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Process control and in silico modeling strategies for enabling high density culture of human pluripotent stem cells in stirred tank bioreactors.

ABSTRACT: The routine therapeutic and industrial applications of human pluripotent stem cells (hPSCs) require their constant mass supply by robust, efficient, and economically viable bioprocesses. Our protocol describes the fully controlled expansion of hPSCs in stirred tank bioreactors (STBRs) enabling cell densities of 35 × 10⁶ cells/mL while reducing culture medium consumption by 75%. This is achieved by in silico process modeling and computable upscaling. We provide a detailed blueprint for systematic process development of hPSCs and their progenies. For complete details on the use and execution of this protocol, please refer to Manstein et al. (2021).

SUBMITTER: Manstein F

PROVIDER: S-EPMC8666714 | biostudies-literature | 2021 Dec

REPOSITORIES: biostudies-literature

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Process control and in silico modeling strategies for enabling high density culture of human pluripotent stem cells in stirred tank bioreactors.

Manstein Felix F Ullmann Kevin K Triebert Wiebke W Zweigerdt Robert R

STAR protocols 20211209 4

The routine therapeutic and industrial applications of human pluripotent stem cells (hPSCs) require their constant mass supply by robust, efficient, and economically viable bioprocesses. Our protocol describes the fully controlled expansion of hPSCs in stirred tank bioreactors (STBRs) enabling cell densities of 35 × 106 cells/mL while reducing culture medium consumption by 75%. This is achieved by in silico process modeling and computable upscaling. We provide a detailed bluepr ...[more]

PMID: 34917976

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Project description:: The routine application of human pluripotent stem cells (hPSCs) and their derivatives in biomedicine and drug discovery will require the constant supply of high-quality cells by defined processes. Culturing hPSCs as cell-only aggregates in (three-dimensional [3D]) suspension has the potential to overcome numerous limitations of conventional surface-adherent (two-dimensional [2D]) cultivation. Utilizing single-use instrumented stirred-tank bioreactors, we showed that perfusion resulted in a more homogeneous culture environment and enabled superior cell densities of 2.85 × 106 cells per milliliter and 47% higher cell yields compared with conventional repeated batch cultures. Flow cytometry, quantitative reverse-transcriptase polymerase chain reaction, and global gene expression analysis revealed a high similarity across 3D suspension and 2D precultures, underscoring that matrix-free hPSC culture efficiently supports maintenance of pluripotency. Interestingly, physiological data and gene expression assessment indicated distinct changes of the cells' energy metabolism, suggesting a culture-induced switch from glycolysis to oxidative phosphorylation in the absence of hPSC differentiation. Our data highlight the plasticity of hPSCs' energy metabolism and provide clear physiological and molecular targets for process monitoring and further development. This study paves the way toward more efficient GMP-compliant cell production and underscores the enormous process development potential of hPSCs in suspension culture.SignificanceHuman pluripotent stem cells (hPSCs) are a unique source for the, in principle, unlimited production of functional human cell types in vitro, which are of high value for therapeutic and industrial applications. This study applied single-use, clinically compliant bioreactor technology to develop advanced, matrix-free, and more efficient culture conditions for the mass production of hPSCs in scalable suspension culture. Using extensive analytical tools to compare established conditions with this novel culture strategy, unexpected physiological features of hPSCs were discovered. These data allow a more rational process development, providing significant progress in the field of translational stem cell research and medicine.

Project description:BackgroundThe fungal natural products illudin S and M have been investigated as precursors for the development of semisynthetic anticancer agents such as Irofulven (illudin S derivative) which is currently in phase II clinical trials. Recently, illudin M derivatives have shown improved in vitro selectivity towards cancer cells encouraging further investigation. This requires a stable supply of the precursor which is produced by Basidiomycota of the genus Omphalotus. We have recently reported a robust shake flask process for the production of gram quantities of illudin M from Omphalotus nidiformis aiming to transfer that process into stirred tank bioreactors, which can be used in a commercial production set-up. However, process transfer across different systems is not straightforward and particularly challenging when the producer is morphologically complex. There are only a few reports that address the development of bioprocesses for the production of compounds from Basidiomycota as these organisms have not been extensively studied because of their complex life cycles and often are difficult to cultivate under laboratory conditions.ResultsThe recently developed shake flask process delivering stable titers of ~ 940 mg L-1 of illudin M was investigated using off-gas analysis to identify critical parameters which facilitated the transfer from shaken into stirred tank bioreactors. Comparable titers to the shake flask process were achieved in 2 L stirred tank bioreactors (1.5 L working volume) by controlling growth of biomass with a carefully timed pH-shift combined with an improved precursor-feeding strategy. A scale-up experiment in a 15 L bioreactor (10 L working volume), resembling the process at 1.5 L resulted in 523 mg L-1 and is the starting point for optimization of the identified parameters at that scale.ConclusionBy identifying and controlling key process parameters, the production process for illudin M was transferred from shake flasks into 2 L stirred tank bioreactors reaching a comparable titer (> 900 mg L-1), which is significantly higher than any previously reported. The insights obtained from 10 L scale pave the way towards further scale-up studies that will enable a sustainable supply of illudin M to support preclinical and clinical development programs.

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Process control and in silico modeling strategies for enabling high density culture of human pluripotent stem cells in stirred tank bioreactors.

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Process control and <i>in silico</i> modeling strategies for enabling high density culture of human pluripotent stem cells in stirred tank bioreactors.

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