10 μM ROCK inhibitor to facilitate attachment to the cell

culture vessel.

5. The L7™hPSC matrix stock solution can be aliquoted in

1.5 mL Eppendorf tubes and stored at 20 ^C until use.

Avoid multiple freeze-thaw cycles.

6. Aliquot the stock solution of ROCK inhibitor and store at

20 ^C until use. Avoid multiple freeze–thaw cycles.

7. Following the autoclave cycle, allow the sterilized materials to

cool before use. Assemble the required tubings with the biore-

actor vessel in a Biological Safety Cabinet.

8. Autoclave the pH probe using the liquid cycle. Allow the probe

to cool after autoclaving.

9. Cell culture vessels may be coated with the extracellular matrix

coating solution for at least an hour and up to 48 h in 37 ^C.

Store in 4 ^C if not used immediately. Use the coated vessels

within 2 weeks.

10. The L7™hPSC culture system allows for an every-other-day

medium change.

11. Ensure to passage or harvest cells at 70–80% confluence.

Higher cell confluence can lead to lower cell viability and

impact attachment to the coated microcarriers.

12. Every 5 min, examine the colonies under the microscope. Over

time, the colonies will form holes indicating that the cells are

ready to be harvested.

13. Cell counts can be performed using a hemocytometer or an

automated cell counter.

14. Tilt the 1-layer cell stack when adding the DPBS^/ to wash,

but not to dislodge the attached cells.

15. Lower cell densities may be used. The cell density of cell

inoculum and rate of expansion are also dependent on the

cell line used in the process.

16. The coated microcarriers may be stored in 4 ^C for up to

7 days. Longer storage times have not been tested.

17. When introducing the coated microcarriers to the bag of L7™

TFO2 hPSC basal medium, periodically shake the bottle to

avoid settlement of microcarriers at the bottom of the bottle.

18. Insert the mesh end of the perfusion dip tube carefully. Do not

bend the mesh against the bottom of the vessel lest it breaks.

19. The LA2 port corresponds to the port from which aliquots of

cell solutions can be drawn.

20. The Sample port is used as an alternative to the LA2 port.

21. Prior to removing the vessel from the Biological Safety Cabi-

net, ensure that the ports are closed and the tubings are

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Marites T. Woon et al.