TY - JOUR

T1 - High density bioprocessing of human pluripotent stem cells by metabolic control and in silico modeling

AU - Manstein, Felix

AU - Ullmann, Kevin

AU - Kropp, Christina

AU - Halloin, Caroline

AU - Triebert, Wiebke

AU - Franke, Annika

AU - Farr, Clara Milena

AU - Sahabian, Anais

AU - Haase, Alexandra

AU - Breitkreuz, Yannik

AU - Peitz, Michael

AU - Brüstle, Oliver

AU - Kalies, Stefan

AU - Martin, Ulrich

AU - Olmer, Ruth

AU - Zweigerdt, Robert

N1 - Funding Information: We would like to thank U. Rinas and T. Scheper from the Institute for Technical Chemistry, Leibniz University, Germany for providing FGF-2; A. Kirschning and G. Dr?ger from the Institute of Organic Chemistry, Leibniz University, Germany for providing Y-27632; T. Schlaeger from Boston Children's Hospital, Harvard Medical School, MA, United States for the idea of measuring aggregate density in a practical way; C. Thiele from the Institute of Reconstructive Neurobiology, University of Bonn Medical Faculty & University Hospital Bonn, Germany for the excellent technical support in neuroectodermal differentiations; G. G?hring and coworkers from the Institute for Human Genetics, Hannover Medical School, Germany for karyotyping; M. Wei? from the Institute for Technical Chemistry, Leibniz University, Germany for amino acid analysis. R.Z. received funding from: the German Research Foundation (DFG; Cluster of Excellence REBIRTH EXC 62/2, ZW64/4-1), the German Ministry for Education and Science (BMBF, grants: 13N14086, 01EK1601A, 01EK1602A), and the European Union H2020 program to the project TECHNOBEAT (grant 668724).

PY - 2021/6/26

Y1 - 2021/6/26

N2 - To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

AB - To harness the full potential of human pluripotent stem cells (hPSCs) we combined instrumented stirred tank bioreactor (STBR) technology with the power of in silico process modeling to overcome substantial, hPSC-specific hurdles toward their mass production. Perfused suspension culture (3D) of matrix-free hPSC aggregates in STBRs was applied to identify and control process-limiting parameters including pH, dissolved oxygen, glucose and lactate levels, and the obviation of osmolality peaks provoked by high density culture. Media supplements promoted single cell-based process inoculation and hydrodynamic aggregate size control. Wet lab-derived process characteristics enabled predictive in silico modeling as a new rational for hPSC cultivation. Consequently, hPSC line-independent maintenance of exponential cell proliferation was achieved. The strategy yielded 70-fold cell expansion in 7 days achieving an unmatched density of 35 × 106 cells/mL equivalent to 5.25 billion hPSC in 150 mL scale while pluripotency, differentiation potential, and karyotype stability was maintained. In parallel, media requirements were reduced by 75% demonstrating the outstanding increase in efficiency. Minimal input to our in silico model accurately predicts all main process parameters; combined with calculation-controlled hPSC aggregation kinetics, linear process upscaling is also enabled and demonstrated for up to 500 mL scale in an independent bioreactor system. Thus, by merging applied stem cell research with recent knowhow from industrial cell fermentation, a new level of hPSC bioprocessing is revealed fueling their automated production for industrial and therapeutic applications.

KW - high density culture

KW - human pluripotent stem cells

KW - in silico process modeling

KW - process scale-up

KW - stirred tank bioreactor

KW - suspension culture

UR - http://www.scopus.com/inward/record.url?scp=85101943087&partnerID=8YFLogxK

U2 - 10.1002/sctm.20-0453

DO - 10.1002/sctm.20-0453

M3 - Article

AN - SCOPUS:85101943087

VL - 10

SP - 1063

EP - 1080

JO - Stem cells translational medicine

JF - Stem cells translational medicine

SN - 2157-6564

IS - 7

ER -