Culture And Expansion Of Mesenchymal Stem Cells In Air-lift Loop Hollow Fiber Membrane Bioreactor

Mesenchymal stem cells (MSCs), having the multi-differentiation potentials of becomingosteoblasts, chondrocytes, adipocytes, cadiocytes and so on, are the promising "seed cells" intissue engineering and ideal target cells in gene therapy for their low implanting response,being easy to be transfected and expressed steadily and function of supporting hematopoiesis.However, MSCs are in limited quantity and only 1/20000～1/30000 of karyocytes in bonemarrow and what's more, the number decreases and viability loses with individual ageincreasing, which can not meet the need of autologous repairation and clinical therapy. Withthe present culture methods, MSCs are in limited expansion fold and lose stem cell characterseasily because of the culture environment far away from the in vivo conditions.In order to solve the above problems, here, an air-lift loop hollow fiber membranebioreactor was developed and used to culture and expand MSCs in vitro. MSCs of passage 4(P4) mixed with typeâ… collagen with bFGF were innoculated into hollow fiber membrane(HFM), where the gel with MSCs formed after an hour in an incubator at 37℃, 5% CO₂ andsaturated humidity. Then the gel with MSCs inside HFM was innoculated into the air-lift loopbioreactor (ALB) to culture and expand MSCs in three dimensions in DMEM supplementedwith bFGF and 15% FBS. During the experiments, the OD value of medium was detectedevery 24 h to calculate the cell number and to obtain the cell growth curves, and the metabolicparameters were also detected. Moreover, the expanded cells were identified with flowcytometer and through inducing them to multi-differentiate.The results showed that in ALHFMB, O₂ concentration kept almost constant; MSCsmetabolized robustly and expanded about 16- and 37-fold within 7 days with the inoculateddensities of 5×10⁵ and 1×10⁵ cells/mL in collagen, respectively, which was obviously morethan that in T-flask about 6- and 12-fold, respectively; the most of the expanded cells wereCD29 and CD44 positive and CD45 negative; after being induced, the expanded cells stillreserved the strong muti-differentiation potential. Therefore, it's safe and effective for MSCsculture and expansion in vitro with the method used here.