securely attached. A sterile environment inside the vessel must

be maintained.

22. Tightly press the DO probe against the membrane at the

bottom of the vessel.

23. Connecting the DO probe to the junction box (J-box) allows

the careful monitoring of DO levels during the expansion

process.

24. The motor may impinge on the DO probe. Therefore, minor

adjustments may be required.

25. Ensure that the electric wire connected to the heating jacket is

connected to the Finesse controller. The heating jacket must

not touch the scale to accurately report the vessel weight.

26. Following this point, the weight recorded on the scale corre-

sponds to the liquid contents inside the vessel.

27. Saturation is achieved after 4–7 h when the DeltaV control

platform shows that the DO levels have plateaued.

28. Offline pH measurement can be done using the BioProfile

FLEX Analyzer (Nova Biomedical) according to manufac-

turer’s instructions.

29. Input the set points for temperature, pH, DO, gas, and agita-

tion speed in the DeltaV control platform.

30. In lieu of obtaining cell inoculum from a 2D seed train, cryo-

preserved cells or a 3D seed train can also be used [9].

31. Pushing air may be required to ensure the entire cell suspen-

sion is introduced into the vessel.

32. The values entered in the DeltaV control platform are depen-

dent on the experiment’s parameters.

33. The 7 L bag of complete L7™TFO2 hPSC medium was

prepared by adding 70 mL of L7™hPSC medium supplement

to the L7™TFO2 hPSC basal medium. This will be used

during the expansion process. Elevate the bag on an intrave-

nous pole, which creates a pressure gradient allowing the

medium to enter the vessel. Cover the bag to protect the

medium from light.

34. Carefully inspect for any leaks in the tubings.

35. 1 mL of the supernatant can be used to check the number of

cells not attached on the microcarriers. This value should be

negligible as it is expected that the cells adhered and grew on

the coated microcarriers.

36. Periodically invert the tube to mix. This ensures the homoge-

neous dissociation of cells from the microcarriers. Larger

aggregates tend to take longer than 20 min to dissociate from

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors

51