3

Methods

Perform the procedures using aseptic technique inside a Biological

Safety Cabinet unless otherwise noted and limit the exposure of the

L7™hPSC culture system to light. To maintain a sterile environ-

ment within the bioreactor vessel and cell culture media bags,

unclamp the tubings only when introducing reagents, media or

cell inoculum or drawing samples. Immediately seal the line and

re-clamp the tubing when finished.

3.1

Extracellular

Matrix Coating of T-75

and 1-Layer Cell Stack

1. Re-suspend 300 μL of the L7™hPSC matrix stock solution in

12 mL of DPBS^+/+ and add to the T-75 flask. To coat a 1-layer

cell stack, resuspend 2.55 mL of the L7™hPSC matrix stock

solution in 125 mL of DPBS^+/+.

2. Allow the coating solution to incubate in a 37 ^C incubator for

at least 1 h (see Note 9).

3.2

hPSC Culture on

T-75 Flask

1. Thaw cryopreserved hPSC cell line of choice in a 37

C

water bath.

2. Transfer the cell solution into a 50 mL conical tube and add

10 mL of L7™TFO2 complete medium dropwise.

3. Centrifuge at 200
g for 5 min.

4. Aspirate the supernatant and re-suspend the cell pellet in

15 mL of L7™TFO2 complete medium supplemented with

10 μM ROCK inhibitor.

5. Before adding the hPSCs to the T-75 flask, aspirate the extra-

cellular matrix coating solution.

6. Replace the medium by aspirating the old medium and adding

new 15 mL of L7™TFO2 complete medium (see Note 10).

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

cells reach 70–80% confluence (see Note 11).

3.3

hPSC Culture on

2D Seed Train

1. Aspirate the old medium from the T-75 flask and wash cells

with 15 mL of DPBS^/.

2. Incubate with 15 mL of pre-warmed L7™hPSC passaging

solution in 37 ^C for 10 min (see Note 12).

3. Aspirate the L7™hPSC passaging solution and add 15 mL of

L7™TFO2 complete medium to harvest the cells into a 50 mL

conical tube.

4. Centrifuge at 200
g for 5 min.

5. Remove the supernatant and re-suspend the cells in 10 mL of

L7™TFO2 complete medium.

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

volume required to seed 0.02–0.03
10⁶ cells/cm².

Expansion of Human Pluripotent Stem Cells in Stirred Tank Bioreactors

43