5. Seal all silicon tubes with hemostatic forceps. Remove the short

silicon tubing from tube 5b and 5c. Connect an air compressor

to tube 5b, and a pressure gauge to tube 5c. Pump in filtered

compressed air until the pressure gage reaches between 0.2 and

0.5 bar. Clamp tube 5b with hemostatic forceps and maintain

air pressure for 15 min. If the vessel is not airtight, the pressure

will drop. Check for air leakage, and tighten any loose bolts. If

vessel is airtight, decompress to room pressure by removing the

air pressure gauge from tube 5c. Disconnect air compressor

from 5b. Connect 5b and 5c together with one short silicon

tubing to form a closed loop.

6. Always stay by the equipment and watch the pressure gauge

closely when performing pressure check. Be careful when intro-

ducing compressed air and do not let the air pressure in the

vessel raise above 1 bar, in case the vessel crack and explode.

7. Robert clamps may open during autoclave due to high temper-

ature. Clamp silicon tubes using hemostatic forceps in conjunc-

tion to Robert clamps as an additional measure.

8. After autoclave, check all bolts on the top plate to make sure

they are tightly secured and check the integrity and dryness of

all air venting filters. If any filter is wet or not intact, change and

autoclave again.

9. All pumps rotate clockwise. Make sure the tubing directing is

fitted for liquid flow in the intended direction.

10. Spray luer connectors with 75% ethanol before connection and

after breaking connection.

11. The objective of this sterility check is to check if the bioreactor

is fully aseptic. Take a sample of the prepared complete DMEM

before feeding into the bioreactor as a control.

12. All microcarriers should be fully dissolved; extend the dissolu-

tion process to not more than 60 min if there is still micro-

carrier particles visible at 30 min.

13. Cells should be counted within 3 min. Non-viable cells are

stained blue and cell viability is counted as percentage of viable

cells in the total cell count, cell viability% ¼ living cells/total

cells
100%.

Acknowledgments

This work is financially supported by Beijing Municipal Science &

Technology Commission (Z181100001818005) and R&D pro-

gram (RC21-01) of Beijing CytoNiche Biotechnology Co. Ltd.

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Huanye Xu et al.