23. Allow the vessel to become saturated with air (see Note 27).

24. Weld a syringe-extension set to the LA(S) tubing and draw

5 mL of sample from the vessel.

25. Measure the pH (see Note 28).

26. Adjust the pH value in the DeltaV control platform if the

online and offline values differ by >0.05.

27. Verify the critical control parameters and leave the system

overnight (see Note 29). Introduction of cell inoculum is

performed the following day.

28. Prior to introducing the cell inoculum, transfer 30 mL of L7™

hPSC medium supplement into a 50 mL syringe and attach an

extension set.

29. Weld the syringe-extension set to the LA(S) port and add the

L7™hPSC medium supplement into the vessel.

30. After 1 h, weld a syringe-extension set to the LA(S) tubing and

draw 5 mL of sample from the vessel.

31. Measure the pH (see Note 28).

32. Adjust the pH value in the DeltaV control platform if the

online and offline values differ by >0.05.

3.6

Cell Inoculum

Introduction

1. Transfer the cell inoculum harvested from the 2D seed train

into an appropriately sized syringe (see Note 30).

2. Connect the syringe to a luer lock extension tubing.

3. Weld the syringe-extension set to tubing corresponding to the

LA(S) port on the vessel. Gently introduce the cell inoculum by

pushing the syringe piston (see Note 31).

4. To the same LA(S) port line, add 3 mL of 10 mM ROCK

inhibitor.

5. Mix the cell inoculum for 30 min to 1 h and draw a 5 mL

sample from the vessel to measure the pH offline (see Note 28).

6. Adjust the pH value in the DeltaV control platform if the

online and offline values differ by >0.05.

7. Set up the appropriate program in the DeltaV control platform

and verify the critical control parameters (see Note 32). This

begins the cell growth and expansion process in a 3 L bioreac-

tor vessel.

3.7

Cell Culture

Medium Perfusion

Setup

1. Weld the C-flex tubing of the media feed line (see Fig. 2) to the

LA (1) tubing.

2. Weld the C-flex tubing of the 7 L bag of complete L7™hPSC

(see Note 33) medium to the far end of the media feed line.

46

Marites T. Woon et al.