3.9

Cell Harvest

1. Pre-warm 1.5 L bag of F3 hPSC passaging solution to 37 ^C.

2. Pre-warm 1.5 L bag of L7™TFO2 hPSC basal medium to

37 ^C.

3. Record the weight of the bioreactor vessel.

4. Using two different syringes, transfer 30 mL of L7™hPSC

medium supplement and 3 mL of 10 mM ROCK inhibitor

solution into the 5 L media bag containing 1.5 L of L7™

TFO2 hPSC basal medium.

5. Weld the C-flex tubing on the 1.5 L bag of L7™TFO2 hPSC

basal medium to the C-flex tubing of the mesh filter bag (see

Note 38).

6. Weld the other end of the mesh filter bag with the C-flex tubing

of the harvest line, which is attached to the Harvest port of the

bioreactor vessel.

7. Attach an empty and sterile 5 L plastic bag to the Perfusion line

via the C-flex tubing ends. This bag will contain the cell culture

supernatant derived from the vessel.

8. When the appropriate tubings are welded to the vessel, remove

the cell culture medium from the bioreactor vessel through the

Perfusion line into the waste bag (see Note 39) while agitating.

9. If cell-microcarrier aggregates on the mesh filter are observed,

then gently tap the vessel or rotate the perfusion dipstick.

10. After 1.5 L of volume has been removed from the vessel,

remove the heating jacket and lower the agitation speed by

10 rpm (see Note 40).

11. Halt the agitation when ~10% of the original vessel volume

remains.

0.00E+00

5.00E+05

1.00E+06

1.50E+06

2.00E+06

2.50E+06

0

3

6

9

12

L

m

/slle

c

e

vi

L

Days in culture

Cell count from samples

Cell count from full harvest

A

B

Fig. 4 Expansion of hPSC in a 3 L bioreactor. (a) Representative cell density plot over time. (b) Representative

image of cell-microcarrier clusters on day 11 of the expansion process as indicated by the yellow arrows; 10

magnification

48

Marites T. Woon et al.