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

Summary

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.

Abstract

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.

Introduction

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 homeostasis^3,4,

Protocol

NOTE: This protocol provides steps to construct a setup that applies static electric currents and perturbs the TEPD of confluent epithelia and to study its effects through live-cell imaging and mechanobiology techniques (Figure 1). The setup consists of devices made of microfluidic chips bonded to cartridges, inserts, and holders to house multiple devices for multiplexed experiments on a microscope, a pair of electrode chambers connected to a source meter and devices, and a live-cell chamber lid for the maintenance of proper live-cell conditions (Figure 1C). The design principles and CAD file drawings of the ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x DPBS	Gibco	14190144
3-(trimethoxysilyl)propyl methacrylate, TPM	Sigma	2530850	Store under nitrogen after opening.
50 mL Falcon tube	Corning	430829	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.
Acetic acid	Sinopharm	10000208
Acrylamide solution, 40%	Sigma	79061	Toxic
Air pump	OKO lab	OKO-AP	Component of microscope stage-top incubator
Ammonium Persulfate, APS	Aladdin	A112447
Cartridge	-	-	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
Clips	-	-	Robert Clip, for tubing  of outer 4.5 mm diameter. https://item.taobao.com/item.htm?id=18717491819&pisk=gAsj0ailDjcjqv-tCE2rVYReT5-1h8rFWA9OKOnqBnKv6A1RBjpxDchRepvBWczD0Ft1wQS2gt-v6c1NQi5NBGJOfnjPucR4ih6OThNUTkrFntYBXWPETzI4tHpikchwH3nJbds8Mz2dntxMXYlT8aBmCOO1qfp968tJCdc9XKnte89wBdK9MFh-ypdJXhdvkYpJBpn9HCnYF3pDKKhv6qK-edJXXCC9X86JZdt96bH_3dwJgtw2FbzzkyxCHQitXiEMlB_i7ci1TKLfXtd5zaSWhEOpWac1uiCPBiSDrzgJjT7C1NCLaqRARp1W8ZNIc61DB6tV4jeJXFQP8E_7McOP0dTpXUMtXTtPPFsWvkhWEaBVWifjCcpc0M86tUwtjF-RYeQdGA2henpOsejatmAAB9jh8HZjtK_Rd3syxDRBL50sFem6FBy7FV0GhSGPYc782SYvEK__F8GxLEpkFBy7FV0MkLvf18wSMv5..&spm=a21xtw.29978518.0.0&skuId=3344063585144
Collagen I	Corning	354236
DI Milli-Q water	Milli-Q	-
Dulbecco's Modified Eagle's Medium culture medium,DMEM (high glucose)	Gibco	11965092	DMEM++ is DMEM with additional 10%  FBS (Sigma-Aldrich), and 100 units/mL Penicillin-Streptomycin (Gibco).
Enclosure black cage	OKO lab	H201	Component of microscope stage-top incubator
Epi-Fluoresence Inverted Microscope	Olympus	IX83
Ethanol absolute	Sinopharm	10009218
Fetal Bovine Serum	Sigma	F8318
Fluorescent beads, 0.1 μL, red (580/605)	Thermo	F8801
Gas chamber	OKO lab	H201-PRIOR-SP200/400/600	Component of microscope stage-top incubator
Gas Manual Mixer	OKO lab	2GF-MIXER	Component of microscope stage-top incubator
Geneticin	Gibco	10131027	GFP–actin and H1–GFP MDCK were maintained using media supplemented with 0.5 mg/mL geneticin (Gibco) to sustain their gene expression
HEPES	Sigma	736459
Isopropanol	Sinopharm	80109218
Kimwipes	Kimberly-Clark Professional	LH-70155	Laboratory wiper
Longer screw	-	-	Nickel-plated round head cross precision machine screw, M3 x 10 mm. https://item.taobao.com/item.htm?id=604899872494&pisk=gbX7s64OI40STNJKAQqqhx58y8d9NoyapDtdjMHrvLpJOvIP5HkeT7xIdEsTqUP3Zw1fJZAyyeRedH_wogkUqgrQdMI9yp5yzHQVRZcPz98FuJsG5QkPv9oliNStUTPkLv9kKpUa7RyZqgvHpfPM7c0okhx_LYdKpLvx7Uiz6RyNqiiy2lSYQ9Sp5stS9vQpwIpvqEhKe3QpMKKBkYKKeH3YcEYv24HKwxnvvh0K9wKdDnKkV03KeBKYHhYvJppdJiEXYELK1OzXm7TRVu-iUlqUdgsJlvHdeJAv5LhELvJw2Q6d2EsV0ttWNFRjAbyCeZSdUTA09X9h01_C9NUqaUC6fZONNPM55axdAHQ7IcTAFM6XZsoZBisfv1Rf3leMFLLRTLjEfzSfFaSlhMoKML9PGC6WBR0J8sS1dH63--_1vGfpG9g54UMw5pb-OMiidnTacoGntEWrjhPgtYw2wnxVQoZjPXAJmn1UcoGntQKDm2rbc4Gh.&spm=tbpc.boughtlist.suborder_itemtitle.1.494a2e8d0lg31n
