Abstract
Controlled large-scale production of human pluripotent stem cells (hPSCs) is indispensable for their envisioned clinical translation. Aiming at advanced process development in suspension culture, the sensitivity of hPSC media to continuous peristaltic pump-based circulation, a well-established technology extensively used in hydraulically-driven bioreactors, was investigated. Unexpectedly, conditioning of low protein media (i.e. E8 and TeSR-E8) in a peristaltic pump circuit induced severe viability loss of hPSCs cultured as aggregates in suspension. Optical, biochemical, and cytological analyses of the media revealed that the applied circulation mode resulted in the reduction of the growth hormone insulin by precipitation of micro-sized particles. Notably, in contrast to insulin depletion, individual withdrawal of other medium protein components (i.e. bFGF, TGFβ1 or transferrin) provoked minor reduction of hPSC viability, if any. Supplementation of the surfactant glycerol or the use of the insulin analogue Aspart did not overcome the issue of insulin precipitation. In contrast, the presence of bovine or human serum albumin (BSA or HSA, respectively) stabilized insulin rescuing its content, possibly by acting as molecular chaperone-like protein, ultimately supporting hPSC maintenance. This study highlights the potential and the requirement of media optimization for automated hPSC processing and has broad implications on media development and bioreactor-based technologies.
Highlights
Human pluripotent stem cells, due to their essentially unlimited proliferative capacity and their potential to differentiate into any somatic cell type in vitro, represent a superior cell source for biomedical sciences[1, 2]
Disruption of human pluripotent stem cells (hPSCs) aggregate culture by peristaltic pump conditioned E8 was mediated by insulin depletion
Light microscopy images of hPSC aggregates cultured in peristaltic pump conditioned E8 medium (PC E8) showed floating single cells and hPSC aggregates with irregular morphology and reduced size compared to the respective static conditioned E8 medium (SC E8) controls already after one day of cultivation, followed by increase in cell debris and disaggregation on days 2–3 (Fig. 2d–f for hiPSCs and Fig. 2j–l for hESCs)
Summary
Human pluripotent stem cells (hPSCs), due to their essentially unlimited proliferative capacity and their potential to differentiate into any somatic cell type in vitro, represent a superior cell source for biomedical sciences[1, 2]. In the perspective of their clinical and industrial applications, it was recently shown that hPSCs, including human embryonic (hESCs) and human induced pluripotent (hiPSCs) cells, can be cultivated as “matrix-free cell only aggregates” in static and dynamic suspension culture[3,4,5] This approach eliminates matrix requirements, enables transition from surface-dependent two-dimensional (2D) culture towards three-dimensional (3D) cultivation, and supports development and up-scaling of good manufacturing practice (GMP)-compliant processes[2, 6,7,8,9]. Thanks to an appositely designed vessel geometry and the connection to a circulation circuit, buoyant vortices form within the culture chamber allowing homogeneous distribution of cells/aggregates in suspension at low-shear conditions[27] Medium flow in this system is achieved by peristaltic pump technology[28], which is extensively used in a broad range of hydraulically driven bioreactors requiring continuous medium circulation[29,30,31,32]
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