12. Connect a sterile waste bottle to tube 2 with luer connector (see

Note 10), unclamp tube 2, and pump out all PBS in the vessel

into the waste bottle at 300 mL/min with pump 2. Clamp and

disconnect the waste bottle from luer connector (see Note 10).

13. Fill a sterile feed bottle with 2 L complete medium and connect

to tube 1 with luer connector, unclamp tube 1 and feed in at

300 mL/min with pump 1. Clamp and disconnect the feed

bottle from luer connector.

14. Remove tube 2 from pump 2 and fit tube 4 on pump

2. Unclamp tube 4 and pump at 100 mL/min to aliquot

10 mL of complete medium into the sampling bottle. Clamp

tube 4 and transfer sample by connecting a syringe to the luer

connector and aspirating the sample from the sampling bottle.

15. Transfer this sample of complete medium to a clean 50-mL

centrifuge tube and incubate in a 37^C orbital shaker for 24 h

to check for the sterility of the bioreactor (see Note 11).

16. Saturate the complete medium in the vessel with air for 24 h.

17. After 24 h, if no bacteria growth is observed in the vessel and

sterility check sample, proceed with inoculation.

3.2

Inoculation on

Day 0

1. On Day 0, calibrate the amount of saturated air as 100% for the

DO sensor.

2. After calibration, set the DO setting value to 70%, pH to 7.2,

temperature to 37^C and agitation to 35 rpm/min. The

Fig. 4 Fully assembled 3D FloTrix^® vivaSPIN bioreactor

118

Huanye Xu et al.