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Quality and Quantity Control Cell Culture with Microgravity increases CD34-positive fraction and angiogenic potential of endothelial progenitor cells
Hiroko Hagiwara, Ph.D.1, Akira Higashibata, Ph.D.2, Shiho Ogawa, Ph.D.2, Shigeyuki Kanazawa, M.D.,Ph.D.1, Hiroshi Mizuno, M.D.,Ph.D.1, Rica Tanaka, M.D.,Ph.D.1.
1Juntendo University School of Medicine, Tokyo, Japan, 2Japan Aerospace Exploration Agency, Ibaraki, Japan.

PURPOSE: We recently developed a serum-free, ex vivo cell expansion system called Mononuclear Cell Quality and Quantity Control Culture System (MNC-QQc), using peripheral blood mononuclear cells (MNCs), to increase the number and the vasculogenic property of endothelial progenitor cells (EPCs) for enhanced vasculogenesis and tissue regeneration from a small amount of peripheral blood. Recently, the effect of microgravity (MG) was reported to enhance the potential of various stem cells. Therefore, we investigated whether combination of MG will enhance the function of our culture system. The purpose of this study was to evaluate the efficacy of MG on MNC-QQc to increase the number and function of peripheral blood EPCs.
METHODS: MG culture condition was established using disposable cell container (DCC) on 3D-clinostat (Mitsubishi Heavy Industries, Ltd., Tokyo, Japan). MNCs were isolated from peripheral blood in healthy volunteers (n = 8). Cells were cultured in MNC-QQc under four different conditions: (1) normal MNC-QQc (Normal Control; NC), (2) earth gravity during 7 days in DCC (Earth Gravity; EG), (3) MG during 7 days in DCC (MG), and (4) MG for 3 days followed by EG for 4 days in DCC (Microgravity and Earth Gravity; ME). After 7 days of MNC-QQc, total cell number and percentage of CD34-positive cells were measured by FACS analysis, as an indicator of EPCs. The vascular regeneration ability of MNC-QQc cells was evaluated by identifying definitive EPC colony-forming units (dEPC-CFU) and primitive EPC CFU (pEPC-CFU) in colony forming assays (EPC-CFA).
RESULTS: While none of the culture conditions changed the total number of cells, the MG and ME groups showed significantly higher number of CD34-positive cells than the NC group [MG vs NC (4.37 ± 2.65 vs 1.32 ± 0.31, p <0.05) and ME vs NC (4.74 ± 2.96 vs 1.32 ± 0.31, p <0.05)]; there was no significant difference between the EG and NC groups (3.76 ± 2.78 vs 1.32 ± 0.31). The total EPC-CFU number did not differ among the four groups (NC: 944.4 ± 646.9, EG: 1043.0 ± 710.2, MG: 1084.7 ± 645.6, ME:1487.4 ± 528.3). However, the dEPC-CFU number, which demonstrates the differentiation potential of EPCs, was significantly increased in ME compared to that in NC (1329.1 ± 573.4 vs 688.3 ± 513.0, p <0.05) and other groups.
CONCLUSION: MNCs that underwent QQ culture under MG give rise to higher CD34-positive fraction compared to those in EG culture. In addition, higher EPC vasculogenic function was seen in ME. This group especially showed increased differentiation potential of EPCs, as revealed by increase in dEPC-CFU. The results indicated that MNC-QQc in combination with MG-EG conditions may be a more effective EPC expansion culture method and is a potentially valuable tool for therapeutic vasculogenesis and tissue regeneration.


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