Overcoming Bioprocess Bottlenecks in the Large-Scale Expansion of High Quality hiPSC Aggregates in Vertical-Wheel Stirred Suspension Bioreactors
Background
Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle and rocking-wave mixing mechanisms have demonstrated unfavourable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production.
Methods
The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20rpm and 100rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times.
Results
CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single-cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function.
Conclusions
Taken together, these protocols provide a feasible solution for the culture of high quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.
Due to technical limitations, Table 1 is only available as a download in the supplementary files section.
This is a list of supplementary files associated with this preprint. Click to download.
Supplementary Figure 1: Representative confocal images are shown for negative control staining. Scale bars = 50 µm.
Supplementary Table 1: Primer sequences used for RT-qPCR analysis.
Posted 10 Dec, 2020
On 13 Jan, 2021
On 16 Dec, 2020
On 28 Nov, 2020
On 28 Nov, 2020
On 28 Nov, 2020
On 10 Oct, 2020
Received 04 Oct, 2020
On 29 Sep, 2020
On 29 Sep, 2020
Received 21 Sep, 2020
On 01 Sep, 2020
Invitations sent on 29 Aug, 2020
On 27 Aug, 2020
On 26 Aug, 2020
On 26 Aug, 2020
On 25 Aug, 2020
Overcoming Bioprocess Bottlenecks in the Large-Scale Expansion of High Quality hiPSC Aggregates in Vertical-Wheel Stirred Suspension Bioreactors
Posted 10 Dec, 2020
On 13 Jan, 2021
On 16 Dec, 2020
On 28 Nov, 2020
On 28 Nov, 2020
On 28 Nov, 2020
On 10 Oct, 2020
Received 04 Oct, 2020
On 29 Sep, 2020
On 29 Sep, 2020
Received 21 Sep, 2020
On 01 Sep, 2020
Invitations sent on 29 Aug, 2020
On 27 Aug, 2020
On 26 Aug, 2020
On 26 Aug, 2020
On 25 Aug, 2020
Background
Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle and rocking-wave mixing mechanisms have demonstrated unfavourable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production.
Methods
The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20rpm and 100rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times.
Results
CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single-cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function.
Conclusions
Taken together, these protocols provide a feasible solution for the culture of high quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Due to technical limitations, full-text HTML conversion of this manuscript could not be completed. However, the manuscript can be downloaded and accessed as a PDF.
Due to technical limitations, Table 1 is only available as a download in the supplementary files section.