12. Connect the C-flex tubing of a 1.5 L bag of pre-warmed F3

hPSC passaging solution to the Sampling port tubing. Allow

the F3 hPSC passaging solution to enter the bioreactor vessel

by gravity (see Note 41).

13. When the vessel is filled with 1.5 L of F3 hPSC passaging

solution, put on the heating jacket and center the bioreactor

vessel on the analytical scale.

14. During the incubation step, turn on the heating jacket at 37 ^C

and begin agitation at 90 rpm for 30 min.

15. Weld a syringe-extension set to the bioreactor and draw 5 mL

of sample. Transfer the solution onto a 10 cm cell culture dish

and confirm the detachment of cells from the microcarriers

using a microscope.

16. Begin the filtration process when more than 90% of cells are

detached from the microcarriers as single cells (see Note 42).

17. While maintaining 90 rpm agitation speed, begin the filtration

process by using the pump (200 mL/min) via the harvest line

into the mesh filter.

18. When the top of the impeller is visible, decrease the agitation

speed to 30 rpm. Tilt the vessel to retrieve as much of the cells

from the vessel as possible (see Note 43).

19. Gently shake the bag to mix the cell suspension.

20. Weld a syringe-extension set to the bioreactor and draw three

5 mL samples.

21. Count the number of cells and determine viability (see Note

13).

22. Downstream processes of interest such as directed differentia-

tion and cryopreservation [9] can now be performed on the

expanded cells (see Note 44).

4

Notes

1. Incomplete medium refers to the L7™TFO2 hPSC basal

medium without the L7™hPSC medium supplement.

2. Other commercially available cell culture medium such as

mTeSR1 [6] have been used in the expansion of stem cells in

bioreactors. During the expansion process in a 3 L bioreactor

vessel, the Lonza L7™TFO2 hPSC basal medium packaged in

7 L media bags were used.

3. The L7™hPSC passaging solution is a non-enzymatic solution

that results in cell clumps.

4. The F3 hPSC passaging solution is a non-enzymatic solution

that results in single cells and may require the addition of

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors

49