Name: Revealing Electromechanical Control of Tissue Homeostasis Using a Two-Layer Microfluidic Device (Video) | JoVE Journal
Uploaded: 2025-09-19T13:30:00.000+00:00
Duration: 11 min 8 s
Description: Discover electromechanical control of tissue homeostasis using a two-layer microfluidic device with controlled electric fields. This method reveals how current direction regulates cell adhesion, proliferation, and extrusion in epithelial tissues.

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XJ, PX, FF, and TBS acknowledge helpful discussions with Dr. Xumei Gao,&#160;Xinru Yu and other group members of the Tissue Biophysics Laboratory. We thank the Customized Technology Service Facility (CTSF) at Westlake University for assisting with our schematic drawing. This work was supported by the Westlake Education Foundation, Westlake Laboratory of Life Sciences and Biomedicine, and the Research Center for Industries of the Future (RCIF) at Westlake University.

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Ensure that all authors have disclosed any and all conflicts of interest.

This protocol provides detailed steps and design logic to fabricate a novel microfluidic device to perform transepithelial electric potential perturbation experiments and to study bioelectric effects through live-cell imaging and mechanobiology methods. The significance of this method is obvious when compared to other related methods. For example, one related method uses a simple microfluidic chip with a single channel design that applies a stationary in-plane current to study galvanotaxis (cell migration as a function o...

Bioelectric signals are important biophysical factors contributing to governing cell behavior. The most famous example is the action potential in neurons and heart cells, which transmit electrical signals over long distances (up to a meter) and at high frequencies (up to hundreds of Hertz)1. Electrical pulses applied with electrodes can be used to stimulate action potential firing and is useful in research and medical applications2. Endogenous bioelectric signals in slow physiological processes such as development, wound healing, and homeostasis3,4,5,6,7,8 have also been measured9,10 over periods of hours to days. An important example is confluent epithelial layers which actively maintain a transepithelial potential difference (TEPD) ranging from a few to tens of millivolts4,11. A cut or wound in these confluent layers generates a short-circuit current, driven by the electrical activity of the tissue12,13. Wound currents up to tens of &#181;A/cm2 and electric fields up to hundreds of mV/mm have been reported with different techniques. Studies suggest that these electric signals produce a directional cue for cells to migrate toward wound or infection sites that is important for the healing process3,6,14. In general, both static (or slow-varying) and fast-varying electric signals are important and trigger different cell or tissue responses.
Within the realm of slow-varying signals, one important phenomenon is the electrically-driven migration process termed &#34;galvanotaxis&#34;. Controlled in-plane galvanic currents can be applied in microfluidic devices with simple channels to mimic the wound currents while cell migration is investigated as a function of current density15,16. Electric fields of 0.1-1000 V/m (corresponding current density of 0.1-1000 A/m2) are found to drive cell migration in many cell types in this in vitro setting17, while signaling pathways canonical to chemotaxis (cell migration guided by chemical gradients), such as PI3K, have been implicated in galvanotaxis7,18. &#34;Bare-bones&#34; microfluidic devices for galvanotaxis study can be fabricated with standard soft lithography and microfabrication techniques19. These devices typically comprise a single-channel polydimethyl-siloxane (PDMS) substrate bonded to a glass slide (channel cross-sectional size: hundreds of micrometers, length: centimeters). Electrodes used to supply the static current are installed far away from the cell region, and connected with or without salt bridges. The salt bridge connection may be made by punched holes through the PDMS substrate, microfluidic connectors, and tubings filled with agar. The simple glass slide (typically 170 &#181;m thick) beneath the channel also supports high-resolution live-cell imaging during the galvanotaxis experiments, which is a crucial feature for studying the process.
