High Density Bioprocessing of Human Pluripotent Stem Cells by Metabolic Control and in Silico Modeling

Abstract

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 × 10 6 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.

The combination of dextran sulphate and polyvinyl alcohol prevents excess aggregation and promotes proliferation of pluripotent stem cells in suspension culture

“…In addition, hiPSCs treated with combination of PVA and DS had a cell density of 1.6 ± 0.33 × 10 6 cells/mL (8‐fold increase) on the 7th days post inoculation, representing a 40.35% increase in the number of cells vs the control culture (Figure 4I, J). Our strategy may be superior to the recent work by Manstein et al They attempted to expand hPSCs in a stirring‐controlled bioreactor by addition of Pluronic F68 as shear protectant 27 . Despite stirring‐controlled reduction in aggregate diameters, the final cell yield did not increase but rather dropped.…”

Improving Human Induced Pluripotent Stem Cell-Derived Megakaryocyte Differentiation and Platelet Production

“…In our study, platelet release from MKs was supported by culturing on an orbital shaker during the final stage III. We plan to scale-up our protocol using bioprocessing in stirred bioreactors [ 41 ].…”

Pluripotent stem cells for skeletal tissue engineering

“…A different model [138] recapitulated in vitro stem cell morphogenesis in silico and demonstrated the possibility of controlling multicellular patterning toward the generation of human organoids and tissues. In an attempt to overcome some of the limitations associated with the in vitro culture of hPSCs and to scale-up their bioprocessing, Manstein and colleagues have combined instrumented stirred tank bioreactor technology with in silico modeling [139]. The authors demonstrate that a 70-fold cell expansion in 7 days (independently of the cell line) is achievable while pluripotency, differentiation potential and karyotype remain unaffected.…”