Revealing Electromechanical Control of Tissue Homeostasis Using a Two-Layer Microfluidic Device

We apply controlled electric fields to study how tissues respond to electrical cues, focusing on how currents or fields regulate cell adhesion, proliferation, and extrusion, impacting tissue homeostasis. We discovered that the direction of physiologically relevant currents dictates tissue states. Apical to basal field induced a proliferative phenotype, while basal to apical field promotes cell extrusion, allowing modulation of overall cell density.

While quantitative bioelectric studies have long focused on migration via gravitaxis, tissue homeostasis remain inaccessible. Our system now enables direct investigation of how physiological cartons regulate tissue organization and maintenance. Our protocol integrates microfluidics, electrical stimulation of tissues, structure and force microscopy, and live imaging to study electromechanic control of tissue homeostasis.

Our work revealed core electromechanical coupling rules. With new tools, we can now explore how native bioelectric patterns emerge and regulate tissues and if we can control complex tissue behavior by tuning these electromechanical cues. To begin, place a clean layer one polydimethylsiloxane, or PDMS mold, face up on a clean plastic dish cap and add 500 microliters of liquid ultraviolet curable adhesive slowly on the mold.

Using a spatula, carefully wet all the protruding structures and remove any air bubbles formed during the wetting process. Gently press a cover slip onto the PDMS mold and use a laboratory wiper to remove any excess ultraviolet curable adhesive, ensuring the top side of the cover slip remains clean and flat. Cure the structure partially under uniformly illuminated ultraviolet light with a wavelength between 363 and 370 nanometers.

Wipe off any marks using 75%ethanol. Then, hold the structure firmly on a flat surface and slowly peel the PDMS mold from the cured layer one. Then, place a clean layer two PDMS mold face down on a flat PDMS slab that is thicker than 2.5 millimeters.

Gently tap the mold features from above using tweezers. Observe the interface beneath the features for darkening or optical contrast, which confirms proper contact. Now, fill the space between the PDMS mold and the slab with liquid ultraviolet curable adhesive by capillary action.

Partially cure the structure under ultraviolet light for 10 seconds, as demonstrated earlier. Use soft padded tweezers to gently peel the composite ultraviolet curable adhesive layer and PDMS mold from the PDMS slab. If necessary, trim the excess edges of the cured adhesive layer with sharp scissors.

Next, place layer one attached to a rectangular glass cover slip on a flat surface. Align layer two, still attached to its PDMS mold, with layer one and press both layers together firmly until shadows appear beneath the features. Cure the aligned structure under ultraviolet light for 10 seconds to bond layers one and two into a two layer microfluidic chip, then gently peel the PDMS mold from the bonded chip.

Immediately pipette 10 microliters of polyacrylamide gel precursor solution onto the middle of the tissue region directly above the slit in layer two. Gently press a round glass cover slip, 10 millimeters in diameter, onto the droplet to form a flat gel. Observe the gel solution flow into layer one through the slit and stop at the row of barrier pillars.

Immediately cure the assembled microfluidic chip under ultraviolet light for five minutes. Then, leave the chip undisturbed for approximately one hour to allow the polyacrylamide gel to fully solidify. Next, incubate the microfluidic chip in 0.1 molar HEPES buffer at pH 7.4 for at least one hour to hydrate the gel and reduce adhesion between the gel and the cover slip.

Then, gently use sharp tweezers to remove the cover slip from the gel surface. Place a medical-grade polycarbonate cartridge on a flat surface with the bottom face up and align a dried chip face down with the cartridge. Pipette UV curable adhesive in the crevices between the chip and cartridge, filling them via capillary effect, and cure it under UV light.

Sterilize the fabricated microfluidic devices by placing them under ultraviolet light at 200 to 280 nanometers inside a biosafety cabinet. Prepare the required volume of 50 micrograms per milliliter collagen one working solution in 1X DPBS on ice. On ice, prepare the Sulfo-SANPAH working solution by diluting two microliters of Sulfo-SANPAH stock solution, previously dissolved in anhydrous dimethyl sulfoxide in 80 microliters of cold 0.1 molar HEPES buffer at pH 7.4 stored at four degrees Celsius.

Dab the edges of the polyacrylamide gel with laboratory wipes to dry it inside the device. Pipette 80 microliters of Sulfo-SANPAH working solution onto the gel surface, ensuring full immersion, and place the chip under ultraviolet light for five minutes to activate the Sulfo-SANPAH. Rinse the gel three times with cold 0.1 molar HEPES buffer at pH 7.4.

Repeat the Sulfo-SANPAH treatment by pipetting 80 microliters of SS working solution over the gel and irradiating it under ultraviolet light for five minutes. This time, rinse the gel three times with cold 1X DPBS. Finally, pipette 100 microliters of collagen one solution onto the polyacrylamide gel, ensuring complete coverage, and leave it for one hour.

Rinse off unattached collagen with 1X DPBS three times, then keep the gel immersed in 1X DPBS until further use. Attach devices to the inserts using screws. Then, secure up to four device insert assemblies to the holder designed for mounting on a microscope stage.

Add two milliliters of culture medium to each main well of the devices and through the waste removal channels to flush out and replace the 1X DPBS. Then, insert the platinum electrodes into the tube containing 45 milliliters of medium, ensuring they are fully submerged in the medium. Pull culture medium through the Tygon tubing with a syringe and clip it tight.

Connect one end of the medium-filled tubing to the electrode chamber and the other end to the device. Now, prepare a cell suspension by following the trypsinization or cell suspension preparation recommendations and seed 0.1 million cells into the main well. Mount the setup onto the microscope with the electrode chambers on the sides and connect the source meter to the electrode chamber.

Use three devices with identical channel resistance. Assign one as a control group. Apply apical to basal current to the second device and basal to apical current to the third device.

Source an electric signal under current clamp mode and supply a total current of 20 microamperes. Choose 20X objective lens for whole tissue imaging with an epifluorescence microscope. Finally, choose channel 561 for red fluorescence and bright-field channels.

Select one one hour time interval during long-time imaging. Electric stimulation was applied perpendicular to the cells to disrupt transepithelial potential to study its role in tissue behavior under different current directions. Under phase contrast imaging, apical to basal, or ATB currents, caused cell junctions to brighten within 10 minutes of application, while control and basal to apical, BTA conditions, showed low contrast and flat apical surfaces.

ATB currents produced an outward convexity at the apical cell surface. Gel deformation was strongly dependent on the direction of current. ATB currents pulled the gel upward, while BTA currents compressed the gel downward.

Junctional E-cadherin intensity increased under BTA currents, but significantly decreased under ATB currents compared to control. Junctional actin intensity decreased under ATB currents, but did not significantly increase under BTA currents relative to control. ATB currents triggered multicellular live cell extrusions across the monolayer, while BTA currents preserved monolayer structure, similar to the control.

Spatial heterogeneity in cell distribution was significantly higher under ATB currents, but remained low and uniform under BTA and control conditions. Cell proliferation rates increased under ATB stimulation, with higher division rates over time. In contrast, BTA currents induced more cell death events.