7. Aspirate the extracellular matrix solution from the 1-layer cell

stack before adding the hPSCs.

8. Culture the cells in an incubator at 37 ^C and 5% CO2 until the

cells reach 70–80% confluence (see Note 11) by aspirating the

old culture medium and replenishing with 150 mL of new

culture medium (see Note 10).

9. To harvest the cell inoculum, aspirate the old medium from the

1-layer cell stack.

10. Wash the cells with 75 mL DPBS^/ (see Note 14).

11. Aspirate and add 75 mL of pre-warmed L7™hPSC passaging

solution. Incubate at 37 ^C for approximately 15 min (see Note

12).

12. Tap the 1-layer cell stack to detach the cells.

13. Transfer the cells into a 250 mL conical tube and add 75 mL

L7™TFO2 complete medium into the tube.

14. Centrifuge the cell suspension at 200
g for 5 min, aspirate

the supernatant and re-suspend the cell pellet with 30 mL of

L7™TFO2 complete medium.

15. Count the number of cells (see Note 13) to determine the

volume

required

to

inoculate

a

3

L

bioreactor

with

120
10⁶ cells harvested with L7™hPSC passaging solution

(see Note 15).

3.4

Microcarrier

Coating

1. In a 1 L bottle, add 20 g of microcarriers, 300 mL DPBS^+/+

and 18 mL of L7™hPSC matrix.

2. Incubate at 37 ^C for 2–4 h with intermittent shaking to mix.

3. Allow the coated microcarriers to settle at the bottom of the

bottle before aspirating the DPBS^+/+.

4. Add 300 mL of L7™TFO2 hPSC basal medium without the

L7™hPSC medium supplement (incomplete) and wrap the

bottle with aluminum foil. Incubate overnight while shaking at

room temperature.

5. Add 700 mL of incomplete L7™TFO2 hPSC medium and

store in 4 ^C until use (see Note 16).

6. When ready to introduce to the bioreactor vessel, replace the

bottle closure. Use a bottle closure pre-mounted with tubing.

7. Weld the pre-mounted tubing of the bottle closure to a 2 L bag

of L7™TFO2 hPSC basal medium and introduce the coated

microcarriers to the medium (see Note 17).

3.5

Bioreactor Setup

1. Carefully unwrap the bioreactor in the Biological Safety Cabi-

net and inspect the vessel to ensure it is not damaged prior

to use.

44

Marites T. Woon et al.