The role of endogenous electric signals in the maintenance of tissue homeostasis is much less understood than that in wound healing. One important reason is the lack of appropriate tools to study these effects in intact tissues. The microfluidic device for galvanotaxis is not suitable for this purpose due to its geometry, which only supports in-plane electric currents. In the context of epithelial homeostasis, a new device is needed to control and perturb the TEPD or out-of-plane currents across a confluent epithelial layer. To achieve the aim of applying a relatively uniform ion current density across a monolayer, we have designed and fabricated a microfluidic device with innovations in device geometry, material usage, and in situ biomechanical measurement capabilities4. The latter capability is important as electric effects may be affecting cell behavior through coupling with biomechanics and mechanobiology pathways, which can be studied with this feature.
This new device exhibits multiple innovative features. It is designed with two layers instead of the single channel found in the galvanotaxis devices (innovation in geometry), while keeping the connection to the electrode chambers similar to that in the latter. The first microfluidic layer has channels to direct ion currents to (or away from) the tissue region, while the second layer has an opening that will direct this current perpendicularly across the epithelium sitting on top of this second microfluidic layer (Figure 1A,B). Microfabrication techniques are used for producing sufficiently thin layers such that high-resolution confocal imaging is possible despite the stacking of microfluidic layers that increases sample thickness. For the other important innovations, a polyacrylamide (PA) synthetic hydrogel is incorporated into the middle of the device, which plays a crucial role (Figure 1A,B). The physico-chemical properties of this gel, such as available chemistry to coat extracellular matrix proteins, permeability to ions, as well as known elasticity, are respectively essential for cell attachment and growth, application of out-of-plane currents, and measurement of mechanical stress20 during electric stimulation. Barrier structures are incorporated in Layer 1 of the tissue region for confinement of the PA gel precursor solution, as done in previous microfluidic devices21 (Figure 1A,B). Last but not least, since PA gel polymerization is oxygen-inhibited at PDMS surfaces permeable to oxygen gas, another material22 is used for device fabrication, which can allow polymerization and tight bonding of PA gel. All the innovative features are crucial for the new device to work.
Overall, the in-depth study of static or slowly varying bioelectric signals in the homeostasis of intact tissues is made possible with this new device, the protocol for which will be detailed below. The protocol and results referred to here are focused on the Madin Darby Canine Kidney II (MDCK II) epithelial cell line, including the cell events (proliferation, death, and extrusion, and migration) and tissue structural changes that are induced by the electric current. These studies are also instructive for researchers who are interested in applying this technique to study the electrical regulation of epithelial transport and induction of cell events and cell fate changes in cell layers and more complex systems, such as primary cells and 2D organoids.

