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

This protocol serves to lower a barrier to entry for cell culture experimentation and enables researchers to evaluate and characterize adherent cell monolayers cultured in dynamic microfluidic environments. The primary advantage of this technique is that it enables both qualitative and quantitative evaluation of cell monolayer characteristics to serve as a basis for further monolayer-dependent experimentation. This method enables the recapitulation of an alveolar epithelium in vitro, permitting exploration of the dynamic responses during acute respiratory distress syndrome, ARDS, as well as ventilator-induced lung injury, VILI.

To begin, obtain a single channel flow array. Clean the cover glass surface in an ultrasonic bath. Immerse the cover glass in poly-d-lysine at room temperature for five minutes.

Then dry it at 60 degrees Celsius for 30 minutes. Next, affix double-sided adhesives in Mylar spacers. Laser cut as described in the manuscript to the cover glass and ensure that the channel cutouts are precisely aligned.

Affix a rectangular cover glass to the bottommost adhesive strip. Use the peeled adhesive papers to cover the portions of adhesive still exposed. After assembly is complete, apply firm and equal pressure to the construction.

Using a syringe filled with deionized water, rinse the channel. Sterilize the channel enclosure in an ultraviolet sterilizer for 30 minutes. Using a sterile technique, treat the channel with human fibronectin solution prepared in PBS and incubate for at least 30 minutes at 37 degrees Celsius.

In the sterile laminar flow hood, prepare NCI H441 cell suspension using RPMI 1640 medium with 10%FBS. Use 0.25 milliliters of this cell suspension to fill the channel and a portion of the ports. Using brightfield microscopy, verify that the cells have been distributed evenly within the channels.

Add the media reservoir and stopcock to the channel. Begin culturing the channel at 37 degrees Celsius with 5%carbon dioxide. Use a programmable syringe pump to draw spent media out from the channel and fresh media into the channel from a sterile media reservoir attached to the channel inlet.

In a chemical fume hood, dilute a 4%formaldehyde solution into DPBS to create a 1%solution and store in an appropriately labeled tube. Prepare the 2%formaldehyde solution in a similar fashion. Transfer the formaldehyde solutions to appropriately labeled five milliliter syringes.

Draw up 20 milliliters of DPBS into a separate 20 milliliter syringe which will be used for the washing steps. Next, remove the microfluidic channel from the culture apparatus and secure it in the chemical fume hood. To assemble the fixation and staining apparatus, attach a segment of transfer tubing to the side port of a three-way stopcock via male Luer lock to hose barb adapter.

Then connect the stopcock to the inlet port of the flow array. Attach another segment of transfer tubing to the outlet port of the existing stopcock using the same type of hose barb adapter. Finally, secure the free ends of both transfer tubes now designated as waste lines into a chemical and biohazard appropriate waste container.

Ensure the stopcock is blocking off the flow array inlet port and flush the waste line with DPBS. Then turn the stopcock to block off the waste line and slowly wash the cells with two milliliters of DPBS. Slowly push two milliliters of 1%fixative through the channel.

Let it sit for five minutes and repeat with 2%fixative. Then wash the cells three times with fresh DPBS as demonstrated previously. Prepare the 0.1%saponin solution as described in the manuscript.

Draw up additional DPBS into a 20 milliliter syringe for use in the washing steps. Cover the tube with aluminum foil. Add the filamentous actin staining phalloidin reagent and a nucleus staining Hoechst reagent to the 0.1%saponin solution.

Then transfer the solution to a foil covered syringe. Flush the waste line with a small amount of permeabilizing and staining solution. Then introduce two milliliters of the solution to the microfluidic channel.

Cover the channel with aluminum foil to block the light. After 30 minutes, flush out the permeabilizing solution with two milliliters of DPBS twice for five minutes each before gently drying the channel. Using a micropipette, introduce a minimal amount of a soft set anti-fade mountant to the microfluidic channel ensuring to cover the bottom surface completely.

Then seal the end to the channel. Using a brightfield microscope, quickly verify the integrity of the cell layer before storing the channel in a container to protect it from light. Test imaging locations by taking reference scans and z-stacks until desired image parameters and conditions have been met.

Using the flow array base plate as a reference, construct z-stacks at various locations along the length of the channel. Finally, analyze cross-sectional data, depth map data, and any other relevant characteristics to evaluate cell layer properties. The successful use of the technique is demonstrated in the microfluidic dynamic culture environment through image acquisition at least one centimeter away from the inlet and the outlet.

In a representative culture duration experiment, successful monolayer production was observed when the cells were cultured for 24 hours. However, when cultured for 48 hours, undesirable multilayer formation was seen. The data collected from each of the five microfluidic channel imaging locations also reveals the relationship between increased culture duration and increased cross-sectional area, suggesting uneven layer formation or overgrowth occurs within 48 hours of culture.

The depth map of a central location within the microfluidic channel cultured for 24 hours was obtained, which is useful for the qualitative evaluation of layer characteristics. This protocol's three most crucial aspects are proper channel construction, accurate culture and media flow conditions, and careful use of the fixation and staining apparatus. Following this procedure, other methods may be used in parallel to validate the experimental viability of the produced cell monolayers such as electric cell-substrate impedance sensing, ECIS, or transepithelial electrical resistance, TEER.