Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture
Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement the...
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| Format: | Article |
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Frontiers Media S.A.
2023-12-01
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| Series: | Frontiers in Bioengineering and Biotechnology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1267007/full |
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| author | Diepiriye G. Iworima Diepiriye G. Iworima Robert K. Baker James M. Piret James M. Piret James M. Piret Timothy J. Kieffer Timothy J. Kieffer Timothy J. Kieffer |
| author_facet | Diepiriye G. Iworima Diepiriye G. Iworima Robert K. Baker James M. Piret James M. Piret James M. Piret Timothy J. Kieffer Timothy J. Kieffer Timothy J. Kieffer |
| author_sort | Diepiriye G. Iworima |
| collection | DOAJ |
| description | Introduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures.Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells.Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: −0.01–0.022 h−1) than cells growing as a monolayer (range: 0.039–0.045 h−1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260 mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types. |
| first_indexed | 2024-03-09T08:27:08Z |
| format | Article |
| id | doaj.art-ea6232bfd13f4492920ebb3a18c3c90b |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-09T08:27:08Z |
| publishDate | 2023-12-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-ea6232bfd13f4492920ebb3a18c3c90b2023-12-02T21:07:00ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852023-12-011110.3389/fbioe.2023.12670071267007Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and cultureDiepiriye G. Iworima0Diepiriye G. Iworima1Robert K. Baker2James M. Piret3James M. Piret4James M. Piret5Timothy J. Kieffer6Timothy J. Kieffer7Timothy J. Kieffer8Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC, CanadaSchool of Biomedical Engineering, The University of British Columbia, Vancouver, BC, CanadaDepartment of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC, CanadaSchool of Biomedical Engineering, The University of British Columbia, Vancouver, BC, CanadaMichael Smith Laboratories, The University of British Columbia, Vancouver, BC, CanadaDepartment of Chemical and Biological Engineering, The University of British Columbia, Vancouver, BC, CanadaDepartment of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC, CanadaSchool of Biomedical Engineering, The University of British Columbia, Vancouver, BC, CanadaDepartment of Surgery, The University of British Columbia, Vancouver, BC, CanadaIntroduction: Human pluripotent stem cells (hPSCs) provide many opportunities for application in regenerative medicine due to their ability to differentiate into cells from all three germ layers, proliferate indefinitely, and replace damaged or dysfunctional cells. However, such cell replacement therapies require the economical generation of clinically relevant cell numbers. Whereas culturing hPSCs as a two-dimensional monolayer is widely used and relatively simple to perform, their culture as suspended three-dimensional aggregates may enable more economical production in large-scale stirred tank bioreactors. To be more relevant to this biomanufacturing, bench-scale differentiation studies should be initiated from aggregated hPSC cultures.Methods: We compared five available bench-scale platforms for generating undifferentiated cell aggregates of human embryonic stem cells (hESCs) using AggreWell™ plates, low attachment plates on an orbital shaker, roller bottles, spinner flasks, and vertical-wheel bioreactors (PBS-Minis). Thereafter, we demonstrated the incorporation of an hPSC aggregation step prior to directed differentiation to pancreatic progenitors and endocrine cells.Results and discussion: The AggreWell™ system had the highest aggregation yield. The initial cell concentrations had an impact on the size of aggregates generated when using AggreWell™ plates as well as in roller bottles. However, aggregates made with low attachment plates, spinner flasks and PBS-Minis were similar regardless of the initial cell number. Aggregate morphology was compact and relatively homogenously distributed in all platforms except for the roller bottles. The size of aggregates formed in PBS-Minis was modulated by the agitation rate during the aggregation. In all cell culture platforms, the net growth rate of cells in 3D aggregates was lower (range: −0.01–0.022 h−1) than cells growing as a monolayer (range: 0.039–0.045 h−1). Overall, this study describes operating ranges that yield high-quality undifferentiated hESC aggregates using several of the most commonly used bench-scale cell culture platforms. In all of these systems, methods were identified to obtain PSC aggregates with greater than 70% viability, and mean diameters between 60 and 260 mm. Finally, we showed the capacity of hPSC aggregates formed with PBS-Minis to differentiate into viable pancreatic progenitors and endocrine cell types.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1267007/fullhuman embryonic stem cellsbioreactor 3D cell cultureaggregationexpansionseed traindifferentiation |
| spellingShingle | Diepiriye G. Iworima Diepiriye G. Iworima Robert K. Baker James M. Piret James M. Piret James M. Piret Timothy J. Kieffer Timothy J. Kieffer Timothy J. Kieffer Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture Frontiers in Bioengineering and Biotechnology human embryonic stem cells bioreactor 3D cell culture aggregation expansion seed train differentiation |
| title | Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture |
| title_full | Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture |
| title_fullStr | Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture |
| title_full_unstemmed | Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture |
| title_short | Analysis of the effects of bench-scale cell culture platforms and inoculum cell concentrations on PSC aggregate formation and culture |
| title_sort | analysis of the effects of bench scale cell culture platforms and inoculum cell concentrations on psc aggregate formation and culture |
| topic | human embryonic stem cells bioreactor 3D cell culture aggregation expansion seed train differentiation |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1267007/full |
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