<table><tbody><tr><td>1x DPBS</td><td>Gibco</td><td>14190144</td><td></td></tr><tr><td>3-(trimethoxysilyl)propyl methacrylate, TPM</td><td>Sigma</td><td>2530850</td><td>Store under nitrogen after opening.</td></tr><tr><td>50 mL Falcon tube</td><td>Corning</td><td>430829</td><td>Punch 4 holes in the 50 mL falcon tube cap, to enable electrode and tubing pass through the cap and immersed in the medium. For a set of microfluidic device, at least 2 caps is needed.</td></tr><tr><td>Acetic acid</td><td>Sinopharm</td><td>10000208</td><td></td></tr><tr><td>Acrylamide solution, 40%</td><td>Sigma</td><td>79061</td><td>Toxic</td></tr><tr><td>Air pump</td><td>OKO lab</td><td>OKO-AP</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Ammonium Persulfate, APS</td><td>Aladdin</td><td>A112447</td><td></td></tr><tr><td>Cartridge</td><td>-</td><td>-</td><td>Cartridge is custom-made, and the design refer to supplementary material. Raw material is PC Makrolon 2858 Medical Grade,and here you can find more details: https://solutions.covestro.com/zh/products/makrolon/2858_000000000000508397?SelectedCountry=US</td></tr><tr><td>Clips</td><td>-</td><td>-</td><td>Robert Clip, for tubing&amp;nbsp; of outer 4.5 mm diameter.&lt;a href=&quot;https://item.taobao.com/item.htm?id=18717491819&amp;amp;pisk=gAsj0ailDjcjqv-tCE2rVYReT5-1h8rFWA9OKOnqBnKv6A1RBjpxDchRepvBWczD0Ft1wQS2gt-v6c1NQi5NBGJOfnjPucR4ih6OThNUTkrFntYBXWPETzI4tHpikchwH3nJbds8Mz2dntxMXYlT8aBmCOO1qfp968tJCdc9XKnte89wBdK9MFh-ypdJXhdvkYpJBpn9HCnYF3pDKKhv6qK-edJXXCC9X86JZdt96bH_3dwJgtw2FbzzkyxCHQitXiEMlB_i7ci1TKLfXtd5zaSWhEOpWac1uiCPBiSDrzgJjT7C1NCLaqRARp1W8ZNIc61DB6tV4jeJXFQP8E_7McOP0dTpXUMtXTtPPFsWvkhWEaBVWifjCcpc0M86tUwtjF-RYeQdGA2henpOsejatmAAB9jh8HZjtK_Rd3syxDRBL50sFem6FBy7FV0GhSGPYc782SYvEK__F8GxLEpkFBy7FV0MkLvf18wSMv5..&amp;amp;spm=a21xtw.29978518.0.0&amp;amp;skuId=3344063585144&quot;&gt; https://item.taobao.com/item.htm?id=18717491819&amp;amp;pisk=gAsj0ailDjcjqv-tCE2rVYReT5-1h8rFWA9OKOnqBnKv6A1RBjpxDchRepvBWczD0Ft1wQS2gt-v6c1NQi5NBGJOfnjPucR4ih6OThNUTkrFntYBXWPETzI4tHpikchwH3nJbds8Mz2dntxMXYlT8aBmCOO1qfp968tJCdc9XKnte89wBdK9MFh-ypdJXhdvkYpJBpn9HCnYF3pDKKhv6qK-edJXXCC9X86JZdt96bH_3dwJgtw2FbzzkyxCHQitXiEMlB_i7ci1TKLfXtd5zaSWhEOpWac1uiCPBiSDrzgJjT7C1NCLaqRARp1W8ZNIc61DB6tV4jeJXFQP8E_7McOP0dTpXUMtXTtPPFsWvkhWEaBVWifjCcpc0M86tUwtjF-RYeQdGA2henpOsejatmAAB9jh8HZjtK_Rd3syxDRBL50sFem6FBy7FV0GhSGPYc782SYvEK__F8GxLEpkFBy7FV0MkLvf18wSMv5..&amp;amp;spm=a21xtw.29978518.0.0&amp;amp;skuId=3344063585144&lt;/a&gt;</td></tr><tr><td>Collagen I</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>DI Milli-Q water</td><td>Milli-Q</td><td>-</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium culture medium,DMEM (high glucose)</td><td>Gibco</td><td>11965092</td><td>DMEM++ is DMEM with additional 10%&amp;nbsp; FBS (Sigma-Aldrich), and 100&amp;thinsp;units/mL Penicillin-Streptomycin (Gibco).