用集成电极直接量化 Transendothelial 电阻的 Organ-on-chip 系统的制作与验证

The overall goal of this video is to show how to fabricate and use micro-fluidic chips with integrated electrodes for transendothelial or transepithelial electrical resistance measurements, or TEER measurements. This is demonstrated using a blood-brain barrier in the micro-fluidic chip. This method can help answer key questions in the field of Organs-on-Chips by enabling direct measurement of the barrier function of, for example, blood-brain barrier tissue using integrated electrodes.

The main advantage of our technique is that electrodes are easily integrated into Organ-on-Chip systems and that the results of this can be compared among the different systems. The implications of this technology extend towards understand blood-brain barrier function in health and disease, drug discovery, and personalized medicine. Though this method can be used to provide insight into blood-brain barrier function, it can also be used in the context of other Organ-on-Chip systems such as the Lung-on-Chip and the Gut-on-Chip.

To begin this procedure, mix 27 grams of PDMS base agent and 2.7 grams of curing agent thoroughly. Then degas the mixture in a dessicator for approximately 45 minutes to remove air bubbles. Meanwhile, prepare the mold for the liquid PDMS mixture by sticking clear tape around the mold, or place the mold in a suitable wafer holder.

Pour the degased PDMS mixture onto the mold. Next, cure the PDMS mixture in an oven at 60 degrees Celsius for four hours and allow it to cool down afterward. In a cross-flow hood, pull the cured PDMS from the mold.

Cut the PDMS replica into separate top and bottom chip parts using cutting lines in the PDMS. Following that, punch four holes in the top parts using a sharp biopsy punch with one millimeter diameter to form inlets and outlets. Punch from inside to outside to prevent PDMS debris from collecting in the chip.

Then, cover the chip parts with clear tape to protect them against dust. Subsequently, cut the polycarbonate membranes from transwell inserts into squares of approximately three by three square millimeters. After that, assemble a porous membrane leakage-free in between two PDMS parts in order to assemble a two-layer device interfaced with a porous membrane.

To that end, prepare a PDMS toluene mortar using 0.7 grams of PDMS base agent, 07 grams of curing agent, and 540 microliters of toluene. Vortex the mortar thoroughly, then spincoat 200 microliters of mortar onto a glass coverslip at 1500 rpm for 60 seconds to acquire a thin, uniform layer of mortar. Next transfer a thin layer of mortar from the coverslip onto the bottom part of the chip with an ink roller.

Put the bottom part in an oven-safe dish and then transfer the mortar onto the top part of the chip. With a set of tweezers, dip the edges of a membrane into the spincoated mortar and place it carefully in the middle of the bottom part. Following that, carefully place the top part on the bottom part while paying attention to the alignment.

Cover the chips inlets with clear tape to prevent dust from entering the chip and bake them at 60 degrees Celsius for three hours. Being careful is very important in this step. Do not exert pressure onto the chip and do not slide the top part over the bottom part, to prevent mortar from entering the channels and clogging the membrane.

To integrate electrodes into the side channels, cut a platinum wire into pieces two centimeters long. Next, submerge them in acetone for 30 minutes. Then rinse them with water and ethanol and allow them to dry.

In a crossflow hood, put a chip on a plastic dish. Insert four platinum wires into the electrode channels of the chip using a pair of tweezers, and bend them down onto the plastic dish to enable fixation to the dish in the next step. Insert the wires of 0.7 to one millimeter into the culture channel past the T-shaped channel junction.

Subsequently, apply a drop of UV-curable glue at the electrode channel entrance and allow the glue to fill the channel by capillary forces. Then, switch on the UV and cure the glue when it reaches the end of the electrode channel. Fixated the four integrated electrodes to the plastic dish with a two-component epoxy adhesive.

Following this, cover the chips with clear tape and bake them at 60 degrees Celsius for two hours. Allow them to cool down and store dust-free until use. To coat the chips to promote cell attachment, first fill both channels with PBS before introducing reagents.

Check under a microscope if there are any air bubbles in the channels. If so, remove them by flushing with extra PBS. Next, fill both channels with 30 microliters of 20 micrograms per milliliter human fibronectin in PBS.

Incubate them at 37 degrees Celsius for three hours. Then flush the chips with endothelial growth medium and incubate them at 37 degrees Celsius for two hours. Afterward, measure TEER of the blank chips to ensure that all of the electrodes are in direct contact with fluid in the channels.

To do so, take a chip from the incubator and allow it to reach room temperature for at least ten minutes. Remove any fluid from the plastic dish around the electrodes to prevent electro-bridging outside of the chip. Next, take the impedance spectrum from 200 hertz to one megahertz for each combination of two electrodes resulting in six impedance spectra per chip in five to ten minutes, from which the TEER can be directly determined.

Check if the impedance spectra have the expected magnitudes and shapes to validate the TEER measurement. Now, prepare a cell suspension to be seated into the top channel by removing the culture medium from a culture flask with confluent monolayer of hcMEC/D3 cells. After that, wash the cells with PBS.

Remove the PBS and then add two milliliters of 05 per cent trypsin EDTA and incubate at 37 degrees Celsius for two to five minutes until the cells have detached from the culture flask. Then, deactivate the trypsin with culture medium supplemented with 20 per cent FBS. Count the cells and calculate their total number in suspension.

Meanwhile, centrifuge the hcMEC/D3 cells at 390 times g for five minutes. Afterward, remove the supernatant and re-suspend the cell pellet in the appropriate volume of endothelial growth medium to result in a concentration of five million cells per milliliter corresponding to a seeding density of 200 thousand cells per square centimeter in the chip. Next, slowly pipette 30 microliters of the well-mixed cell suspension into the top channel and remove the pipette from the inlet in a fluent motion while still exerting pressure.

Check the seeding density under a microscope. A uniform distribution of cells throughout the top channel should be achieved. Then incubate the chips at 37 degrees Celsius and five percent carbon dioxide for at least one hour.

Afterward, flush out any non-attached cells with endothelial culture medium. Keep in mind that TEER is monitored during the culture period. These are the typical schematic impedance spectrum showing impedance magnitude and phase shift versus frequency for electrical impedance spectroscopy on chips without cells and with cells.

There are four main regions which are dominated by the double-layer capacitance at the electrodes, the culture medium resistance, the cell-barrier resistance or the cell membrane capacitance. The region of interest indicates where the contribution of the cell layer can be quantified. And this is the formula to calculate TEER from the measured resistances between all six combinations of four electrodes.

Shown here is the average TEER of four BBBs on chips during the culture period of three days reaching a plateau of 22 plus or minus 1.3 ohm square centimeter. For comparison, data of blank chips is included, showing marginal variation and deviation from zero ohm square centimeter in the same period compared to the variation in TEER value of chips with cells. The fluorescence microcopy of stained nuclei revealed a continuous monolayer of endothelium both on PDMS and the membrane at the location indicated in the inset.

Immunofluorescence revealed the presence of tight junction protein zonula occludens one indicating that BBB-specific tight junctions between the cells give rise to the measured TEER. After watching this video, you should have a good understanding of the fabrication and use of chips with integrated electrodes for TEER measurements in Organs-on-Chips. This technique can be used in the field of blood-brain barrier research to study the direct delivery and disease characteristics in, for example, Alzheimer's disease.

Following this procedure, the barrier function of brain endothelium differentiated from human-derived induced pluripotent stem cells can also be monitored for applications in personalized medicine.