三维共培养对髓核细胞与骨髓间充质干细胞增殖的影响

Effects of 3D coculture system on the proliferation of nucleus pulposus cells and bone marrow mesenchymal stem cells

目的建立椎间盘髓核细胞(NPCs)和骨髓间充质干细胞(BMSCs)体外聚乳酸-乙醇酸共聚物(PLGA)支架三维共培养系统。方法分别培养乳兔NPCs和BMSCs至原代融合和第3代,将两种细胞按2∶1(BMSCs∶NPCs)比例混匀,以6×106/ml的终浓度分别接种于培养板作为平面共培养组(A组)和PLGA支架(直径10mm,厚3mm)作为三维共培养组(B组);每组各接种6例标本,每例接种细胞悬液100μl,体外培养2周,利用倒置显微镜、扫描电镜进行细胞形态学观察;MTT法测定各组细胞增殖情况。结果细胞与材料支架黏附良好。经过2周的培养,A组和B组细胞均能够增殖;与A组相比,B组细胞增殖率增高(P<0.01)。结论共培养的NPCs和BMSCs均能够与PLGA支架很好地黏附并增殖;PLGA支架三维共培养NPCs和BMSCs较平面共培养更能促进细胞增殖。

Objective To establish a 3D coculture system of nucleus pulposus cells（NPCs） and bone marrow mesenchymal stem cells（BMSCs） in polylactic-co-glycolic acid（PLGA） scaffold in vitro for intervertebral disc tissue engineering.Methods Rabbit NPCs and BMSCs were in vitro expanded,respectively,to the primary NPCs fusion and the third generation of BMSCs.The cells mixed at a ratio of 2∶1（BMSCs∶NPCs）were assigned into two groups of A（monolayer coculture） and B（3D coculture）.The mixed cells 100 μl（6×106/ml） were seeded into a 6-well culture plate in group A or seeded into a PLGA scaffold（10 mm in diameter and 3 mm in thickness） in group B with 6 specimens each.All specimens were cultured in vitro for 2 weeks.The morphological changes were observed by inverted microscope and scanning electron microscope.The cell proliferation was tested by MTT.Results The cells of two groups had fine adhesion to the scaffolds and the proliferation of cells was observed after two weeks in both groups.Compared with group A,the rate of cell proliferation was increased in group B（P0.01）.Conclusion Both of rabbit NPCs and BMSCs can adhere to PLGA scaffolds and proliferate.Compared with monolayer coculture,3D coculture system is better in promoting the cell proliferation.