000 EDTA 0.5 Molarity (M) in distilled water; e.g. 5 mL EDTA 0.5 Molarity (M) in 995 mL distilled water. 
Filter sterile using 0.22 µm filter.
Store at RT.




 

[GUIDELINES]
In this protocol, hPSC passaging is done in aggregate/clumps format. The protocol is adaptable to different enzyme-free reagents described in Materials section and variable plate formats.

[STEPS]
SECTION: Preparation of destination vessel
1. Prepare coated tissue culture vessels and culture media according to hPSC line requirements and desired format. 
Refer to protocols: Coating of tissue Culture Vessels for hPSC and Maintenance of hPSC.
SECTION: hPSC non-enzymatic passaging
6. Aspirate and discard media from selected source vessel. 
SECTION: hPSC non-enzymatic passaging
7. Wash the well once using DPBS (no Calcium/no Magnesium).
SECTION: Preparation of destination vessel
4. Keep prepared destination vessel at 37 °C until usage.
SECTION: Preparation of destination vessel
3. Add hPSC maintenance media per destination vessel, refer to Table 1 for recommended volumes.
 
 
SECTION: hPSC non-enzymatic passaging
5. Prepare required volume of the reagents and media according to the Table 1.  Equilibrate the media to Room temperature.
 

SECTION: hPSC non-enzymatic passaging
8. Add enzyme-free reagent, refer to Table 1 for recommended working volumes.
SECTION: hPSC non-enzymatic passaging
9. Incubate the vessel at 37 °Cmonitoring hPSC detachment using a microscope. Refer to Table 2 for recommended incubation times. 

Cells will be ready to harvest when colonies start to be loosen though still attached. 
 
 
 

SECTION: hPSC non-enzymatic passaging
10. Aspirate and discard enzyme-free reagent.
SECTION: hPSC non-enzymatic passaging
11. Add fresh hPSC maintenance media to the wells, refer to Table 1 for recommended harvesting volumes.
SECTION: hPSC non-enzymatic passaging
12. Pipette up and down using 5 mL serological pipette against the bottom of the well to dislodge cells from the culture surface.
 

 


SECTION: hPSC non-enzymatic passaging
13. Collect hPSC aggregates suspension using 5 mL pipette and directly distribute drop-wise the approximate volume for splitting ratios: 1/10, 1/15, 1/20 (or as desired) to the destination vessel/s.
 

SECTION: hPSC non-enzymatic passaging
14. Gently move destination vessels with freshly passed aggregates in cross shape to distribute them evenly.
SECTION: hPSC non-enzymatic passaging
15. Incubate the vessels at 37 °C  and at 5 % volume CO2.
SECTION: hPSC non-enzymatic passaging
16. After 1440 min perform full media change. For further hPSC culture refer to protocol Maintenance of hPSC and protocol Reference pictures of hPSC cultured in defined conditions
SECTION: Preparation of destination vessel
2. Aspirate and discard coating solution from wells of destination vessel.