Department of Biomedical Engineering, School of Science and Engineering, Tulane University;
Department of Biomedical Engineering, School of Science and Engineering, Tulane University.
J Vis Exp. 2022 Jul 1(185). doi: 10.3791/64148.
In vitro microfluidic experimentation holds great potential to reveal many insights into the microphysiological phenomena occurring in conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). However, studies in microfluidic channels with dimensions physiologically relevant to the terminal bronchioles of the human lung currently face several challenges, especially due to difficulties in establishing appropriate cell culture conditions, including media flow rates, within a given culture environment. The presented protocol describes an image-based approach to evaluate the structure of NCI-H441 human lung epithelial cells cultured in an oxygen-impermeable microfluidic channel with dimensions physiologically relevant to the terminal bronchioles of the human lung. Using phalloidin-based filamentous-actin staining, the cytoskeletal structures of the cells are revealed by confocal laser scanning microscopy, allowing for the visualization of individual as well as layered cells. Subsequent quantification determines whether the cell culture conditions being employed are producing uniform monolayers suitable for further experimentation. The protocol describes cell culture and layer evaluation methods in microfluidic channels and traditional fixed-well environments. This includes channel construction, cell culture and requisite conditions, fixation, permeabilization and staining, confocal microscopic imaging, image processing, and data analysis.
体外微流控实验在揭示急性呼吸窘迫综合征（ARDS）和呼吸机诱导性肺损伤（VILI）等情况下发生的微生理现象方面具有很大的潜力。然而，目前在与人类终末细支气管生理相关的微流控通道中进行的研究面临着几个挑战，特别是由于在给定的培养环境中难以建立适当的细胞培养条件，包括培养基流速。本方案描述了一种基于图像的方法，用于评估在与人类终末细支气管生理相关的尺寸的氧气不可渗透的微流控通道中培养的 NCI-H441 人肺上皮细胞的结构。使用鬼笔环肽基丝状肌动蛋白染色，通过共焦激光扫描显微镜揭示细胞的细胞骨架结构，允许对单个和分层细胞进行可视化。随后的定量确定所采用的细胞培养条件是否产生适合进一步实验的均匀单层。该方案描述了微流控通道和传统固定孔环境中的细胞培养和层评估方法。这包括通道构建、细胞培养和必要条件、固定、透化和染色、共焦显微镜成像、图像处理和数据分析。