</td></tr><tr><td>Enclosure black cage</td><td>OKO lab</td><td>H201</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Epi-Fluoresence Inverted Microscope</td><td>Olympus</td><td>IX83</td><td></td></tr><tr><td>Ethanol absolute</td><td>Sinopharm</td><td>10009218</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>F8318</td><td></td></tr><tr><td>Fluorescent beads, 0.1 &amp;mu;L, red (580/605)</td><td>Thermo</td><td>F8801</td><td></td></tr><tr><td>Gas chamber</td><td>OKO lab</td><td>H201-PRIOR-SP200/400/600</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Gas Manual Mixer</td><td>OKO lab</td><td>2GF-MIXER</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Geneticin</td><td>Gibco</td><td>10131027</td><td>GFP&amp;ndash;actin and H1&amp;ndash;GFP MDCK were maintained using media supplemented with 0.5&amp;thinsp;mg/mL geneticin (Gibco) to sustain their gene expression</td></tr><tr><td>HEPES</td><td>Sigma</td><td>736459</td><td></td></tr><tr><td>Isopropanol</td><td>Sinopharm</td><td>80109218</td><td></td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>LH-70155</td><td>Laboratory wiper</td></tr><tr><td>Longer screw</td><td>-</td><td>-</td><td>Nickel-plated round head cross precision machine screw, M3 x 10 mm. &lt;a href=&quot;https://item.taobao.com/item.htm?id=604899872494&amp;amp;pisk=gbX7s64OI40STNJKAQqqhx58y8d9NoyapDtdjMHrvLpJOvIP5HkeT7xIdEsTqUP3Zw1fJZAyyeRedH_wogkUqgrQdMI9yp5yzHQVRZcPz98FuJsG5QkPv9oliNStUTPkLv9kKpUa7RyZqgvHpfPM7c0okhx_LYdKpLvx7Uiz6RyNqiiy2lSYQ9Sp5stS9vQpwIpvqEhKe3QpMKKBkYKKeH3YcEYv24HKwxnvvh0K9wKdDnKkV03KeBKYHhYvJppdJiEXYELK1OzXm7TRVu-iUlqUdgsJlvHdeJAv5LhELvJw2Q6d2EsV0ttWNFRjAbyCeZSdUTA09X9h01_C9NUqaUC6fZONNPM55axdAHQ7IcTAFM6XZsoZBisfv1Rf3leMFLLRTLjEfzSfFaSlhMoKML9PGC6WBR0J8sS1dH63--_1vGfpG9g54UMw5pb-OMiidnTacoGntEWrjhPgtYw2wnxVQoZjPXAJmn1UcoGntQKDm2rbc4Gh.&amp;amp;spm=tbpc.boughtlist.suborder_itemtitle.1.494a2e8d0lg31n&quot;&gt; https://item.taobao.com/item.htm?id=604899872494&amp;amp;pisk=gbX7s64OI40STNJKAQqqhx58y8d9NoyapDtdjMHrvLpJOvIP5HkeT7xIdEsTqUP3Zw1fJZAyyeRedH_wogkUqgrQdMI9yp5yzHQVRZcPz98FuJsG5QkPv9oliNStUTPkLv9kKpUa7RyZqgvHpfPM7c0okhx_LYdKpLvx7Uiz6RyNqiiy2lSYQ9Sp5stS9vQpwIpvqEhKe3QpMKKBkYKKeH3YcEYv24HKwxnvvh0K9wKdDnKkV03KeBKYHhYvJppdJiEXYELK1OzXm7TRVu-iUlqUdgsJlvHdeJAv5LhELvJw2Q6d2EsV0ttWNFRjAbyCeZSdUTA09X9h01_C9NUqaUC6fZONNPM55axdAHQ7IcTAFM6XZsoZBisfv1Rf3leMFLLRTLjEfzSfFaSlhMoKML9PGC6WBR0J8sS1dH63--_1vGfpG9g54UMw5pb-OMiidnTacoGntEWrjhPgtYw2wnxVQoZjPXAJmn1UcoGntQKDm2rbc4Gh.