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45,80 €ISBN 978-3-8440-6858-0Softcover134 pages32 figures185 g21 x 14,8 cmEnglishThesis
August 2019
Jasmin Leber
Investigation on the scale-up strategy and harvest for human mesenchymal stromal cell production in a dynamic bioreactor system
Influences of medium, microcarrier choice and new harvest enzymes on the cell quality
Many clinical trials in the field of regenerative medicine using human mesenchymal stromal/stem cells (hMSC) underline the therapeutic potential of these cells. To enable clinical applications with hMSC, an optimal large-scale expansion process is required. Advantageous for these adherent growing cells is the use of suspended microcarriers in a dynamic system (stirred tank reactor). Influencing parameters which are considered in this work are the choice of microcarriers for cell expansion, a defined growth medium, the scale-up procedure, the harvest and the quality analysis.
The scale-up of the dynamic expansion of the cells including the harvest were carried out in up to 3 L stirred tank bioreactors. The seed train from small spinner flask to bioreactor scale was carried out via bead-to-bead transfer without the enzymatic detachment of the cells. A chemically defined medium was used for the expansion in static and dynamic culture conditions.
For the detachment of the cells, new proteases from non-mammalian origin were investigated. The cells expanded in static and dynamic conditions showed cell surface marker profiles and differentiability into adipocytes, osteoblasts and chondrocytes as stated by the International Society for Cellular Therapy (ISCT). The expansion in the chemically defined medium did not impair the tested cell quality attributes or indicate cell stress on gene level. A theoretical process analysis of parameters like mixing and oxygen transfer in the stirred tank reactor were used as basis for scale-up design. The microcarrier-based stirred tank reactor offers a good opportunity to generate large amounts of high-quality hMSC under monitored process conditions without the need for uncontrolled pre-cultures in tissue flaks.
The scale-up of the dynamic expansion of the cells including the harvest were carried out in up to 3 L stirred tank bioreactors. The seed train from small spinner flask to bioreactor scale was carried out via bead-to-bead transfer without the enzymatic detachment of the cells. A chemically defined medium was used for the expansion in static and dynamic culture conditions.
For the detachment of the cells, new proteases from non-mammalian origin were investigated. The cells expanded in static and dynamic conditions showed cell surface marker profiles and differentiability into adipocytes, osteoblasts and chondrocytes as stated by the International Society for Cellular Therapy (ISCT). The expansion in the chemically defined medium did not impair the tested cell quality attributes or indicate cell stress on gene level. A theoretical process analysis of parameters like mixing and oxygen transfer in the stirred tank reactor were used as basis for scale-up design. The microcarrier-based stirred tank reactor offers a good opportunity to generate large amounts of high-quality hMSC under monitored process conditions without the need for uncontrolled pre-cultures in tissue flaks.
Keywords: Advanced Therapy Medicinal Products; bead-to-bead transfer; cell harvest; chemically defined medium; hMSC-TERT; mesenchymal stem/stromal cells; microcarriers; proteases; scale-up; stirred tank bioreactor
Schriftenreihe des Institutes für Bioverfahrenstechnik und Pharmazeutische Technologie
Edited by Prof. Dr.-Ing Peter Czermak, Gießen
Volume 13
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