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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session C

Poster I11 - Optimizing Lung Epithelial Cell Culture in a Microfluidic Respiratory Airway Model

Friday, October 25, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Mechanical ventilation is a life-saving treatment for patients with acute respiratory distress syndrome (ARDS). However, barrier function disruption inherent in ARDS can deactivate lung surfactant and increase the surface tension of the lining fluid. This leads to highly non-uniform inflation and deflation of sensitive alveoli. While ventilation is essential, it can induce repetitive recruitment and derecruitment (RD) of the alveoli as the air-liquid interface propagates through airways and separates epithelial surfaces held together by surface tension. Consequently, prolonged mechanical ventilation can result in ventilator-induced lung injury (VILI) in already damaged lungs, contributing to a mortality rate of nearly 40%. We hypothesize that the repetitive stress caused by the air-liquid interface during RD events leads to atelectrauma. Our goal is to elucidate the mechanisms and risks of atelectrauma through in vitro cell-biological experimental data.
This study establishes culturing conditions for alveolar epithelial cell monolayers (NCI-H441) and the formation of tight junctions within the non-air permeable microchannel designed to work with Electric Cell-substate Impedance Sensing (ECIS) for time-course damage monitoring. We will investigate the optimal culture conditions in this system using alveolar epithelial cells (NCI-H441). This analysis will be conducted using immunofluorescent imaging of Zonula occludens-1 (ZO-1), since tight junctions are critical indicators of ideal epithelial layer formation.

Materials and

Methods:  Custom microfluidic flow channels for RD damage and Electric Cell-substrate Impedance Sensing (ECIS) time course monitoring having dimensions (50 mm x 5.0 mm x 0.6 mm) were used to culture NCI-H441 cells. The flow rate of the growth media is important because the channel is non-air permeable. We have tested various flow rates with constant (Q = 10 μL/min) and pulse flow (On: Q = 100 μL/min for 2 minutes, Off: for 60 minutes) patterns. Different culturing conditions were tested with incubation times of 3, 4, and 7 days at cell densities of 75k, 90k, 120k, and 150k cells/cm2 with dexamethasone (DXM) concentrations of 0μM, 200μM, 1000μM, and 2000μM.
NCI-H441 (American Type Culture Collection) cells were seeded into flow and ECIS channels and cultured with RPMI Medium 1640 (1X) with 10% FBS and 1% Anti-Anti (A/A) for an initial 24-hour incubation. After 24 hours, the media was replaced with Advanced RMPI 1640 with 5% FBS, 1% A/A, 1% GlutaMax, and DXM for the remaining time.
After incubation, immunofluorescence imaging of tight junction protein ZO-1 (Alexa FluorTM 488, Invitrogen) was performed on the cell layer in the flow channels to visualize the tight junctions through confocal microscopy. ImageJ (National Institutes for Health) was used to perform quantitative analysis based on the number of pixels near peak fluorescent intensity by comparing the total amount (6 images) of fully formed inter-cellular tight junctions in each image, since fully formed tight junctions may be correlated with the highest local density of ZO-1 protein.

Results, Conclusions, and Discussions: The representative fluorescent confocal image of a successful NCI-H441 layer is shown in Figure 1. Currently, we have maintained the most stable with the pulse feeding flow cycle. The most uniform monolayer was formed using the following conditions: 75k cells/cm2 density and 200 μM DXM concentration for a 4-day incubation period. The quantitative analysis in Figure 2 shows that the highest amount of tight junctions was formed in images of the 7-day incubation, even though our qualitative observation indicates the monolayer uniformity declines after the 4-day incubation.
The visual image analysis indicates that we have achieved the formation of a uniform epithelial layer in the non-air permeable microfluidic flow channel. Although the 7-day incubation period has the highest amount of fully formed tight junctions, the NCI-H441 cell layer formed after a 4-day incubation period using pulse flow with 75k cells/cm2 seeding density and 200 μM DXM has the preferable uniformity of cell monolayer with no stacking. These conditions provide sufficient consistency to be a subject for the RD damage and recovery tests with the ECIS time-course observation to investigate atelectrauma. In further experimentation, we will focus on continuing to investigate the relationship between layer uniformity and the incubation length beyond 7 days, and the relationship between immunofluorescence image quantification and barrier-function analysis using ECIS.

Acknowledgements (Optional):  NIH R01 HL0142702