&amp;amp;spm=tbpc.boughtlist.suborder_itemtitle.1.494a2e8d0lg31n&lt;/a&gt;</td></tr><tr><td>Microfluidic connector</td><td>-</td><td>-</td><td>Polypropylene Straight Union Fitting, 1/16&quot;-10-32UNF. &lt;a href=&quot;https://item.taobao.com/item.htm?abbucket=16&amp;amp;detail_redpacket_pop=true&amp;amp;id=555839592609&amp;amp;ltk2=1752310108435i7ldxj7ekdgu9qhnphlmj&amp;amp;ns=1&amp;amp;priceTId=2150470617523101021033708e1a4b&amp;amp;query=10-32unf%E8%9E%BA%E7%BA%B9%E7%9B%B4%E9%80%9A%E6%8E%A5%E5%A4%B4%20%E5%A1%91%E6%96%99%E5%A4%96%E8%9E%BA%E7%BA%B9%E6%8E%A5%E5%A4%B4&amp;amp;skuId=3600497427939&amp;amp;spm=a21n57.1.hoverItem.1&amp;amp;utparam=%7B%22aplus_abtest%22%3A%2245243a364caf5527e8392ded0656451a%22%7D&amp;amp;xxc=taobaoSearch&quot;&gt; https://item.taobao.com/item.htm?abbucket=16&amp;amp;detail_redpacket_pop=true&amp;amp;id=555839592609&amp;amp;ltk2=1752310108435i7ldxj7ekdgu9qhnphlmj&amp;amp;ns=1&amp;amp;priceTId=2150470617523101021033708e1a4b&amp;amp;query=10-32unf%E8%9E%BA%E7%BA%B9%E7%9B%B4%E9%80%9A%E6%8E%A5%E5%A4%B4%20%E5%A1%91%E6%96%99%E5%A4%96%E8%9E%BA%E7%BA%B9%E6%8E%A5%E5%A4%B4&amp;amp;skuId=3600497427939&amp;amp;spm=a21n57.1.hoverItem.1&amp;amp;utparam=%7B%22aplus_abtest%22%3A%2245243a364caf5527e8392ded0656451a%22%7D&amp;amp;xxc=taobaoSearch&lt;/a&gt;</td></tr><tr><td>Multiwell plates holder</td><td>OKO lab</td><td>H201-MW-HOLDER-NZ500</td><td>Component of microscope stage-top incubator</td></tr><tr><td>N&#039;,N&#039;-Methylenebisacrylamide, 2%</td><td>Sigma</td><td>110269</td><td>Toxic</td></tr><tr><td>NOA 73</td><td>Norland Products</td><td>17-345</td><td>UV-curable adhesive</td></tr><tr><td>Penicillin-Streptomycin, PS (10,000 U/mL)</td><td>Gibco</td><td>15140122</td><td></td></tr><tr><td>Petri dish (150 mm &amp;acute; 25 mm)</td><td>Corning</td><td>430599</td><td>Resist to at least 80 &amp;deg;C for 2 h</td></tr><tr><td>Pico Plasma tool</td><td>Diener Electronic GmbH + Co. KG</td><td>Pico Plasma</td><td>For O&lt;sub&gt;2&lt;/sub&gt; plasma treatment</td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-002</td><td>For air plasma treatment</td></tr><tr><td>Plastic dish (diameter 60 mm, height 15 mm)</td><td>Nest</td><td>705001</td><td></td></tr><tr><td>Platinum electrodes</td><td>Tianjin Aida Hengsheng</td><td>Pt005</td><td>Diameter 0.5 mm, length 37 mm&amp;nbsp;</td></tr><tr><td>Rain-X Original Glass Water Repellent</td><td>ITW Chemical Products</td><td>800002242</td><td></td></tr><tr><td>Rectangle coverslip</td><td>Marienfeld</td><td>0107222</td><td>&amp;nbsp;1.5H, 24 &amp;acute; 50 mm&lt;sup&gt;2&lt;/sup&gt;, 170 &amp;mu;m thickness</td></tr><tr><td>Round glass coverslip</td><td>Marienfeld</td><td>0111500</td><td>&amp;nbsp;1.5H, round 10 mm diameter</td></tr><tr><td>Shorter screw</td><td>-</td><td>-</td><td>Nickel-plated round head cross precision machine screw, M3 x 4 mm. &lt;a href=&quot;https://item.taobao.com/item.htm?id=604899872494&amp;amp;pisk=gbX7s64OI40STNJKAQqqhx58y8d9NoyapDtdjMHrvLpJOvIP5HkeT7xIdEsTqUP3Zw1fJZAyyeRedH_wogkUqgrQdMI9yp5yzHQVRZcPz98FuJsG5QkPv9oliNStUTPkLv9kKpUa7RyZqgvHpfPM7c0okhx_LYdKpLvx7Uiz6RyNqiiy2lSYQ9Sp5stS9vQpwIpvqEhKe3QpMKKBkYKKeH3YcEYv24HKwxnvvh0K9wKdDnKkV03KeBKYHhYvJppdJiEXYELK1OzXm7TRVu-iUlqUdgsJlvHdeJAv5LhELvJw2Q6d2EsV0ttWNFRjAbyCeZSdUTA09X9h01_C9NUqaUC6fZONNPM55axdAHQ7IcTAFM6XZsoZBisfv1Rf3leMFLLRTLjEfzSfFaSlhMoKML9PGC6WBR0J8sS1dH63--_1vGfpG9g54UMw5pb-OMiidnTacoGntEWrjhPgtYw2wnxVQoZjPXAJmn1UcoGntQKDm2rbc4Gh.