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Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors
BACKGROUND: Since their inception, human induced pluripotent stem cells (hiPSCs) have held much promise for pharmacological applications and cell-based therapies. However, their potential can only be realised if large numbers of cells can be produced reproducibly on-demand. While bioreactors are ide...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744632/ https://www.ncbi.nlm.nih.gov/pubmed/31534477 http://dx.doi.org/10.1186/s13036-019-0204-1 |
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author | Nogueira, Diogo E. S. Rodrigues, Carlos A. V. Carvalho, Marta S. Miranda, Cláudia C. Hashimura, Yas Jung, Sunghoon Lee, Brian Cabral, Joaquim M. S. |
author_facet | Nogueira, Diogo E. S. Rodrigues, Carlos A. V. Carvalho, Marta S. Miranda, Cláudia C. Hashimura, Yas Jung, Sunghoon Lee, Brian Cabral, Joaquim M. S. |
author_sort | Nogueira, Diogo E. S. |
collection | PubMed |
description | BACKGROUND: Since their inception, human induced pluripotent stem cells (hiPSCs) have held much promise for pharmacological applications and cell-based therapies. However, their potential can only be realised if large numbers of cells can be produced reproducibly on-demand. While bioreactors are ideal systems for this task, due to providing agitation and control of the culture parameters, the common impeller geometries were not designed for the expansion of mammalian cells, potentially leading to sub-optimal results. RESULTS: This work reports for the first time the usage of the novel Vertical-Wheel single-use bioreactors for the expansion of hiPSCs as floating aggregates. Cultures were performed in the PBS MINI 0.1 bioreactor with 60 mL of working volume. Two different culture media were tested, mTeSR1 and mTeSR3D, in a repeated batch or fed-batch mode, respectively, as well as dextran sulfate (DS) supplementation. mTeSR3D was shown to sustain hiPSC expansion, although with lower maximum cell density than mTeSR1. Dextran sulfate supplementation led to an increase in 97 and 106% in maximum cell number when using mTeSR1 or mTeSR3D, respectively. For supplemented media, mTeSR1 + DS allowed for a higher cell density to be obtained with one less day of culture. A maximum cell density of (2.3 ± 0.2) × 10(6) cells∙mL(− 1) and a volumetric productivity of (4.6 ± 0.3) × 10(5) cells∙mL(− 1)∙d(− 1) were obtained after 5 days with mTeSR1 + DS, resulting in aggregates with an average diameter of 346 ± 11 μm. The generated hiPSCs were analysed by flow cytometry and qRT-PCR and their differentiation potential was assayed, revealing the maintenance of their pluripotency after expansion. CONCLUSIONS: The results here described present the Vertical-Wheel bioreactor as a promising technology for hiPSC bioprocessing. The specific characteristics of this bioreactor, namely in terms of the innovative agitation mechanism, can make it an important system in the development of hiPSC-derived products under current Good Manufacturing Practices. |
format | Online Article Text |
id | pubmed-6744632 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-67446322019-09-18 Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors Nogueira, Diogo E. S. Rodrigues, Carlos A. V. Carvalho, Marta S. Miranda, Cláudia C. Hashimura, Yas Jung, Sunghoon Lee, Brian Cabral, Joaquim M. S. J Biol Eng Research BACKGROUND: Since their inception, human induced pluripotent stem cells (hiPSCs) have held much promise for pharmacological applications and cell-based therapies. However, their potential can only be realised if large numbers of cells can be produced reproducibly on-demand. While bioreactors are ideal systems for this task, due to providing agitation and control of the culture parameters, the common impeller geometries were not designed for the expansion of mammalian cells, potentially leading to sub-optimal results. RESULTS: This work reports for the first time the usage of the novel Vertical-Wheel single-use bioreactors for the expansion of hiPSCs as floating aggregates. Cultures were performed in the PBS MINI 0.1 bioreactor with 60 mL of working volume. Two different culture media were tested, mTeSR1 and mTeSR3D, in a repeated batch or fed-batch mode, respectively, as well as dextran sulfate (DS) supplementation. mTeSR3D was shown to sustain hiPSC expansion, although with lower maximum cell density than mTeSR1. Dextran sulfate supplementation led to an increase in 97 and 106% in maximum cell number when using mTeSR1 or mTeSR3D, respectively. For supplemented media, mTeSR1 + DS allowed for a higher cell density to be obtained with one less day of culture. A maximum cell density of (2.3 ± 0.2) × 10(6) cells∙mL(− 1) and a volumetric productivity of (4.6 ± 0.3) × 10(5) cells∙mL(− 1)∙d(− 1) were obtained after 5 days with mTeSR1 + DS, resulting in aggregates with an average diameter of 346 ± 11 μm. The generated hiPSCs were analysed by flow cytometry and qRT-PCR and their differentiation potential was assayed, revealing the maintenance of their pluripotency after expansion. CONCLUSIONS: The results here described present the Vertical-Wheel bioreactor as a promising technology for hiPSC bioprocessing. The specific characteristics of this bioreactor, namely in terms of the innovative agitation mechanism, can make it an important system in the development of hiPSC-derived products under current Good Manufacturing Practices. BioMed Central 2019-09-14 /pmc/articles/PMC6744632/ /pubmed/31534477 http://dx.doi.org/10.1186/s13036-019-0204-1 Text en © The Author(s). 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Nogueira, Diogo E. S. Rodrigues, Carlos A. V. Carvalho, Marta S. Miranda, Cláudia C. Hashimura, Yas Jung, Sunghoon Lee, Brian Cabral, Joaquim M. S. Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors |
title | Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors |
title_full | Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors |
title_fullStr | Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors |
title_full_unstemmed | Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors |
title_short | Strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use Vertical-Wheel™ bioreactors |
title_sort | strategies for the expansion of human induced pluripotent stem cells as aggregates in single-use vertical-wheel™ bioreactors |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6744632/ https://www.ncbi.nlm.nih.gov/pubmed/31534477 http://dx.doi.org/10.1186/s13036-019-0204-1 |
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