Cell Culture Process Scale-Up Challenges for Commercial-Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products

Abstract

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to manufacturing PSCs in single-use bioreactors, particularly at larger volumetric scales. This manuscript addresses these challenges and presents potential solutions to alleviate the anticipated bottlenecks for commercial-scale manufacturing of high-quality therapeutic cells derived from PSCs.

Keywords: allogeneic cell therapy; cell aggregate; computational fluid dynamics; differentiation; expansion; homogeneous hydrodynamic environment; human embryonic stem cell; induced pluripotent stem cell; medium exchange; oxygenation; scalable manufacturing; scale up; shear stress; single-use bioreactor; turbulent energy dissipation rate; vertical-wheel.

Figure 1

CFD models of velocity streamlines for (A) horizontal-blade spinner and (B) VW bioreactors. All agitation rates are sufficient to fully suspend large particles such as cell aggregates. An identical modeling method was used for both fluid simulation softwares (COMSOL and ANSYS Fluent). Adapted from Borys et al. (2018) [27].

Figure 2

CFD models of EDR contours for various scales and agitation rates in VW bioreactors. A narrow distribution of EDR, which promotes formation of uniformly spherical aggregates, can be maintained during scale up in VW bioreactors. Adapted from Dang et al. (2021) [28].

Figure 3

Curves to determine average EDR based on VW bioreactor working volume and agitation rate. Average EDR can be used to predict aggregate morphology for a particular combination of volume and agitation, with desired spherical aggregates as consequence of average EDR within an optimal range. Adapted from Dang et al. (2021) [28].

Figure 4

Observed morphologies of iPSC aggregates for different combinations of VW working volume and agitation rate. Uniformly spherical aggregates correspond to average EDRs that fall within the optimal range. There is also an inverse correlation between average EDR and aggregate size. Photomicrographs were taken at 10× magnification. Scale bars: 100 μm. Adapted from Dang et al. (2021) [28].

Figure 5

Generation of human iPSC-derived cerebellar organoids using 0.1 L VW bioreactors. (A) Process for differentiation of iPSCs to cerebellar organoids. (B) Brightfield photomicrographs of spherical iPSC-derived organoids during cerebellar differentiation process. Scale bar: 100 μm. Graphs of organoid diameter distributions indicate homogeneous sizes were maintained. (C) Immunofluorescence for NESTIN, PAX6, and TUJ1 during cerebellar differentiation indicate efficient neural induction in iPSC-derived organoids. Scale bar: 50 μm. Adapted from Silva et al. (2020) [29].

Figure 6

A methodology for using an external retention and separation device in conjunction with multiple bioreactors to facilitate rapid, complete, and scalable medium exchange.