2. Insert the sterile pH probe into the appropriate port.

3. Insert the sterile perfusion dip tube into the appropriate port

(see Note 18).

4. Screw the pH probe and perfusion dip tube in the vessel tightly

to ensure they are held in place.

5. Wrap parafilm around the ports to close any gaps and maintain

a sterile environment.

6. To the Harvest port on the vessel, remove the male MPC

connection. Attach the harvest line extension assembly (see

Fig. 3) by removing the MPC coupling body part and inserting

the male type connection on the assembly line to the female

connection on the vessel.

7. Attach extension sets onto two luer lock female type connec-

tions on the vessel ports labeled LA2 (see Note 19) and Sample

(see Note 20).

8. Close the Roberts clamp on the vessel and close the hemostat at

the far end of the tubing.

9. Carefully remove the vessel from the Biological Safety Cabinet

(see Note 21).

10. Weld the Media-In line to the LA1 line.

11. Tare the scale, which is connected to the DeltaV control

platform.

12. Place the vessel on the scale.

13. Insert the DO probe to the appropriate port (see Note 22) and

connect it to the junction box (see Note 23).

14. Uncap the pH probe and connect it to the Finesse controller.

15. Insert the temperature probe in the appropriate port and con-

nect to the Finesse controller.

16. Attach the motor to the center of the vessel headplate (see Note

24).

17. Affix the heating jacket around the vessel (see Note 25).

18. Connect the gas line from the Mass Flow Controller labeled

headspace to the gas inlet line.

19. Record the weight of the vessel with added probes and acces-

sories. Tare the scale (see Note 26).

20. On the DeltaV control platform, confirm that the vessel weight

is 0 g. Enter a specific Batch ID and click ‘Batch Start.’

21. Weld the 3 L L7™TFO2 hPSC basal medium with coated

microcarriers to the LA2 port tubing and allow contents to

enter the vessel by gravity.

22. Verify the appropriate process parameters on the DeltaV con-

trol platform: 37 ^C temperature, 100 RPM agitation and 2 L/

min air flow rate.

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors

45