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

Abstract

Human induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self‐renewal and differentiation into the three germ layers of the body. Their discovery has facilitated a new era in biomedicine for understanding human development, drug screening, disease modeling, and cell therapy while reducing ethical issues and risks of immune rejection associated with traditional embryonic stem cells. Bioreactor‐based processes have been the method of choice for the efficient expansion and differentiation of stem cells in controlled environments. Current protocols for the expansion of hiPSCs use horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell culture starting populations and achieve only moderate cell fold increases. This study focused on optimizing inoculation, agitation, oxygen, and nutrient availability for the culture of hiPSCs as aggregates in single‐use, low‐shear, vertical‐wheel bioreactors. Under optimized conditions, we achieved an expansion of more than 30‐fold in 6 days using a small starting population of cells and minimal media resources throughout. Importantly, we showed that that this optimized bioreactor expansion protocol could be replicated over four serial passages resulting in a cumulative cell expansion of 1.06E6‐fold in 28 days. Cells from the final day of the serial passage were of high quality, maintaining a normal karyotype, pluripotent marker staining, and the ability to form teratomas in vivo. These findings demonstrate that a vertical‐wheel bioreactor‐based bioprocess can provide optimal conditions for efficient, rapid generation of high‐quality hiPSCs to meet the demands for clinical manufacturing of therapeutic cell products.

Challenges and Solutions for Commercial Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products

“…Biological studies in Vertical-Wheel bioreactors have confirmed the inverse correlation between agitation rate and average size for iPSC aggregates ( Figure 3 A). The uniformity of cell aggregate sizes at different agitation rates in small-scale Vertical-Wheel bioreactors, compared to those produced in horizontal spinner flasks, was also confirmed ( Figure 3 B) [ 22 ]. The observed narrow size distributions and uniform morphology also indicate that unwanted agglomeration into overly large or irregular spheroids is minimized across various agitation rates.…”

“…The cells harvested at the end of the serial passage were of high quality, maintaining a normal karyotype, pluripotency, and the ability to form teratomas in vivo. This scale-down model process suggests that commercial scale production of over one trillion high quality iPSCs could feasibly be generated in a Vertical-Wheel bioreactor with 50 L working volume [22]. While these various experiments were performed at relatively small scales (0.1 to 15 L) in Vertical-Wheel bioreactors, the homogeneous hydrodynamic environment created by the Vertical-Wheel impeller and U-shaped vessel has been modelled by CFD analysis up to 80 L scale and beyond.…”

“…The cells harvested at the end of the serial passage were of high quality, maintaining a normal karyotype, pluripotency, and the ability to form teratomas in vivo. This scale-down model process suggests that commercial scale production of over one trillion high quality iPSCs could feasibly be generated in a Vertical-Wheel bioreactor with 50 L working volume [22].…”

Single-Use Bioreactors for Human Pluripotent and Adult Stem Cells: Towards Regenerative Medicine Applications

“…Borys et al tested hiPSC expansion in 100 mL VWBR and found normoxic conditions using repeated batch to lead to a higher cell growth than experiments with hypoxia (3%) and/or batch conditions. By performing a low-density inoculation with pre-formed aggregates, they obtained up to (6.3 ± 0.4) × 10 8 cells in 6 days, and about 2.1 × 10 12 cells in 28 days, over the course of four consecutive passages [ 65 ]. The same group then optimised the culture protocol, in terms of inoculation (pre-formed aggregates vs. single cell), agitation speed and harvesting enzyme, and exposure time.…”

Transcriptome profiling of human pluripotent stem cell-derived cerebellar organoids reveals faster commitment under dynamic conditions

“…By using the novel single-use Vertical-Wheel TM bioreactors (VWBRs, PBS Biotech), we are able to mimic later stages of human cerebellar development in vitro, by easily generating high numbers of human iPSC-derived aggregates and efficiently differentiating them into mature cerebellar organoids, which contain diverse types of cerebellar neurons including Purkinje cells and granule cells. VWBRs were already successfully used for human iPSC (Borys et al, 2020;Nogueira et al, 2019;Rodrigues et al, 2018) and mesenchymal stem cell expansion (Sousa et al, 2015;de Sousa Pinto et al, 2019). The VWBRs combine a large vertical impeller and a U-shaped bottom to provide a more homogeneous shear distribution inside the bioreactor, allowing a gentle and uniform mixing and particle suspension with reduced power input and agitation speeds (Croughan et al, 2016).…”