Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation
Abstract Background Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such co...
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doaj-6390564226624f02b6b5f0bb091bfb1f2020-11-24T22:20:27ZengBMCStem Cell Research & Therapy1757-65122017-05-018111710.1186/s13287-017-0538-xCritical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiationYoushan Melissa Lin0Jialing Lee1Jessica Fang Yan Lim2Mahesh Choolani3Jerry Kok Yen Chan4Shaul Reuveny5Steve Kah Weng Oh6Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR)Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR)Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR)Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health SystemExperimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health SystemBioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR)Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR)Abstract Background Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types. Methods A screen was performed to evaluate whether 1) cell confluency, 2) cell numbers, 3) cell density, 4) centrifugation, or 5) agitation are crucial in driving effective chondrogenic differentiation of human early mesenchymal stromal cell (heMSC)-laden Cytodex 1 microcarrier (heMSC-Cytodex 1) constructs. Results Firstly, we found that seeding 10 × 103 cells at 70% cell confluency with 300 microcarriers per construct resulted in substantial increase in cell growth (76.8-fold increase in DNA) and chondrogenic protein generation (78.3- and 686-fold increase in GAG and Collagen II, respectively). Reducing cell density by adding empty microcarriers at seeding and indirectly compacting constructs by applying centrifugation at seeding or agitation throughout differentiation caused reduced cell growth and chondrogenic differentiation. Secondly, we showed that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes since critically defined heMSC-Cytodex 1 constructs developed larger diameters (2.6-fold), and produced more DNA (13.8-fold), GAG (11.0-fold), and Collagen II (6.6-fold) than their equivalent cell-only counterparts. Thirdly, heMSC-Cytodex 1/3 constructs generated with cell-laden microcarriers from 1-day attachment in shake flask cultures were more efficient than those from 5-day expansion in spinner cultures in promoting cell growth and chondrogenic output per construct and per cell. Lastly, we demonstrate that these critically defined parameters can be applied across multiple microcarrier types, such as Cytodex 3, SphereCol and Cultispher-S, achieving similar trends in enhancing cell growth and chondrogenic differentiation. Conclusions This is the first study that has identified a set of critical attributes that enables efficient chondrogenic differentiation of heMSC-microcarrier constructs across multiple microcarrier types. It is also the first to demonstrate that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes across different microcarrier types, including biodegradable gelatin-based microcarriers, making heMSC-microcarrier constructs applicable for use in allogeneic cartilage cell therapy.http://link.springer.com/article/10.1186/s13287-017-0538-xCartilageCell therapyChondrogenic differentiationMesenchymal stromal cellsMicrocarrier |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Youshan Melissa Lin Jialing Lee Jessica Fang Yan Lim Mahesh Choolani Jerry Kok Yen Chan Shaul Reuveny Steve Kah Weng Oh |
spellingShingle |
Youshan Melissa Lin Jialing Lee Jessica Fang Yan Lim Mahesh Choolani Jerry Kok Yen Chan Shaul Reuveny Steve Kah Weng Oh Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation Stem Cell Research & Therapy Cartilage Cell therapy Chondrogenic differentiation Mesenchymal stromal cells Microcarrier |
author_facet |
Youshan Melissa Lin Jialing Lee Jessica Fang Yan Lim Mahesh Choolani Jerry Kok Yen Chan Shaul Reuveny Steve Kah Weng Oh |
author_sort |
Youshan Melissa Lin |
title |
Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation |
title_short |
Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation |
title_full |
Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation |
title_fullStr |
Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation |
title_full_unstemmed |
Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation |
title_sort |
critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation |
publisher |
BMC |
series |
Stem Cell Research & Therapy |
issn |
1757-6512 |
publishDate |
2017-05-01 |
description |
Abstract Background Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types. Methods A screen was performed to evaluate whether 1) cell confluency, 2) cell numbers, 3) cell density, 4) centrifugation, or 5) agitation are crucial in driving effective chondrogenic differentiation of human early mesenchymal stromal cell (heMSC)-laden Cytodex 1 microcarrier (heMSC-Cytodex 1) constructs. Results Firstly, we found that seeding 10 × 103 cells at 70% cell confluency with 300 microcarriers per construct resulted in substantial increase in cell growth (76.8-fold increase in DNA) and chondrogenic protein generation (78.3- and 686-fold increase in GAG and Collagen II, respectively). Reducing cell density by adding empty microcarriers at seeding and indirectly compacting constructs by applying centrifugation at seeding or agitation throughout differentiation caused reduced cell growth and chondrogenic differentiation. Secondly, we showed that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes since critically defined heMSC-Cytodex 1 constructs developed larger diameters (2.6-fold), and produced more DNA (13.8-fold), GAG (11.0-fold), and Collagen II (6.6-fold) than their equivalent cell-only counterparts. Thirdly, heMSC-Cytodex 1/3 constructs generated with cell-laden microcarriers from 1-day attachment in shake flask cultures were more efficient than those from 5-day expansion in spinner cultures in promoting cell growth and chondrogenic output per construct and per cell. Lastly, we demonstrate that these critically defined parameters can be applied across multiple microcarrier types, such as Cytodex 3, SphereCol and Cultispher-S, achieving similar trends in enhancing cell growth and chondrogenic differentiation. Conclusions This is the first study that has identified a set of critical attributes that enables efficient chondrogenic differentiation of heMSC-microcarrier constructs across multiple microcarrier types. It is also the first to demonstrate that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes across different microcarrier types, including biodegradable gelatin-based microcarriers, making heMSC-microcarrier constructs applicable for use in allogeneic cartilage cell therapy. |
topic |
Cartilage Cell therapy Chondrogenic differentiation Mesenchymal stromal cells Microcarrier |
url |
http://link.springer.com/article/10.1186/s13287-017-0538-x |
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