&amp;amp;spm=tbpc.boughtlist.suborder_itemtitle.1.494a2e8d0lg31n&quot;&gt; https://item.taobao.com/item.htm?id=604899872494&amp;amp;pisk=gbX7s64OI40STNJKAQqqhx58y8d9NoyapDtdjMHrvLpJOvIP5HkeT7xIdEsTqUP3Zw1fJZAyyeRedH_wogkUqgrQdMI9yp5yzHQVRZcPz98FuJsG5QkPv9oliNStUTPkLv9kKpUa7RyZqgvHpfPM7c0okhx_LYdKpLvx7Uiz6RyNqiiy2lSYQ9Sp5stS9vQpwIpvqEhKe3QpMKKBkYKKeH3YcEYv24HKwxnvvh0K9wKdDnKkV03KeBKYHhYvJppdJiEXYELK1OzXm7TRVu-iUlqUdgsJlvHdeJAv5LhELvJw2Q6d2EsV0ttWNFRjAbyCeZSdUTA09X9h01_C9NUqaUC6fZONNPM55axdAHQ7IcTAFM6XZsoZBisfv1Rf3leMFLLRTLjEfzSfFaSlhMoKML9PGC6WBR0J8sS1dH63--_1vGfpG9g54UMw5pb-OMiidnTacoGntEWrjhPgtYw2wnxVQoZjPXAJmn1UcoGntQKDm2rbc4Gh.&amp;amp;spm=tbpc.boughtlist.suborder_itemtitle.1.494a2e8d0lg31n&lt;/a&gt;</td></tr><tr><td>Sulfo-SANPAH, no weigh</td><td>Thermo</td><td>A35395</td><td>SS stock solution: 20 mg/mL in anhydrous DMSO, 2 &amp;mu;L for each; SS working solution: 0.5 mg/mL SS in HEPES (0.1 M, pH 7.4).</td></tr><tr><td>SYLGARD 184 Silicone Elastomer Kit</td><td>Dow Corning</td><td>H052KCM147</td><td>The final product is polydimethylsiloxane, PDMS. This Kit includes base PDMS resin and crosslinking agent.&amp;nbsp;</td></tr><tr><td>Temperature Unit</td><td>OKO lab</td><td>H201-T-UNIT-BL</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Tetramethylethylenediamine, TEMED</td><td>Sigma</td><td>110189</td><td>Toxic</td></tr><tr><td>Touch Screen Display</td><td>OKO lab</td><td>OKO-TOUCH</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane</td><td>Sigma</td><td>78560459</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>gibco</td><td>25200072</td><td></td></tr><tr><td>Tygon tubings</td><td>Tygon</td><td>AJK00002</td><td>Outer diameter (OD) = 3.2mm, Inner diameter (ID) = 1.6 mm</td></tr><tr><td>Ultrasonic cleaner</td><td>Supmile</td><td>KD-300DE</td><td></td></tr><tr><td>UV LED curing machine</td><td>GHUV</td><td>GHS-MFLG</td><td>UV-LED light source, Emision range: 363&amp;ndash;370 nm, maxsium power density: 230 mW/cm&lt;sup&gt;2&lt;/sup&gt;</td></tr><tr><td>Vibration Free Humidifying Module</td><td>OKO lab</td><td>HM-VF</td><td>Component of microscope stage-top incubator</td></tr><tr><td>Visiview</td><td>Visitron Systems GmbH</td><td>-</td><td>Imaging softer ware</td></tr><tr><td>Wafer-layer1</td><td>National University of Singapore, Mechanobiology institute microfabrication facility</td><td>-</td><td>The wafer is custom-made, and the design refer to supplementary material.</td></tr><tr><td>Wafer-layer2</td><td>National University of Singapore, Mechanobiology institute microfabrication facility</td><td>-</td><td>The wafer is custom-made, and the design refer to supplementary material.</td></tr></tbody></table>

