See the published version of this preprint at Stem Cell Research & Therapy.
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Research
Michael S. Kallos
University of Calgary
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1480-8022
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
Induced pluripotent stem cells, vertical-wheel bioreactor, computational fluid dynamics, scale-up, single-cell, harvest, serial-passage
Figure 1
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This is a list of supplementary files associated with this preprint. Click to download.
- hiPSCPaper2GraphicAbstractDraftPage1.tiff
- Table1hiPSCPBSPaper2FiguresRevision1Page01T1.tiff
- fIGs1hiPSCPBSPaper2FiguresRevision1Page11SF1.tiff
Supplementary Figure 1: Representative confocal images are shown for negative control staining. Scale bars = 50 µm.
- TableS1hiPSCPBSPaper2FiguresRevision1Page10ST1.tiff
Supplementary Table 1: Primer sequences used for RT-qPCR analysis.
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CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 2
Posted 10 Dec, 2020
No community comments so far
Editorial decision: Accept
On 16 Dec, 2020
Editor assigned
On 28 Nov, 2020
Submission checks complete
On 28 Nov, 2020
Editor invited
On 28 Nov, 2020
No comments provided
Editorial decision: Minor Revision
On 10 Oct, 2020
Review #2 received
Received 04 Oct, 2020
Reviewer #2 agreed
On 29 Sep, 2020
Reviewer #3 agreed
On 29 Sep, 2020
Review #1 received
Received 21 Sep, 2020
Reviewer #1 agreed
On 01 Sep, 2020
Reviewers invited
Invitations sent on 29 Aug, 2020
Editor assigned
On 27 Aug, 2020
Submission checks complete
On 26 Aug, 2020
Editor invited
On 26 Aug, 2020
First submitted
On 25 Aug, 2020
Metrics
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HTML Views: ...
Subject Areas
Stem Cell & Developmental Cell Biology
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See the published version of this preprint at Stem Cell Research & Therapy.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Michael S. Kallos
University of Calgary
Corresponding Author
ORCiD: https://orcid.org/0000-0002-1480-8022
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
View the published article at Stem Cell Research & Therapy.
Version 2
Posted 10 Dec, 2020
No community comments so far
Editorial decision: Accept
On 16 Dec, 2020
Editor assigned
On 28 Nov, 2020
Submission checks complete
On 28 Nov, 2020
Editor invited
On 28 Nov, 2020
No comments provided
Editorial decision: Minor Revision
On 10 Oct, 2020
Review #2 received
Received 04 Oct, 2020
Reviewer #2 agreed
On 29 Sep, 2020
Reviewer #3 agreed
On 29 Sep, 2020
Review #1 received
Received 21 Sep, 2020
Reviewer #1 agreed
On 01 Sep, 2020
Reviewers invited
Invitations sent on 29 Aug, 2020
Editor assigned
On 27 Aug, 2020
Submission checks complete
On 26 Aug, 2020
Editor invited
On 26 Aug, 2020
First submitted
On 25 Aug, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Stem Cell & Developmental Cell Biology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published article at Stem Cell Research & Therapy.
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
This is a list of supplementary files associated with this preprint. Click to download.
- hiPSCPaper2GraphicAbstractDraftPage1.tiff
- Table1hiPSCPBSPaper2FiguresRevision1Page01T1.tiff
- fIGs1hiPSCPBSPaper2FiguresRevision1Page11SF1.tiff
Supplementary Figure 1: Representative confocal images are shown for negative control staining. Scale bars = 50 µm.
- TableS1hiPSCPBSPaper2FiguresRevision1Page10ST1.tiff
Supplementary Table 1: Primer sequences used for RT-qPCR analysis.
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