March 15, 2013, Hyderabad, India – Disposable bioreactors are increasingly used for Cell Cultivation because they improve process flexibility, Shorten turnover times and eliminate the requirement for cleaning and sterilization Numerous cell lines such as ‘CHO, HEK 293, NSO, S2 and SF9’ have been cultivated successfully in wave Bioreactors systems, demonstrating the flexibility of this device. In a Wave Bioreactor system (Fig. 1) the cells are grown in a pre-sterilized, inflated cultivation chamber, a Cell-bag bioreactor. The Culture is agitated by rocking the bag attached to the rocker tray. Heating occurs via a heater pad located below the tray and gas supply is provided via the headspace that can be filled with a mixture of ambient air, CO2, an N2. Additional O2 can be blended in to support high cell concentration. The reactors equipped with DOOPT II and pHOPT patch sensors that are integrated in the bottom film of the Cell bag bioreactor and optically measure the level of dissolved oxygen and the pH in the Cell medium (Fig. 2). Attaching the optical fiber cables to the sensors is easy and the resulting data is provided fast and with high resolution. The parameters are controlled by the control units integrated in the WAVE Bioreactor 200 system.
Wave Bioreactor 200 system was used with a disposable Cellbag 200L with embedded DOOPT II and pHOPT sensors to successfully cultivate Chinese Ovary (Cho) cells.
Materials and methods Cell line cultivation medium:
CHO DG44 cells (Celica) were used in this study. The Cellbag bioreactor was inoculated at density of 0.176 MCV/mL. The cells were pre-cultured in ActiCHO medium and grown in ActiCHO PCD medium. Nutrient concentrates ActiCHO Feed-A CD and ActiCHo Feed-B CD were used for feed A and feed B respectively.
Bioreactor cultivations :
The cultivation was performed in fed batch mode using WAVE Bioreactor 200 system. The experiment was based on a modified fed-batch protocol. Cellbag 200L with DOOPT II and pHOPT patch sensors was used with a working volume after inoculation of 62 liters of ACtiCHO P CD medium. Set points were 7 for pH and 60% air saturation for dissolved oxygen (DO). THE CO2 level was set to 7% until the culture reached a high density and no longer required this level of CO2. The control was then set to maintain a pH of 7.2. During the experiment O2 level, air flow, and agitation angle and speed were changed to provoke changes in DO level, to evaluate how well the DOOPT sensor system could handle disturbances during rum(Fig 3). 2.4 L Feed A and 0.24 L feed B were added daily from daily day 8 and extra glucose when needed. Cultures were monitored by daily analysis of samples with a Mob Select Surety lag assay. The culture was prematurely terminated at over 95% viability.
Results and discussion :
CHO cells were grown in fed-batch culture, following a modified culture protocol, resulting in an LgG end-concentrations of 3.96 g/L Figure 4 shows that the cell viability high throughout the experiment (Over 95%) and remained high at culture termination, indicating that the concentration could have been increased had the culture been allowed to continue. Extrapolation of the linearized titer data suggests an end concentration of 5 g/L after two more days of growth.
Figure 3 shows that the DO regulation quickly adjusts the DO to the set level(60%) after disturbances resulting from the intentional provocations.
Conclusion:
This study shows that WAVE Bioreactor 200 system can successfully be used with a disposable Cellbag 200L with embedded DOOPT II and pHOPT patch sensors to cultivate cells. The patch sensors were used to control the Do and pH levels during the culture, offering a simple and easy-to-use solution.
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