以微流控芯片为载体的神经血管单元三细胞共培养模型的构建与鉴定

Establishing and identification of neurovascular unit in a tri-culture model using microfluidic chip

目的在微流控芯片中构建神经元-星形胶质细胞-脑微血管内皮细胞直接接触生长的体外共培养模型。方法(1)确定芯片内神经血管单元体外三细胞共培养模型的接种细胞密度、接种时间和接种顺序;(2)采用重复测量方差分析分别比较神经元、星形胶质细胞、脑微血管内皮细胞共培养和单独培养的细胞活力有无差异。结果(1)新型神经血管单元模型体外共培养时神经元、星形胶质细胞、脑微血管内皮细胞的接种密度分别为5×10^(6)~10×10^(6)个/ml、3×10^(5)~8×10^(5)个/ml、1×10^(5)~5×10^(5)个/ml;接种顺序依次为神经元、星形胶质细胞、脑微血管内皮细胞;接种时间为先前接种的细胞进入指数增长期后接种另一种细胞。(2)神经元、星形胶质细胞和脑微血管内皮细胞各自的细胞活力在所构建的模型中共培养和各自单种培养进行比较,细胞活力均无统计学差异。结论本研究以微流控芯片为载体实现了神经元、星形胶质细胞和脑微血管内皮细胞的共培养,在此共培养模型中3种要素细胞拥有较高的细胞活力。

Objective We established a model by using microfluidic chip that is a horizontal side by side diffusion system through microporous separating three adjacent channels in a triple-culture using neurons,astrocytes,and bEnd.3.Methods(1)In the in vitro microphysiological system of neurovascular unit,we identified the seed density,seed time,seed sequence and seed method.(2)We adopt the repeated measures data of ANOVA to see whether there is significant difference in the viability when the three cell types are tri-cultured and separately mono-cultured.Results(1)In the novel in vitro NVU model,the seed density of the neurons,astrocytes and bEnd.3 is 5×10^(6)~10×10^(6)/ml,3×10^(5)~8×10^(5)个/ml and 1×10^(5)~5×10^(5)/ml.The seed sequence is from neurons to astrocytes to bEnd.3.The seed time is that after the previous seeded cells enter the exponential phase,the next cells will be seeded.(2)We adopt the repeated measures data of ANOVA to see whether there is significant difference in the viability when the three cell types are tri-cultured and separately mono-cultured.Conclusion In this study,neurons,astrocytes and bEnd.3 were tri-cultured by using microfluidic chip.In this tri-culture model,these three kinds of cells have high cell viability.