基于双层光刻技术制备神经血管单元三细胞共培养微流控芯片  

Fabrication of microfluidic chip for tri-culture of neurovascular unit using double-layered lithography template

作　　者：罗翰 舒伟良[2] 蔡川 陈艳[2] 褚晓凡 LUO Han;SHU Weiliang;CAI Chuan;CHEN Yan;CHU Xiaofan(Department of Neurology,Shenzhen Longhua District Central Hospital,Shenzhen 518110,China;Centre for Micro Nano Systems and Bionic Medicine,Shenzhen Institutes Of Advanced Technology Giinese Academy Of Sciences,Shenzhen 518055,China;Department of Neurology,The University of Hong Kong-Shenzhen Hospital,Shenahen 518053,China)

机构地区：[1]深圳市龙华区中心医院神经内科,广东深圳518110 [2]中国科学院深圳先进技术研究院,微纳系统与仿生医学研究中心,广东深圳518055 [3]香港大学深圳医院神经内科,广东深圳518053

出　　处：《暨南大学学报（自然科学与医学版）》2020年第6期560-568,共9页Journal of Jinan University(Natural Science & Medicine Edition)

摘　　要：目的:应用双层软光刻技术和微流控芯片技术,制备以星形胶质细胞为桥梁,神经元-星形胶质细胞-脑微血管内皮细胞在同一立体空间或平面内肩并肩生长的微流控芯片.方法:(1)设计一种贯通三通道神经血管单元的体外共培养微流控芯片模型.(2)基于双层光刻技术制备芯片模板及神经血管单元共培养微流控芯片.(3)测算不同通道进样流速比.(4)在芯片内培养神经血管单元3种要素细胞.结果:(1)芯片内3个主流道的长度均为1cm,高度均为45μm,中间主流道的宽度为570μm,两侧主流道的宽度均为500μm.设计流道间孔径的长度均为30μm,宽度和高度均为10μm,孔径的间距均为15μm.(2)甩两层光刻胶,第1层结构高度为10μm、第2层结构高度为35~40μm、总高度约为45μm的芯片模板进行后续试验.(3)A、B、C三通道进样流速比为5∶6∶5.(4)明场下神经元、星形胶质细胞和脑微血管内皮细胞在微流控芯片内以星形胶质细胞为桥梁背靠背生长.结论:本研究设计一种神经血管单元三细胞共培养微流控芯片,使3种要素细胞以星形胶质细胞为桥梁在同一平面肩并肩生长,能更好地模拟体内细胞所处的微环境,有助于细胞间相互作用、信号传导和信息交流的研究.Objective:To utilize soft lithography and microfluidic technologies to prepare a chip that can enable the growth of neurons-astrocytes-cerebral microvascular endothelial cells within one stereoscopic space or parallelly on the plane,using astrocytes as the bridge.Methods:(1)We designed a kind of 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(2)Double layered lithography technology was used to prepare the chip template as well as tri-culture microfluidic chip for the neurovascular unit.(3)The flow rate ratio of liquid of different channels was calculated.(4)Three component cells of the neurovascular unit were cultured within the chip.Results:(1)The length and height of the three channels within the chip were 1 cm and 45μm,respectively.The width of the channel in the center was 570μm and width of the channels on the two sides was 500μm.The length of the aperture between the channels was 30μm,the width and height were 10μm and the aperture spacing was 15μm.(2)A double-layered photoresist was prepared with height of the first and second structure being 10μm and 35-40μm,respectively.A surface profiler was used to measure the height of the template and chip template,and a total height of 45μm was selected for the follow-up test.(3)The flow rate ratio of sample of A,B,and C channels was 5:6:5.(4)The back-to-back growth of neurons,astrocytes,and bEnd.3 in the microfluidic chip using astrocytes as a bridge,was observed in the bright field.Conclusion:We successfully designed a tri-culture microfluidic chip for the neurovascular unit.The new microfluidic chip can simulate parallel growth of three component cells within one plane using astrocytes as the bridge,which can better simulate the microenvironment of the in vivo cell,and is conducive for the study of interactions among cells,signal transduction,and information exchange.

关 键 词：软光刻 微流控芯片 神经血管单元 三细胞共培养 

分 类 号：R318.08[医药卫生—生物医学工程]