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

• Published in: Biochemical engineering journal 2014-01, Vol.82, p.81-90
• Main Authors: Fernandes-Platzgummer, Ana, Diogo, Maria M., Lobato da Silva, Cláudia, Cabral, Joaquim M.S.
• Format: Article
• Language: English
• Subjects: Animal cell culture; Biological and medical sciences; Bioreactors; Biotechnology; Dissolved oxygen; Expansion; Fundamental and applied biological sciences. Psychology; Methods. Procedures. Technologies; Microcarriers; Mouse embryonic stem cells; Various methods and equipments
• ISSN: 1369-703X; 1873-295X
• DOI: 10.1016/j.bej.2013.11.014

•mESC were cultivated on microcarriers in a fully controlled stirred tank reactor.•The influence of dissolved oxygen (DO) on cell growth and pluripotency was investigated.•The influence of different residence times (12, 24, 32, 48 and 96h) was evaluated.•Continuous perfusion with cell retention under 20% DO maximized cell production/numbers.•Cells retained pluripotency and differentiation potential throughout the process. 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 microcarriers 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 7.9–9.2×106cells/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 32–48h. 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.