Successful Cell–Cultivation in Single Use Bioreactors

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DÜPERTHAL Sicherheitstechnik GmbH & Co. KG

Glatt Ingenieurtechnik GmbH

Angst+Pfister AG

Sartorius AG

Staying Alive: Sf9 Culture in the Bioreactor

11.87 + 10^6 cells/ml at ninth day of cultivation (average of three consecutive trials). Cell viability decreased from ninth day of cultivation and dropped to 50 per cent till day 12. Glucose was consumed from 10.2gm/l to 4gm/l during cultivation and lactate accumulation was 3mg/ml at the end of cultivation. Temperature was maintained at 28°C constantly and pH around 6.2.

Further the process was scaled up and cultivation was done in 5L media in 10L Cultibag RM. The peak cell density was 12.9 + 10^6 cells/ml similar to 2L Cultibag RM.

How to Avoid Media Evaporation

DO was maintained at 60 percent through out cultivation period. The variation in DO was +/- 5 per cent. It was maintained by using nitrogen, gas flow and oxygen in cascade mode. Oxygen was required to maintain DO after the cell reaches the exponential log phase. The evaporation of media can also be curtailed by intermittent supply of oxygen instead of continuous supply of air. The pH was maintained at 6.2 by using 0.1 N alkalis(NaHCo3) and CO2 in cascade mode. CO2 is not necessary at the start of the cultivation but can be used in toward the end of cultivation when pH tends to rise as result of toxic accumulation.

Shaken, Not Stirred: Selcting the Right Rocking Speed

The rocking speed is dependent on both the culture volume and cell density. At low volumes i.e. 20-30 per cent of the Cultibag working volume, an initial rocks of 15 is sufficient for mixing. It would benecessary to increase the number of rocks to 18-25 at 100 per cent of Cultibag working volume or at high cell density. It is advisable to monitor the oxygen demand and adjust the rocks and rocking angle as needed. Rocking speed of 18 to 23 was suitable for cultivation of Sf9 in 2L and 10L Cultibag RM systems.

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