3. Place the Pharmed tubing portion of the media feed line (see

Fig. 2) into the pump controller (pump #3) located on the G3

model tower.

4. Connect a 20 L empty bag with a 1/8⁰⁰
1/4⁰⁰ C-flex tubing

by welding it to the C-flex tubing of the perfusion out line.

This is the waste bag.

5. Place the waste bag on a scale located on the floor and tare the

scale.

6. Place the Pharmed tubing portion of the perfusion out line (see

Fig. 1) into the pump controller (pump #4) located on the G3

model tower.

7. Unclamp the tubings and prime the waste line by pressing the

button next to pump #4. Media will travel from the vessel to

the waste bag. To prime the feed line, press the button next to

pump #3 until media can be observed entering the vessel (see

Note 34).

8. Initiate perfusion by setting the value of pump #3 to cascade on

‘vessel weight output’ and the value of pump #4 to 2.08 g/min

on the DeltaV control platform.

3.8

Cell Count during

Expansion

1. Aliquot 15 mL of F3 hPSC passaging solution into two 50 mL

conical tubes and allow to warm up in the 37 ^C incubator for

10 min.

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

of cell solution. Take two 15 mL samples.

3. Deposit the cell solution into 50 mL conical tubes and allow

the microcarriers to settle (see Note 35).

4. Aspirate the media carefully and resuspend the microcarriers in

7.5 mL F3 hPSC passaging solution.

5. Invert the conical tube twice to mix and incubate in the 37 ^C

incubator for 15–20 min (see Note 36).

6. Gently pipette the cell solution.

7. Prepare a single cell suspension by straining the microcarrier-

cell solution through a 70 μM cell strainer into another 50 mL

conical tube.

8. Add 7.5 mL of pre-warmed incomplete or complete L7™

TFO2 hPSC medium.

9. Centrifuge the cell only solution at 200
g for 5 min.

10. Re-suspend the cell pellet in 1 mL or 15 mL (see Note 37)

L7™TFO2 hPSC medium.

11. Perform cell count (see Note 13) throughout the expansion

process (see Fig. 4).

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors

47