一种简易的单细胞图案化微流控芯片方法与条件优化  

An Optimized Confined Single-Cell Patterning in Simple Reversible Microfluidic Chip

作　　者：黄柯 陈志强 孙园迪 董媛 孟桂先 HUANG Ke;CHEN Zhiqiang;SUN Yuandi;DONG Yuan;MENG Guixian(Jilin Medical University,Department of Laboratory Medicine,Jilin Province 132013,China;Jilin Medical University,Department of Clinical Medical School,Jilin City,Jilin Province 132013,China)

机构地区：[1]吉林医药学院检验学院,吉林吉林132013 [2]吉林医药学院临床医学院,吉林吉林132013

出　　处：《吉林医药学院学报》2024年第3期188-193,共6页Journal of Jilin Medical University

基　　金：吉林省科技厅自然科学基金面上项目(YDZJ202201ZYTS193);吉林省大学生创新创业训练项目(S202113706035)。

摘　　要：目的 解决可控流体刺激下单细胞水平图案化的精准控制问题。方法 结合微接触印刷图案化方法与微流控芯片技术,优化一种简易可拆卸芯片中单细胞图案化策略。首先利用单因素法优化蛋白图案化与细胞图案化步骤,分别改变离子体处理时间、FN包被时间、清洗次数、印章接触时间、F127浓度、F127处理时间、细胞接种时间和细胞密度等,最终得到单细胞图案化最佳参数。然后利用3D打印与真空负压封装,实现微流控芯片可拆可密封,保证图案化细胞与流体剪切力二者都精准可控。结果 根据单因素法条件配比,得到蛋白图案化最佳参数:等离子体氧处理1 min、FN包被30 min、印章接触时间6~24 h、清洗2次。细胞图案化最佳参数:F127浓度2%,处理时间1 h,细胞密度2×10~6mL^(-1),细胞接种时间为15~30 min,单细胞图案有效维持时间6~8 h。基于3D打印模具与真空负压封装的微流控芯片可同时提供两种强度不同的流体剪切力。结论 优化后的单细胞图案化微流控芯片方法为图案化驱动的多细胞体系与力学环境耦合机制提供较为理想的实验平台。ObjectiveTo obtain the confined single-cell patterned multiple cellular arrays exposed to exactly controllable fluidic shear stress stimuli.Methods Based on micro-contact printing(μCP),3D printed mold and vacuum negative bonding,a simple microfluidic chip with single-cell patterned osteoblasts was developed.Meanwhile,μCP-controled cell micro-patterns were optimized and determined exactly,from two main aspects of protein patterning and cell patterning,with altering related key parameters in terms of plasma treatment time,FN coating time,cleaning times,printing time,F127 concentration,F127 treatment time,cell seeding time and cell density.Results The optimized parameters for good FN micro-patterns were plasma oxygen treatment for 1 minute,FN coating for 30 minutes,stamp contact time of 6-24 hours,and 2 washes.The optimal parameters for confined micro-patterned cellular arrays were 2%F127,F127 incubation for 1 hour,cell density of 2×10~6mL~(-1),and cell seeding for 15-30 minutes.The confined ostoeblastic patterns can maintain for 6-8 hours.The microfluidic chip based on 3D printed molds and vacuum sealing could provide two different fluid shear forces simultaneously.Conclusion This new and developed method provides a promising experimental platform for studying the reaction between patterned multicellular system and mechanical microenvironment.

关 键 词：单细胞图案化 微流控芯片 微接触印刷 力学刺激 钙信号 3D打印 

分 类 号：Q6[生物学—生物物理学]