Immunocompetent Alveolus-on-Chip Model for Studying Alveolar Mucosal Immune Responses

We developed physiologically relevant in vitro models to answer mechanistic question in human infections. Our focus is on the host human response where we use this model to dissect the complex interaction of the pathogen to the host to identify molecular and cellular targets for therapeutic options in human infections. Organ-on-a-chip model like lung-on-chip model balance biological complexity with specific research needs, offering insights into the human host response regulation.

This system mimic in vivo cellular composition and 3D structure, providing more defined conditions and high throughput than the animal experiments. In lung-on-chip technology, one of the primary challenges is selecting the right cell types that mimics lungs'complex functionality for infection study results. Moreover, extending the timeframe of the experiment beyond traditional in vitro models is crucial.

Leveraging is the chip ability to maintain cell viability with continuous nutrient flow. For longer observation, the experiment is also necessary. We developed the micro fluidic based model of human alveolus as an effective tool to mimic human alveolar environment.

It was achieved by applying perfusion to mimic blood flow and also by a mechanical stimulation to the endothelial cells, and also integrating air-liquid interface for the epithelial cells by exposing them to the air. The next step is to extend this model to a more advanced platform such as induced pluripotent stem cell-based model. By doing so, we aim to bring personalized medicine closer to application, particularly in the context of antiviral drug testing and as a tool for biomedical research and pharmaceutical development.

To begin, place the Biochip model BBC002 in a glass Petri dish and flush all cavities with sterile 70%ethanol for 45 minutes. Then, flush cavities twice with sterile double distilled water. Using a 1000-microliter pipet with blue tips, coat the Biochip membrane of upper cavity with sterile collagen IV solution.

Incubate the chip for 30 minutes at 37 degrees Celsius and 5%carbon dioxide. After incubation, flush the lower and upper chambers twice with sterile PBS containing magnesium and calcium. Fill the upper chamber of each cavity with 250 microliters of endothelial cell or EC medium containing penicillin streptomycin and the lower chamber with 150 microliters.

Wash the confluent human umbilical vein endothelial cell culture in a T-25 flask with five milliliters of DPBS without magnesium and calcium PBS. Add one milliliter of 0.25 trypsin and one millimolar EDTA in PBS and incubate for three minutes at 37 degrees Celsius. After incubation, add five milliliters of PBS and 5%FCS to stop the trypsin activity.

In a 50-milliliter conical tube, centrifuge the suspension at 350 G for five minutes and resuspend the pellet in one milliliter of EC medium. Add 10 microliters of one to 10 diluted cell suspension to a micro centrifuge tube containing 10 microliters of Trypan blue stain. Count the cells manually with a Neubauer chamber.

After counting, replace the endothelial cell medium in the upper chamber of the Biochip with 200 microliters of EC medium containing 0.4 times 10 to the power of six human umbilical vein endothelial cell culture. Place the glass Petri dish with the Biochips at 37 degrees Celsius and 5%carbon dioxide. Perform daily medium exchange with 300 microliters of EC medium for the upper chamber and 200 microliters of RPMI+medium for the lower chamber.

After washing the monocytes with one milliliter of PBS, incubate them in prewarmed PBS containing lidocaine and EDTA at 37 degrees Celsius and 5%carbon dioxide for seven minutes. Collect and centrifuge the cells at 350 G for seven minutes. After resuspending in one milliliter of RPMI+count the cells as demonstrated previously.

Seed 0.1 times 10 to the power of six monocytes suspended in 200 microliters of RPMI+in the lower chamber. Place the Biochip ports facing downwards so the cells attach to the membrane. Before connecting the sterilized tubing to the Biochip, flush the tubing first with 200 microliters of PBS followed by 200 microliters of EC medium.

After flushing the upper and lower chamber with freshly prepared cell culture medium, insert the reservoir on the outlet side of the Biochip and connect the reservoir with the tubing to the inlet of the Biochip. For circular medium perfusion, connect the tubing to the reservoir and the Biochip. Now fill the reservoir with 500 microliters of EC medium.

Connect the Biochip with the tubing to start profusion at a flow rate of 21 microliters per minute. The next day, stop the profusion and empty the reservoirs. Under a sterile safety cabinet, pipette 500 microliters of EC medium into the reservoirs and gently flush the lower chamber with 200 microliters of RPMI+medium.

Then restart the perfusion at a flow rate of 21 microliters per minute. To establish an air-liquid interface from day 12 to day 14, open the plugs at the lower chamber and carefully soak up the medium until an air bubble is visible. After ensuring all liquid from the channel and chamber is removed, carefully close the ports.

fill the reservoir with freshly prepared vascular EC medium and continue profusion culture only in the upper chamber until day 14. Disconnect the Biochip from the flow system. After removing the tubing, examine the cell layers from the Biochip under a microscope for cell confluency.

Wash the Biochip cavities with PBS to remove the cell culture medium. Add 300 microliters of 4%paraformaldehyde solution in PBS to the upper chamber and 200 microliters to the lower chamber. After 10 minutes of incubation, wash the chamber thrice with PBS.

For permeabilization, add 300 microliters of 0.25%Triton X-100 in PBS and incubate for 30 minutes. Following washing with PBS, pipette 300 microliters of 3%bovine serum albumin in PBS in both chambers and incubate for one hour. For immunofluorescent staining, prepare 50 microliters of antibody solutions using 3%BSA for each membrane using appropriate dilution ratios.

To access the cells within the Biochip, make an exact cut across the outside of the cavity and remove the bonding foil. Using a scalpel, cut the membrane out by removing the edges sealed to the Biochip. After dividing the membrane into two pieces, collect the membrane pieces with a tweezer and place them on a microscopic slide for staining.

Add 50 microliters of antibody solution to each membrane piece and incubate overnight at four degrees Celsius, protecting from light. After washing the membrane thrice with PBS, add a drop of mounting medium on the labeled slide and place the corresponding membrane. Add a drop of the mounting medium on the top of the membrane and place a cover glass.

Immunofluorescence staining of human umbilical vein endothelial cells revealed morphological alterations and marker protein expression after 14 days of co-culture. On the vascular side, VE-cadherin expression at endothelial cell borders suggested confluency and endothelial barrier formation. The epithelial side was assessed for E-cadherin and surfactant protein A expression, indicating alveolar epithelial integrity and function.