Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System
Sarah Alsobaie,1 Tamador Alsobaie,2 Amal F Alshammary,1 Manal Abudawood,1 Athanasios Mantalaris3 1Department of Clinical Laboratory Science, King Saud University, Riyadh, Saudi Arabia; 2Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK...
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| Format: | Article |
| Language: | English |
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Dove Medical Press
2023-09-01
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| Series: | Stem Cells and Cloning: Advances and Applications |
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| Online Access: | https://www.dovepress.com/alginate-beads-as-a-promising-tool-for-successful-production-of-viable-peer-reviewed-fulltext-article-SCCAA |
| _version_ | 1797671531707367424 |
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| author | Alsobaie S Alsobaie T Alshammary AF Abudawood M Mantalaris A |
| author_facet | Alsobaie S Alsobaie T Alshammary AF Abudawood M Mantalaris A |
| author_sort | Alsobaie S |
| collection | DOAJ |
| description | Sarah Alsobaie,1 Tamador Alsobaie,2 Amal F Alshammary,1 Manal Abudawood,1 Athanasios Mantalaris3 1Department of Clinical Laboratory Science, King Saud University, Riyadh, Saudi Arabia; 2Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK; 3Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USACorrespondence: Sarah Alsobaie, Department of Clinical Laboratory Science, King Saud University, Prince Turki Alawal Street, Riyadh, Saudi Arabia, Tel +966507191011, Fax +966114567888, Email salsobaie@ksu.edu.saPurpose: Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs).Patients and Methods: Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction.Results: Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period.Conclusion: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.Keywords: stem cell culture, large-scale expansion, HARV bioreactor, self-renewal |
| first_indexed | 2024-03-11T21:16:54Z |
| format | Article |
| id | doaj.art-699e2ba0c0d34e5f8ac00a12dbb7d88d |
| institution | Directory Open Access Journal |
| issn | 1178-6957 |
| language | English |
| last_indexed | 2024-03-11T21:16:54Z |
| publishDate | 2023-09-01 |
| publisher | Dove Medical Press |
| record_format | Article |
| series | Stem Cells and Cloning: Advances and Applications |
| spelling | doaj.art-699e2ba0c0d34e5f8ac00a12dbb7d88d2023-09-28T18:12:52ZengDove Medical PressStem Cells and Cloning: Advances and Applications1178-69572023-09-01Volume 16617387024Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture SystemAlsobaie SAlsobaie TAlshammary AFAbudawood MMantalaris ASarah Alsobaie,1 Tamador Alsobaie,2 Amal F Alshammary,1 Manal Abudawood,1 Athanasios Mantalaris3 1Department of Clinical Laboratory Science, King Saud University, Riyadh, Saudi Arabia; 2Biological Systems Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK; 3Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USACorrespondence: Sarah Alsobaie, Department of Clinical Laboratory Science, King Saud University, Prince Turki Alawal Street, Riyadh, Saudi Arabia, Tel +966507191011, Fax +966114567888, Email salsobaie@ksu.edu.saPurpose: Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs).Patients and Methods: Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction.Results: Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period.Conclusion: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs.Keywords: stem cell culture, large-scale expansion, HARV bioreactor, self-renewalhttps://www.dovepress.com/alginate-beads-as-a-promising-tool-for-successful-production-of-viable-peer-reviewed-fulltext-article-SCCAAstem cell culturelarge-scale expansionharv bioreactorself-renewal |
| spellingShingle | Alsobaie S Alsobaie T Alshammary AF Abudawood M Mantalaris A Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System Stem Cells and Cloning: Advances and Applications stem cell culture large-scale expansion harv bioreactor self-renewal |
| title | Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System |
| title_full | Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System |
| title_fullStr | Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System |
| title_full_unstemmed | Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System |
| title_short | Alginate Beads as a Promising Tool for Successful Production of Viable and Pluripotent Human-Induced Pluripotent Stem Cells in a 3D Culture System |
| title_sort | alginate beads as a promising tool for successful production of viable and pluripotent human induced pluripotent stem cells in a 3d culture system |
| topic | stem cell culture large-scale expansion harv bioreactor self-renewal |
| url | https://www.dovepress.com/alginate-beads-as-a-promising-tool-for-successful-production-of-viable-peer-reviewed-fulltext-article-SCCAA |
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