Objective To investigate whether co culture of mouse corneal limbal stem cells (LSCs) with fibrin and mouse embryonic fibroblasts(MEFs)can improve viability of LSCs and help maintain their phenotypes in a 3D cell culture model. Methods Cells were cultured in 96 well bottom plates,and assigned into three groups: ① LSCs group(group A) , which was com posed of 3×104 LSCs;②LSCs and fibrin co culture group(group B) :LSCs were resuspended in 10 mg/mL fibrin solution at a concentration of 3× 105/mL,and 100μL of the mixture was aspirated to one well of 96 well bottom plate; ③LSCs,fibrin and MEF co culture group(group C) :LSCs and MEFs were cultured at the ratio of 1 :0. 5. Cell mixture was resuspended in 10 mg/ mL fibrin solution at a concentration of 4. 5 ×105/mL,and 100 /μL of the mixture was aspirated to one well of 96 well bottom plate. After being cultured for 4,24 and 48 h,LCSs were stained with Annexin V ,and cell viability and apoptosis were evalua ted by using flow cytometry. Western blot was used to detect the expression of LSC differentiation marker:Keratin K3/K76. Results LSCs in group C had highest viability rate after being cultured for 4,24 and 48 h [(95.7±2. 0) % ,(95. 1 ± 1. 2)% ,and (92. 0±1. 7)%, respectively],followed by group B [(95. 2±3. 0)%,(89. 8±1. 5 ) % ,and(80. 0±0. 8) %, respectively] , and group A [(95. 0 ± 1. 2)%, (84. 5 ± 1. 2)%,and(70. 0 ± 0. 9) %, respectively]. The apoptosis rate had a reverse tendency com pared to the viability. There were no statistically significant differences in the Keratin K3/K76 expression at 4 h among groups, but at 48 h,the expression of Keratin K3/K76 in group C(70+4)was significantly lower than in group B(97 + 6)and group A (102 + 4). Conclusion Co culture of LSCs with fibrin and MEF improved LSCs' viability,decreased their differentiation and helped maintain their phenotypes in a 3D culture model.%目的 探讨纤维蛋白凝胶、鼠胚胎成纤维细胞与鼠角膜缘干细胞的3D共培养模型是否有助于角膜缘干细胞的生存率提高和细胞表型的维持.方法 细胞采用96孔圆底培养板培养,并分为3组:①鼠角膜缘干细胞单独培养组(A组),含3×104个角膜缘干细胞;(2)鼠角膜缘干细胞与纤维蛋白共培养组(B组),将纤维蛋白原配成10 mg/mL的磷酸盐溶液,与鼠角膜缘干细胞混合成3×105/mL的混合悬液,取100 μL 细胞悬液培养;③鼠角膜缘干细胞、纤维蛋白和鼠胚胎成纤维细胞共培养组(C组),将鼠角膜缘干细胞与鼠胚胎成纤维细胞以1:0.5混合,配成含有10 mg/mL纤维蛋白原的4.5×105/mL混合悬液,取100 μL 进行培养.于培养后4、24和48 h,以Annexin Ⅴ染色,采用流式细胞仪检测其细胞生存率和凋亡率.以Western blot检测角膜缘干细胞分化蛋白Keratin K3/K76的表达.结果 角膜缘干细胞在4、24 和48 h的生存率最高者为C组,分别是(95.7±2.0)%、(95.1±1.2)% 和(92.0±1.7)%;其次为B组,分别是(95.2±3.0)%、(89.8±1.5)% 和(80.0±0.8)%;最低为A组,分别是(95.0±1.2)%,(84.5±1.2)% 和(70.0±0.9)%.细胞凋亡率的趋势与此相反.角膜干细胞分化蛋白的表达强度,在4 h时各组之间的表达强度无统计学差异,但在48 h时,C组的表达强度(70±4)明显低于B组(97±6)和A组(102±4).结论 鼠角膜缘干细胞与纤维蛋白和鼠胚胎成纤维细胞的共同培养模式有助于提高角膜缘干细胞在3D培养中的存活率,可减少鼠角膜缘干细胞的分化和有益于其表型的维持.
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