Impact of fluidic agitation on human pluripotent stem cells in stirred suspension culture

Abstract

The success of human pluripotent stem cells (hPSCs) as a source of future cell therapies hinges, in part, on the availability of a robust and scalable culture system that can readily produce a clinically relevant number of cells and their derivatives. Stirred suspension culture has been identified as one such promising platform due to its ease of use, scalability, and widespread use in the pharmaceutical industry (e.g., CHO cell-based production of therapeutic proteins) among others. However, culture of undifferentiated hPSCs in stirred suspension is a relatively new development within the past several years, and little is known beyond empirically optimized culture parameters. In particular, detailed characterizations of different agitation rates and their influence on the propagation of hPSCs are often not reported in the literature. In the current study, we systematically investigated various agitation rates to characterize their impact on cell yield, viability, and the maintenance of pluripotency. Additionally, we closely examined the distribution of cell aggregates and how the observed culture outcomes are attributed to their size distribution. Overall, our results showed that moderate agitation maximized the propagation of hPSCs to approximately 38-fold over 7 days by keeping the cell aggregates below the critical size, beyond which the cells are impacted by the diffusion limit, while limiting cell death caused by excessive fluidic forces. Furthermore, we observed that fluidic agitation could regulate not only cell aggregation, but also expression of some key signaling proteins in hPSCs. This indicates a new possibility to guide stem cell fate determination by fluidic agitation in stirred suspension cultures. Biotechnol. Bioeng. 2017;114: 2109-2120. © 2017 Wiley Periodicals, Inc.

Hydrodynamic characterization within a spinner flask and a rotary wall vessel for stem cell culture

“…1). Although the standard working volume of this spinner flask is 125 mL, a 50 mL working volume was used to be consistent with our previous experimental study [21]. The second culture vessel was a rotary wall vessel designed to impose more uniform shear stress on the stem cells (Fig.…”

“…In particular, researchers from CSIRO Australia have successfully employed Large Eddy Simulation (LES) to study the flow field within stirred suspension culture systems, including the Corning ProCulture spinner flask, and experimentally validated the simulation results using particle image velocimetry (PIV) [13,14]. Here we take a similar LES approach to simulate complex flow fields corresponding to our previous report [21] and compute key flow properties such as shear stress and the Kolmogorov length scale [25,26]. We compared the distribution of the Kolmogorov length scale to that of stem cell aggregate size which we measured in our previous study [21].…”

“…Understanding how fluid shear stresses and other physical forces affect stem cell behavior such as protein expression and folding, contractile activity, proliferation, maintenance of pluripotency or differentiation would enable new designs of scalable stem cell culture systems that utilize fluid shear as a key input parameter. With such an ambitious goal, we recently reported our initial experimental study where we investigated effects of varying impeller speeds on undifferentiated hPSCs using Corning ProCulture spinner flasks [21]. Interestingly, varying impeller speeds (0, 40, 60, 80, and 100 rpm) resulted in significant differences in stem cell aggregate size, population growth, maintenance of pluripotency, spontaneous differentiation, and protein kinase expressions.…”

“…Here we take a similar LES approach to simulate complex flow fields corresponding to our previous report [21] and compute key flow properties such as shear stress and the Kolmogorov length scale [25,26]. We compared the distribution of the Kolmogorov length scale to that of stem cell aggregate size which we measured in our previous study [21]. Finally, we performed CFD simulations of a newly developed custom rotary wall vessel, which features a pair of concentric rotating cylindrical walls designed to produce a laminar, circular Couette flow, resulting in a narrow and controlled distribution of shear stress.…”

Computational fluid dynamic analysis reveals the underlying physical forces playing a role in 3D multiplex brain organoid cultures

“…Historically, cerebral organoids have been cultured in spinner 64 flasks (Lancaster and Knoblich, 2014b). These flasks have the advantage of providing 65 a low-shear environment (Wang et al, 2013), which is important because hPSCs have 66 been shown to be sensitive to shear stress (Nampe et al, 2017;Wang et al, 2013). printed scalable mini-bioreactor, the SpinΩ, which would be cost effective and provide 72 a feasible, reproducible platform for chemical compound testing.…”