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Serum-free culture system for spontaneous human mesenchymal stem cell spheroids formation

By Guoyi Dong, Shengpeng Wang, Yuping Ge, Qiuting Deng, Qi Cao, Quanlei Wang, Zhouchun Shang, Wenjie OuYang, Jing Li, Chao Liu, Jie Tang, Weihua Zhao, Ying Gu

Posted 11 Jun 2019
bioRxiv DOI: 10.1101/666313 (published DOI: 10.1155/2019/6041816)

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSCs spheroids, but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stems medium (MiPS), adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR derived spheroids and that genes related to inflammatory response, immune response, and angiogenesis were up-regulated in spheroids at day 6. qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were re-plated in normal FBS medium, cells formed a typical spindle-shaped morphology, and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSCs spheroids for clinical research and therapy.

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