Maximizing mouse embryonic stem cell production in a stirred tank reactor by controlling dissolved oxygen concentration and continuous perfusion operation

摘要

One of the key challenges in stem cell bioprocessing is the large-scale cultivation of stem cells in order to meet the demanding meaningful cell numbers needed for biomedical applications, especially for clinical settings. Mouse embryonic stem cells [1], used as a model system herein, were cultivated on micro-carriers in a fully controlled stirred tank reactor (STR) [2]. The impact of varying the concentration of dissolved oxygen (at 5%, 10%, 20% and 30% DO) and operating under a continuous perfusion mode on cell growth and pluripotency maintenance was investigated. In addition, in order to further optimize the feeding strategy of the STR operating under continuous perfusion toward maximal cell production, the influence of different medium residences times (12h, 24h, 32h, 48h and 96h) was evaluated. Overall, the maximal cell concentration of 7.9-9.2 × 10~6 cells/mL were attained after 11 days, with no passaging required, under a DO of 10-20% in the continuous perfused bioreactor with cell retention and medium residences times of 32-48 h. Importantly, mESC expanded under these conditions/retained the expression of pluripotency markers (Oct4, Nanog and Ssea-1), as well as their differentiation potential into cells of the three embryonic germ layers. The STR-based cultivation platform optimized herein represents a major contribution toward the development of large-volume production systems of differentiated cell derivatives for a wide range of biomedical applications.