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Characterization of Hematopoietic Stem Cells Cultured in 3D-clinostat. R. Yoshimoto¹, T. Kajiume², Y. Kawahara^1,3, A. Sasaki¹, S.L. Wu¹, T. Manabe¹, M. Takeda², T. Magaki² and L. Yuge^{1,3  1}Graduate School of Health Sciences, ²Graduate School of Biomedical Sciences, ³Space Bio-Laboratories Y. K., Hiroshima University, Japan. 

   Expansion and/or maintenance of hematopoietic stem cells (HSCs) in vitro are key issue for clinical application of HSCs.  To this end, cytokines and growth factors combination, co-culture system with bone marrow stromal cells, and gene transfection were studied, and new method is required to achieve it.  Microgravity is known to control cell cycle, cell proliferation, and differentiation.  A 3D-clinostat is a multi-directional gravity device for simulating microgravity.  By controlling rotation of two axes, a 3D-clinostat minimizes the cumulative gravity vector in cells cultured at the center of the device and makes 10^-3 G average over time.  In this study, we investigated the application of microgravity to HSCs culture using a 3D-clinostat.

   Mouse bone marrow cells (BMCs) were cultured for 3 to 7 days in a 3D-clinostat (group CL), or in control cultures at 1G (group C) in the presence of optimal cytokine combination.  The number of total BMCs was counted and that of highly purified HSCs, defined as lineage marker negative and Sca1 and c-Kit positive cells (Lin(-) Sca1(+) c-Kit(+) (LSKs)) were assessed by flow cytometry on day 3 and day 7.  For the purpose of evaluating function of the cultured BMCs, colony-forming cell assay (CFC assay) was performed twice (2nd CFC assay), and telomere length was assessed.  The fold increase of BMCs expanded from day 3 to day 7 was significantly greater in group C than in group CL. Otherwise, group CL included larger proportion of LSKs than group C at day 7.  The number of colony-forming cells (CFCs) in 2nd CFC assay was also more abundant in group CL.  In conclusion, we demonstrated that HSCs were well proliferated and maintained their multipotency in culture in simulated microgravity by 3D-clinostat.