controller will automatically regulate the air and CO2 input as

well as the heat mat to maintain the system at the set points.

3. Connect a sterile waste bottle to tube 2 with luer connector,

unclamp the Robert clamp and pump out all medium in the

vessel into the waste bottle at 300 mL/min with pump

2. Clamp and disconnect the waste bottle from luer connector.

4. Disperse 500 tablets 3D TableTrix^® microcarrier tablets (F01,

equivalent to 10 g) with 500 mL hUCMSC complete medium

(20 mg/mL) in a sterile feed bottle.

5. Thaw 10 tubes of 2.5
10⁷/tubes cryopreserved hUCMSCs

seed cells in a 37 ^C water bath, dilute to 50 mL by slowly

adding warm complete hUCMSC culture medium. Centrifuge

at 179
g, 5 min. Discard supernatant and resuspend with

50 mL warm complete medium. Transfer to the feed bottle.

6. Top up with 2.45 L warm complete medium to a final volume

of 3 L.

7. Connect the feed bottle via luer connector to tube 1. Unclamp

the Robert clamp and transfer this microcarrier and cell sus-

pension into the culture vessel via tube 1 on pump 1 at

300 mL/min. Clamp tube 1 and remove the feed bottle.

3.3

Cell Culture,

Medium

Replenishment, and

Growth Monitoring on

Days 1–3

1. At 24 h, fill 2 L of warm complete medium in a new sterile feed

bottle, connect the bottle to tube 1 via luer connector.

Unclamp and feed into the culture vessel by setting pump

1–300 mL/min. The total culture volume is now 5 L. Clamp

tube 1 and remove the feed bottle.

2. At 72 h, connect a sterile waste bottle via luer connector on

tube 3. Fit tube 3 to pump 2 in the desired direction. Unclamp

the Robert clamp and pump out 3 L of spent medium in the

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

tube 3 and remove the waste bottle.

3. Fill a sterile feed bottle with 3 L of warm fresh complete

medium and connect to tube 1 via luer connector. Unclamp

and feed in the fresh medium using pump 1 at 300 mL/min.

Clamp tube 1 and remove the feed bottle.

4. Increase agitation speed to 40 rpm.

5. At 24 h, 48 h, 72 h, aliquot 10 mL of cell-microcarrier suspension

into the sampling bottle according to step 14, Subheading 3.1.

6. Transfer sample to a clean 15-mL centrifuge tube for cell

enumeration by connecting a syringe to the luer connector

and aspirating the sample from the sampling bottle. Discard

the syringe after each sampling.

7. Aspirate 50 μL of cell-microcarrier suspension to a 96-well

plate for cell staining. Leave the rest for cell enumeration.

Large-Scale Expansion of UCMSCs with Microcarrier Tablets

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