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Expansion of Fetal Human Mesenchymal Stem Cells in Microcarrier-Spinner Cultures Improves their Chondrogenic Potential
22nd Annual ISCT Meeting
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214 WILL NOT BE PRESENTED 215 EXPANSION OF FETAL HUMAN MESENCHYMAL STEM CELLS IN MICROCARRIER-SPINNER CULTURES IMPROVES THEIR CHONDROGENIC POTENTIAL Y. Lin1, J. Lim1, J. Lee1, M. Cholani2, J.K. Chan2,3, S. Reuveny1, S. Oh1 1 Stem Cell Group, Bioprocessing Technology Institute, Singapore, 2 Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, Singapore, 3Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore The inability of conventional 2D monolayer cultures to provide the large cell numbers required to meet potential market demand for allogeneic cartilage cell therapy has renewed interest in propagating hMSC with 3D microcarrier-based cultures that are scalable for bioreactor use. Yet, it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential differ from that of monolayer systems. Thus, our study aims to compare between the chon-
drogenic potential of fetal hMSC (fhMSC) cultured in 3D microcarrier-spinner and 2D monolayer modes. To identify the most effective chondrogenic inducer for fhMSC, we screened multiple inducers including TGFβ-1/3 and BMP2, using chondrogenic pellets generated with high-throughput, ultra-low attachment 96 well plates. We found that BMP2 at 100 ng/ml was most effective as it induced the greatest increase in pellet diameter (4.6 fold), DNA (3.8 fold), GAG (524 fold), GAG/DNA (161 fold), Collagen II (1626 fold), and Collagen II/DNA (681 fold) after 28 days, as compared to those grown without inducer. We grew fhMSC either as static monolayer cultures, or on collagen-coated Cytodex 3 microcarriers in agitated spinner cultures, and used them for chondrogenic pellet differentiation with BMP2. We found that chondrogenic pellets derived from 3D microcarrier cultures developed larger pellet diameters (1.6 fold), and produced more DNA (1.8 fold), GAG (7.5 fold) and Collagen II (12 fold) per pellet with greater GAG/DNA (3.7 fold) and Collagen II/DNA (6.7 fold) ratios, as compared to that of monolayer cultures (all p < 0.05). Moreover, these pellets induced expression of chondrogenic genes (e.g. S100β) but not of hypertrophic genes (e.g. MMP13 and ALPL). Similar trends were obtained with positivelycharged Cytodex 1 microcarriers. Our results show that scalable 3D microcarrier cultures have an additional advantage of enhancing chondrogenic potential, supporting their use for cell expansion in cartilage cell therapy.
S73
Figure 2.
Figure 3.
214 WILL NOT BE PRESENTED 215 EXPANSION OF FETAL HUMAN MESENCHYMAL STEM CELLS IN MICROCARRIER-SPINNER CULTURES IMPROVES THEIR CHONDROGENIC POTENTIAL Y. Lin1, J. Lim1, J. Lee1, M. Cholani2, J.K. Chan2,3, S. Reuveny1, S. Oh1 1 Stem Cell Group, Bioprocessing Technology Institute, Singapore, 2 Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, Singapore, 3Department of Reproductive Medicine, KK Women’s and Children’s Hospital, Singapore The inability of conventional 2D monolayer cultures to provide the large cell numbers required to meet potential market demand for allogeneic cartilage cell therapy has renewed interest in propagating hMSC with 3D microcarrier-based cultures that are scalable for bioreactor use. Yet, it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential differ from that of monolayer systems. Thus, our study aims to compare between the chon-
drogenic potential of fetal hMSC (fhMSC) cultured in 3D microcarrier-spinner and 2D monolayer modes. To identify the most effective chondrogenic inducer for fhMSC, we screened multiple inducers including TGFβ-1/3 and BMP2, using chondrogenic pellets generated with high-throughput, ultra-low attachment 96 well plates. We found that BMP2 at 100 ng/ml was most effective as it induced the greatest increase in pellet diameter (4.6 fold), DNA (3.8 fold), GAG (524 fold), GAG/DNA (161 fold), Collagen II (1626 fold), and Collagen II/DNA (681 fold) after 28 days, as compared to those grown without inducer. We grew fhMSC either as static monolayer cultures, or on collagen-coated Cytodex 3 microcarriers in agitated spinner cultures, and used them for chondrogenic pellet differentiation with BMP2. We found that chondrogenic pellets derived from 3D microcarrier cultures developed larger pellet diameters (1.6 fold), and produced more DNA (1.8 fold), GAG (7.5 fold) and Collagen II (12 fold) per pellet with greater GAG/DNA (3.7 fold) and Collagen II/DNA (6.7 fold) ratios, as compared to that of monolayer cultures (all p < 0.05). Moreover, these pellets induced expression of chondrogenic genes (e.g. S100β) but not of hypertrophic genes (e.g. MMP13 and ALPL). Similar trends were obtained with positivelycharged Cytodex 1 microcarriers. Our results show that scalable 3D microcarrier cultures have an additional advantage of enhancing chondrogenic potential, supporting their use for cell expansion in cartilage cell therapy.