Oxygen Transport and Stem Cell Aggregation in Stirred-Suspension Bioreactor Cultures

Abstract

Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC) aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC) and human ESC (hESC) clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE) for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.

Inverse problem analysis of pluripotent stem cell aggregation dynamics in stirred-suspension cultures

“…This is in line with the doubling time of 11.7 hours for mESCs in spinner flask cultures (Wu et al, 2014). Thus, equation 9 becomes: …”

“…After background subtraction (rolling ball method), the diameter of each aggregate was obtained as the mean of two perpendicular diameters. The number of cells per aggregate was estimated assuming that the aggregates are spherical and taking into account aggregate porosity (Wu et al, 2014). …”

“…We assumed a batch process with randomly mixed aggregates which form by the combination of two smaller clusters/cells. Negligible attrition is also accepted given the high viability of cultured cells (typically >90% (Kehoe et al, 2008; Wu et al, 2014)).…”

“…Aggregation promoted through agitation-induced collisions among cells and/or cell clusters, is a central determinant of the process outcome. Cluster size influences the transport of nutrients, O 2 , metabolites and growth factors while cells residing within large clusters potentially experience hypoxic conditions with poor nutrient supplementation (Van Winkle et al, 2012; Wu et al, 2014). In addition to the growth and viability, the aggregate dimensions strongly influence the differentiation trajectory and heterogeneity of cultured cell populations (Niebruegge et al, 2009).…”

Scalable stirred suspension culture for the generation of billions of human induced pluripotent stem cells using single‐use bioreactors

“…The survival and maintenance of pluripotency of hiPSC culture relies on the access of hiPSCs to oxygen and medium components, an important factor especially in stirred suspension culture. In aggregates >400 μm in diameter, oxygen distribution to cells closer to the core of the aggregate may be impaired (Wu, Rostami, Cadavid Olaya, & Tzanakakis, 2014), and access to medium components for the maintenance of pluripotency may be similarly affected.…”