Microfluidic connector	-	-	Polypropylene Straight Union Fitting, 1/16"-10-32UNF. https://item.taobao.com/item.htm?abbucket=16&detail_redpacket_pop=true&id=555839592609<k2=1752310108435i7ldxj7ekdgu9qhnphlmj&ns=1&priceTId=2150470617523101021033708e1a4b&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&skuId=3600497427939&spm=a21n57.1.hoverItem.1&utparam=%7B%22aplus_abtest%22%3A%2245243a364caf5527e8392ded0656451a%22%7D&xxc=taobaoSearch
Multiwell plates holder	OKO lab	H201-MW-HOLDER-NZ500	Component of microscope stage-top incubator
N',N'-Methylenebisacrylamide, 2%	Sigma	110269	Toxic
NOA 73	Norland Products	17-345	UV-curable adhesive
Penicillin-Streptomycin, PS (10,000 U/mL)	Gibco	15140122
Petri dish (150 mm ´ 25 mm)	Corning	430599	Resist to at least 80 °C for 2 h
Pico Plasma tool	Diener Electronic GmbH + Co. KG	Pico Plasma	For O2 plasma treatment
Plasma cleaner	Harrick Plasma	PDC-002	For air plasma treatment
Plastic dish (diameter 60 mm, height 15 mm)	Nest	705001
Platinum electrodes	Tianjin Aida Hengsheng	Pt005	Diameter 0.5 mm, length 37 mm
Rain-X Original Glass Water Repellent	ITW Chemical Products	800002242
Rectangle coverslip	Marienfeld	0107222	1.5H, 24 ´ 50 mm2, 170 μm thickness
Round glass coverslip	Marienfeld	0111500	1.5H, round 10 mm diameter
Shorter screw	-	-	Nickel-plated round head cross precision machine screw, M3 x 4 mm. https://item.taobao.com/item.htm?id=604899872494&pisk=gbX7s64OI40STNJKAQqqhx58y8d9NoyapDtdjMHrvLpJOvIP5HkeT7xIdEsTqUP3Zw1fJZAyyeRedH_wogkUqgrQdMI9yp5yzHQVRZcPz98FuJsG5QkPv9oliNStUTPkLv9kKpUa7RyZqgvHpfPM7c0okhx_LYdKpLvx7Uiz6RyNqiiy2lSYQ9Sp5stS9vQpwIpvqEhKe3QpMKKBkYKKeH3YcEYv24HKwxnvvh0K9wKdDnKkV03KeBKYHhYvJppdJiEXYELK1OzXm7TRVu-iUlqUdgsJlvHdeJAv5LhELvJw2Q6d2EsV0ttWNFRjAbyCeZSdUTA09X9h01_C9NUqaUC6fZONNPM55axdAHQ7IcTAFM6XZsoZBisfv1Rf3leMFLLRTLjEfzSfFaSlhMoKML9PGC6WBR0J8sS1dH63--_1vGfpG9g54UMw5pb-OMiidnTacoGntEWrjhPgtYw2wnxVQoZjPXAJmn1UcoGntQKDm2rbc4Gh.&spm=tbpc.boughtlist.suborder_itemtitle.1.494a2e8d0lg31n
Sulfo-SANPAH, no weigh	Thermo	A35395	SS stock solution: 20 mg/mL in anhydrous DMSO, 2 μL for each; SS working solution: 0.5 mg/mL SS in HEPES (0.1 M, pH 7.4).
SYLGARD 184 Silicone Elastomer Kit	Dow Corning	H052KCM147	The final product is polydimethylsiloxane, PDMS. This Kit includes base PDMS resin and crosslinking agent.
Temperature Unit	OKO lab	H201-T-UNIT-BL	Component of microscope stage-top incubator
Tetramethylethylenediamine, TEMED	Sigma	110189	Toxic
Touch Screen Display	OKO lab	OKO-TOUCH	Component of microscope stage-top incubator
Tridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane	Sigma	78560459
Trypsin-EDTA (0.25%)	gibco	25200072
Tygon tubings	Tygon	AJK00002	Outer diameter (OD) = 3.2mm, Inner diameter (ID) = 1.6 mm
Ultrasonic cleaner	Supmile	KD-300DE
UV LED curing machine	GHUV	GHS-MFLG	UV-LED light source, Emision range: 363–370 nm, maxsium power density: 230 mW/cm2
Vibration Free Humidifying Module	OKO lab	HM-VF	Component of microscope stage-top incubator
Visiview	Visitron Systems GmbH	-	Imaging softer ware
Wafer-layer1	National University of Singapore, Mechanobiology institute microfabrication facility	-	The wafer is custom-made, and the design refer to supplementary material.
Wafer-layer2	National University of Singapore, Mechanobiology institute microfabrication facility	-	The wafer is custom-made, and the design refer to supplementary material.