(G-280) In-Vitro Study of Epithelial Cell Tight Junction Responses from an Acid and Atelectrauma Lung Injury Model

Introduction:: Acute respiratory distress syndrome (ARDS) is a common life-threatening medical condition caused by underlying ailments such as COVID-19, pneumonia, or inspiration of vomit. Damaged tight junctions (TJ) of endothelial and epithelial cell layers leak blood plasma proteins into the airway, deactivating surfactant, flooding the airspaces and increasing the surface tension. The resulting increase in pulmonary elastance makes normal breathing impossible, so mechanical ventilation is required for life support. However, long-term ventilation can cause progressive failure that induces ventilator-induced lung injury (VILI), leading to a high mortality rate of ~40%. We hypothesize that the repetitive stress caused by the air-liquid interface propagating across the epithelium during recruitment/derecruitment (RD) events causes atelectrauma, with the goal of clarifying the mechanisms and risks of atelectrauma through in-vitro cell-biological experimental data. This study will establish culturing conditions for H441 cell monolayers and the formation of tight junctions within the microchannel. Additionally, it will analyze how chemical and physical stressors impact cell survival rate and tight junction integrity within the model system.


Materials and Methods:: Physiologically consistent microchannels (50 mm x 5 mm x 0.6 mm) custom flow channels and ECIS Flow Arrays (1F8x10E-PC, Applied Biophysics, Troy, NY, USA) were used with an actuator pump to simulate airway reopening and closing. An H441 human epithelial lung cell monolayer (ATCC® HTB-174™) is then cultured and monitored in the flow-chamber where weak HCl solutions and RD events were introduced to simulate ARDS and atelectrauma. Immunofluorescence imaging on cell-cell tight junction proteins, live/dead assay, and Electric Cell-Substrate Impedance Sensing (ECIS) were used to investigate the integrity of the monolayer.

NCI-H441 epithelial lung cells were cultured and incubated in RPMI Medium 1640 (1X) with 10% of fetal bovine serum (FBS) within After incubation, the cell culture medium in a microchannel was replaced by a solution of pH4 HCl solutions to induce ARDS-like damage. A computer-controlled actuator pump was used to pass a bubble through the flow chamber to achieve consistent RD counts, mimicking the effects of mechanical ventilation. The ECIS ZӨ (Applied Biophysics, Troy, NY, USA) used impedance to monitor the time-course of cell growth, maturation, damage, and recovery. This powerful non-harmful method of measuring cell morphology allowed for the analysis of different culturing and damaging conditions. Immunofluorescence ZO-1 protein staining visualizes the status of tight junctions. Live/Dead assays using DAPI (4’,6-diamidino-2-phenylindole) for live cells, and propidium iodide (PI) for dead cells provides evidence of the monolayer viability. Western Blot analysis will be performed in the future to identify surfactant protein B (SPB) that signifies tight junction maturity.


Results, Conclusions, and Discussions:: Results and Discussion: Figure 1 demonstrates tight junction behavior after A) 3-days of culture and B) after the addition of 60 min of pH4 HCl exposure with 50 Rec/Der events. The three-day incubation showed prominent signs of tight junction formation as high intensity and sharp ZO-1 barrier function. In contrast, When the layer was exposed to pH 4 HCl and Rec/Der, immunofluorescent images of ZO-1 showed very few tight junctions present. ECIS measurements quantitatively confirm this visualization behavior.

Conclusions: This research study on H441 epithelial lung cells can be used to expand our knowledge cellular responses to mechanical ventilation techniques. The ECIS data quantify our visualization observations. Our in-vitro ARDS model demonstrates that the durability of H441 epithelial cell monolayers depend on the strength of tight junctions and the repetitive reopening and closing of airways break down those tight junctions. Further experimentation to study culture conditions and monolayer health will utilize Western Blots to detect SPB protein release as a marker of tight-junction maturity and adjustments to flow rates within the microchannel to see its effects on cell growth and polarity.


Acknowledgements (Optional): : NIH R01 HL0142702

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