3.4

Cell Harvesting

on Day 4

1. At 96 h, prepare 1.5 L concentrated 3D FloTrix^® Digest solu-

tion and transfer to a sterile feed bottle. Connect this bottle via

luer connector to tube 1.

2. Connect a sterile waste bottle via luer connector to tube 3. Fit

silicon tubing of tube 3 to pump 2 in the desired direction.

Unclamp the Robert clamp and pump out 4 L of spent medium

in the vessel to the waste bottle using pump 2 at 300 mL/min.

Clamp and remove the waste bottle.

3. Feed in the concentrated 3D FloTrix^® Digest solution using

pump 1 at 300 mL/min.

4. Increase agitation speed to 50 rpm during microcarrier disso-

lution process.

5. Connect a sterile harvest bottle via luer connector to tube 2. Fit

the silicon tubing of tube 2 in the desired direction on pump

2. When all microcarriers are fully dissolved (usually within

30–40 min, see Note 12), pump the cell suspension in the

culture vessel to the harvest bottle at 300 mL/min. A total of

2.5 L cell suspension could be harvested.

6. Centrifuge cell suspension at 179
g, 5 min, discard superna-

tant and resuspend in 400 mL PBS.

7. Take a sample and mix with 0.1% Trypan blue solution at 1:1

ratio. Count cells to calculate the total cell number in the

culture vessel and cell viability (Figs. 7 and 8).

8. Repeat step 6 for another two times but resuspend cell pellets

with PBS at 2
10⁶/mL instead. Discard supernatant and

resuspend in complete medium or cells cryopreservation solu-

tion at desired density.

Fig. 8 Cell viability of hUCMSCs on 3D TableTrix^® microcarriers in 3D FloTrix^®

vivaSPIN bioreactor

122

Huanye Xu et al.