revealing electromechanical control of tissue homeostasis using a two-layer microfluidic device

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

Cell behavior and cell fate are impacted by electric currents or fields that are endogenous or externally applied. Static electric signals can be applied in customized microfluidic devices to mimic the electric environment in slow physiological processes such as development, wound healing, and homeostasis. An important class of cellular electric studies is the control of cell migration by static in-plane galvanic currents in simple microfluidic channels mimicking wound currents, with current densities of ~0.1-1000 A/m2. However, due to incompatible geometry, these devices are not appropriate to study electric effects in tissue homeostasis, where cells adopt apico-basal polarity and a transepithelial potential difference (TEPD). Here, we detail a unique microfluidic-based device that applies physiological ion currents perpendicular to the plane of confluent epithelial cell layers to perturb the TEPD and investigate electrical regulation of tissue steady states. The setup is made from a two-layer UV-curable polymer embedded with soft, polyacrylamide gel substrate coated with extracellular-matrix protein of choice. This microfluidic device provides the correct geometry and permeable substrate to induce a relatively uniform ion current across the cell layers of centimetric-scale. The setup is compatible with confocal live-cell imaging and Traction Force Microscopy to infer mechanical stresses induced by the transepithelial currents. Strikingly, the proliferation, extrusion and migration of cells are collectively influenced within the confluent epithelium depending on the direction of ion current, inducing a new tissue state characterized by different cell-cell interaction strengths, cell events (death and proliferation), and tissue structures. The electrically controlled cell behaviors can be understood as an electrically induced mechanical stress and cell response. This novel microfluidic device and protocol provide the tool and documentation required for the mechanobiology and bioengineering communities to study electric effects in tissue homeostasis and develop novel tissue engineering applications.

All results presented here are adapted from the previous publication4; for more details, refer to this publication.
This protocol allows the application of an external ion current or electric field perpendicular to the plane of cells or tissues. This electric stimulation perturbs the endogenous transepithelial electric potential difference (TEPD) and allows one to study the role of TEPD in governing tissue behavior. Following the protocol, wild-type and genetically edit...

Here, we present a protocol to fabricate a unique two-layer microfluidic device to study the electromechanical regulation of epithelial tissue homeostasis. The device applies static physiological electric currents perpendicular to the tissue plane, impacting cell-cell adhesion, proliferation, and extrusion. Live-cell imaging and mechanical stress measurements reveal mechanisms of these processes.

Cell behavior and cell fate are impacted by electric currents or fields that are endogenous or externally applied. Static electric signals can be applied ...

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.

revealing-electromechanical-control-tissue-homeostasis-using-two

Research

JoVE Journal

Bioengineering

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes.   A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

We demonstrate fabrication of a simple microfluidic device that can be integrated with standard electrophysiology setups to expose microscale surfaces of a brain slice in a well controlled manner to different neurotransmitters.

Brain Slice Stimulation Using a Microfluidic Network and Standard Perfusion Chamber

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

A Microfluidic Device for Studying Multiple Distinct Strains

Source: Kurz, D. L., et al. Microfluidic Platform to Study Bioclogging in Porous Media. J. Vis. Exp. (2022)The video demonstrates the use of a microfluidic device to study biofilm formation. A bacterial suspension is introduced into the microchannel, followed by incubation to allow biofilm development. Flow is then resumed, and pressure monitoring along with imaging is used to detect biofilm formation and pore blockage over time.

A Microfluidic Device for Real-Time Imaging and Pressure Tracking During Biofilm Formation

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The goal of this protocol is to explain and demonstrate the development of a three-dimensional (3D) microfluidic model of highly aligned human cardiac tissue, composed of stem cell-derived cardiomyocytes co-cultured with cardiac fibroblasts (CFs) within a biomimetic, collagen-based hydrogel, for applications in cardiac tissue engineering, drug screening, and disease modeling.

Developing 3D Organized Human Cardiac Tissue within a Microfluidic Platform

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

The fabrication of a polydimethylsiloxane (PDMS)-based bilayer device for the production of combinatorial libraries in water-in-oil emulsions (plugs) is presented here. The necessary hardware and software required to automate plug production are detailed in the protocol, and the production of a quantitative library of fluorescent plugs is also demonstrated.

Bilayer Microfluidic Device for Combinatorial Plug Production

Here, we present a protocol to fabricate and operate a microfluidic system that exposes endothelial cells to synchronized oscillatory hyperglycemia and pulsatile shear stress. This approach provides a physiologically relevant in vitro model for studying diabetic endothelial dysfunction. This protocol enables quantitative measurement of oxidative stress and endothelial cell responses.

A Microfluidic System for Modeling Endothelial Dysfunction under Combined Physiological Pulsatile Shear Stress and Oscillatory Hyperglycemia

Discover electromechanical control of tissue homeostasis using a two-layer microfluidic device with controlled electric fields. This method reveals how current direction regulates cell adhesion, proliferation, and extrusion in epithelial tissues.

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

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