Preparation and Structural Evaluation of Epithelial Cell Monolayers in a Physiologically Sized Microfluidic Culture Device

Abstract

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.

Figure 1:. Exploded-view schematic of the microfluidic channel construction.

The top element is the top portion of the flow array, thin grey elements are adhesive strips, thin blue elements are mylar spacers, and the bottom element is the rectangular coverglass.

Figure 2:. Five imaging locations along the consistently layer-producing region of the microfluidic culture channel.

Imaging locations are as follows: inlet-side, near where the first electrode would be on the intact flow array; halfway between inlet-side location and the center of the channel; center of the channel; halfway between the center and the outlet-side location, and outlet-side, near where the last electrode would be on the intact flow array.

Figure 3:

Diagram of the 8-well chambered coverglass used for the fixed-well culture, staining, and imaging experiment comparing the effects of initial cell seeding density and culture duration on the formation of cell layers.

Figure 4:. NCI-H441 cell layers cultured in the microfluidic channel for 24 and 48 h and imaged at the inlet-side and outlet-side locations as depicted in Figure 2.

(A) 24 h, inlet-side. (B) 24 h, outlet-side. (C) 48 h, inlet-side. (D) 48 h, outlet-side. Blue represents the nuclei staining and green represents the staining of filamentous-actin.

Figure 5:. Graphical representation of the data collected during the 24–48 h microfluidic channel imaging experiment.

This includes 6 cross-sectional area samples from each of 5 locations along the relevant length of the microfluidic channel (as depicted in Figure 2). Error bars represent standard deviations.

Figure 6:. 2D image taken in the XY plane in a central location within the microfluidic channel.

Blue represents the nuclei staining and green represents the staining of filamentous-actin.

Figure 7:. 3D model of the same microfluidic channel location as Figure 6.

The color-coding depicts depth data.

Figure 8:. NCI-H441 cell layers cultured in the 8-well chambered coverglass.

For 24 h (A), 48 h (B), and 96 h (C) h at initial seeding densities of 180,000 (X); 90,000 (Y); and 45,000 (Z) cells/cm². Blue represents the nuclei staining and green represents the staining of filamentous-actin.

Figure 9:. Graphical representation of the data collected during the density-duration 8-well culture experiment.

This includes 6 cross-sectional area samples from each density-duration match-up. Error bars represent standard deviations.