Scalable culture of human induced pluripotent cells on microcarriers under xeno‐free conditions using single‐use vertical‐wheel™ bioreactors

Abstract

BACKGROUND: Human induced pluripotent stem cells (hiPSC) are expected to become powerful tools for disease modelling, for the discovery and testing of new drugs and, ultimately, for regenerative therapies. The success of these applications depends on the development of scalable bioprocesses capable of generating large numbers of hiPSC and derivatives. RESULTS:In this work, the novel vertical-wheel single-use bioreactors were used for the first time for the expansion of hiPSC under xeno-free conditions. Cultures were performed on microcarriers in two different scales of vessels (100 and 500 mL with 80 and 300 mL working volumes, respectively), leading to maximum cell densities up to 1.21 ± 0.02 × 10 6 cells mL −1 and volumetric productivities of 2.01 ± 0.04 × 10 5 cells mL −1 day −1 . The pluripotency as well as a normal karyotype were maintained after cell expansion. Consistency of the processes was confirmed with a different hiPSC line, which is an important aspect for a personalized medicine approach. CONCLUSION: The results here described demonstrate the feasibility of scalable production of hiPSC in a microcarrier-based system using vertical-wheel bioreactors. The protocols developed in this study provide a Good Manufacturing Practices (GMP)-compliant system for hiPSC manufacturing which may be an important step towards the successful implementation of hiPSC-based products.

Optimized serial expansion of human induced pluripotent stem cells using low-density inoculation to generate clinically relevant quantities in vertical-wheel bioreactors

“…7,12,36 While the suspension culture of human pluripotent stem cells (hPSCs) has been studied extensively using rocking platforms 37,38 and traditional horizontal-blade, stirred suspension bioreactors, 17,19,[22][23][24][25] the use of vertical-wheel bioreactors for culture of hPSCs has not been studied in great detail. 33,39 The geometry of the scalable, single-use bioreactor consists of a large vertical-wheel with pitched inner blades that rotate along the vertical plane about a horizontal axis. 40 The system combines radial and axial flows that provide agitation and particle suspension with a more uniform distribution of hydrodynamic forces.…”

“…This results in efficient mixing, reduced shear rates and power input compared to traditional horizontal-blade, stirred suspension bioreactors. 33,40 It was, therefore, our hypothesis that hiPSC expansion as uniform aggregates from low inoculation densities could be greatly enhanced using the novel vertical-wheel bioreactor.…”

“…A graphical outline highlighting the experimental design leading to an optimized process for hiPSC bioreactor serial passaging and quality testing is shown in Figure 1. The PBS Biotech verticalwheel bioreactor, is a novel system that combines radial and axial flow components producing more uniform distributions of hydrodynamic forces, as well as lower shear stress, compared with traditional horizontal-blade based bioreactors, 32,33 shown in Figure 1. We hypothesized that this unique bioreactor system would provide a more optimal environment for the efficient, rapid generation of highquality hiPSCs to meet the demands for manufacturing of therapeutic cell products.…”

Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System

“…In addition, the Vertical-Wheel TM impeller can fully suspend microcarriers with minimal power input and thus minimize shear stress effects (Croughan et al, 2016). Moreover, this technology is scalable, being available at working volumes that range from 100 mL up to 500 L. Recently, VWBR have been successfully applied in microcarrier-based cell culture processes for the expansion of MSC from multiple sources (Sousa et al, 2015;de Sousa Pinto et al, 2019), as well as for human induced pluripotent stem cells (Rodrigues et al, 2018;Nogueira et al, 2019).…”

Overcoming Bioprocess Bottlenecks in the Large-Scale Expansion of High Quality hiPSC Aggregates in Vertical-Wheel Stirred Suspension Bioreactors

“…This in turn affect cell viability, proliferation, pluripotency and differentiation. The CFD model allowed us to confirm that the vertical-wheel platform uniquely combines radial and axial flow components producing more uniform distributions of hydrodynamic forces and better scalability compared to traditional bioreactor geometries [34,35].…”