Youshan Melissa Lin1, Jessica Fang Yan Lim2, Jialing Lee2, Mahesh Choolani3, Jerry Kok Yen Chan4, Shaul Reuveny2, Steve Kah Weng Oh5. 1. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore. Electronic address: Melissa_Lin@bti.a-star.edu.sg. 2. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore. 3. Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, Singapore. 4. Experimental Fetal Medicine Group, Department of Obstetrics and Gynaecology, Yong Loo Lin School of Medicine, National University Health System, Singapore; Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore; Cancer and Stem Cell Biology Program, Duke-NUS Graduate Medical School, Singapore. 5. Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore. Electronic address: Steve_Oh@bti.a-star.edu.sg.
Abstract
BACKGROUND AIMS: Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. METHODS: heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. RESULTS: BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. CONCLUSIONS: This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy.
BACKGROUND AIMS: Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. METHODS: heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100β, MMP13 and ALPL, were investigated and compared in both conditions. RESULTS:BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100β) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. CONCLUSIONS: This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy.