Name: fabrication and validation of an organ-on-chip system with integrated electrodes to directly quantify transendothelial electrical resistance
Uploaded: 2017-09-26T15:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance at JoVE.com

We gratefully acknowledge Johan Bomer for fabrication of the mold and Mathijs Bronkhorst for fruitful discussions and assistance with data representation.
This research was funded by: SRO Biomedical Microdevices of L.I. Segerink, MIRA Institute for Biomedical Engineering and Technical Medicine, University of Twente; SRO Organs-on-Chips of A.D. van der Meer, MIRA Institute for Biomedical Engineering and Technical Medicine, University of Twente; and VESCEL, ERC Advanced Grant to A. van den Berg (grant no. 669768).

1. Fabrication of the organ-on-chip device
<ol>
	<li>Make a PDMS replica of a mold with the channel designs, fabricated using standard photolithography techniques, and obtain the PDMS parts of the microfluidic chip.
	<ol>
		<li>Weigh approximately 27 g PDMS base agent and 2.7 g curing agent; the mass ratio needs to be 10:1. This is good for a 3 mm thick PDMS slab on a mold with 130 mm (5 inch) diameter. Mix these components thoroughly.</li>
		<li>Degas the mixture in a desiccator for approximately 45 min to remove air bubbles.</li>
		<li>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.</li>
		<li>Pour the degassed PDMS mixture onto the mold. If any air bubbles remained in the PDMS mixture or at the mold surface, degas it again for approximately 30 min.</li>
		<li>Cure the PDMS mixture in an oven at 60 &#176;C for 4 h. Allow to cool down.</li>
		<li>In a cross-flow hood, pull the cured PDMS from the mold; the mold can be reused immediately to make more PDMS replicas.</li>
		<li>Cut the PDMS replica into separate top and bottom chip parts using cutting lines in the PDMS.</li>
		<li>Punch four holes in the top parts using a sharp biopsy punch with 1 mm diameter to form inlets and outlets. Punch from inside to outside to prevent PDMS debris from collecting in the chip. Cover the chip parts with clear tape to protect them against dust.</li>
		<li>Cut polycarbonate membranes from Transwell inserts into squares of approximately 3&#215;3 mm2.</li>
	</ol>
	</li>
	<li>Assemble a porous membrane leakage-free in between two PDMS parts in order to assemble a two-layer device interfaced with a porous membrane. 
	NOTE: This protocol is adapted from Griep et al.19 and Chueh et al.26
	<ol>
		<li>Prepare a PDMS/toluene mortar using 0.7 g of PDMS base agent, 0.07 g of curing agent and 540 &#181;L of toluene, resulting in a 5:3 weight ratio. Vortex the mortar thoroughly.</li>
		<li>Spin-coat 200 &#181;L of mortar onto a glass coverslip at 1500 rpm for 60 s (ramped at 1000 rpm/s) to acquire a thin, uniform layer of mortar.</li>
		<li>Transfer a thin layer of mortar from the coverslip onto a bottom part and a top part of the chip with an ink roller. Put the bottom part in an oven-safe dish.</li>
		<li>With a set of tweezers, dip the edges of a membrane into the spin-coated mortar and place it carefully in the middle of the bottom part.</li>
		<li>Carefully place the top part on the bottom part while paying attention to the alignment. 
		NOTE: 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.</li>
		<li>Cover the chips inlets with clear tape to prevent dust from entering the chip and bake them at 60 &#176;C for 3 h.</li>
	</ol>
	</li>
	<li>Integrate electrodes into the side channels. 
	NOTE: This protocol is adapted from Griep et al.19 and Douville et al.18
	<ol>
		<li>Cut platinum wire into pieces of approximately 2 cm. Clean by submerging them in acetone for 30 min. Rinse with water and ethanol and allow to dry.</li>
		<li>In a cross-flow hood, put a chip on a plastic dish. Insert four platinum wires into the electrode channels of a chip using a pair of tweezers and bend them down onto the plastic dish to enable fixation to the dish in a subsequent step. Insert the wires 0.7-1 mm into the culture channel, past the T-shaped channel junction.</li>
		<li>Apply a drop of UV-curable glue at the electrode channel entrance and allow the glue to fill the channel around the electrode by capillary forces.</li>
		<li>Switch on UV and cure the glue when it reaches the end of the electrode channel. 
		Caution: Do not look into the UV light source as this may harm your eyes.</li>
		<li>Fixate the four integrated electrodes to the plastic dish with a two-component epoxy adhesive. This allows easier handling of the electrodes during measurements without the risk of pulling the electrodes from the chip.</li>
		<li>Cover the chips with clear tape and bake them at 60 &#176;C for 2 h. Allow to cool down and store dust-free until use. In this way they can be safely stored for up to a month.</li>
	</ol>
	</li>
</ol>
2. On-chip culture of brain-derived endothelial cells
<ol>
	<li>Coat the chips to promote cell attachment.
	<ol>
		<li>Fill both channels with phosphate buffered saline (PBS) to wet them 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.</li>
		<li>Fill both channels with 30 &#181;L of 20 &#181;g/mL human fibronectin in PBS. Incubate at 37 &#176;C for 3 hours. Check for air bubbles and, if needed, flush the channels with PBS and refill with fibronectin solution.</li>
		<li>Flush the chips with endothelial growth medium and incubate them at 37 &#176;C and 5% CO2 in an incubator for 2 h.</li>
		<li>Measure the TEER of the blank chips as described in step 3 to ensure that all of the electrodes are in direct contact with fluid in the channels. In empty chips, typical TEER values are 0-1 &#8486; cm2.</li>
	</ol>
	</li>
	<li>Seed cells into the top channel.
	<ol>
		<li>Remove the culture medium from a culture flask (75 cm2 culture area) with a confluent monolayer of human cerebral microvascular endothelial cells (hCMEC/D3).</li>
		<li>Wash the cells with PBS. Add 2 mL of 0.05% trypsin-EDTA and incubate at 37 &#176;C and 5% CO2 for 2-5 minutes until the cells have detached from the culture flask.</li>
		<li>Deactivate the trypsin with culture medium supplemented with 20% fetal bovine serum (FBS). Count the cells and calculate the total number of cells in suspension.</li>
		<li>Meanwhile, centrifuge the hCMEC/D3 cells at 390 x g for 5 min and remove supernatant.</li>
		<li>Resuspend the cell pellet in the appropriate volume of endothelial growth medium (EGM-2) to arrive at a concentration of 5 x 106 cells/mL. This results in a seeding density of 2 x 105 cells/cm2 in the chip.</li>
		<li>Slowly pipette 30 &#181;L 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.</li>
		<li>Check the seeding density under a microscope. A uniform distribution of cells throughout the top channel should be achieved.</li>
		<li>Incubate the chips at 37 &#176;C and 5% CO2 for at least 1 h. Flush out any non-attached cells with endothelial culture medium.</li>
	</ol>
	</li>
	<li>Maintain the on-chip endothelial culture.
	<ol>
		<li>Twice per day, insert culture medium-filled pipette tips as reservoirs in the inlets and replace the medium inside the chip by gravity-driven flow. 
		NOTE: To avoid air bubbles from entering the microchannels and destroying the cell layer, make sure to make fluid-fluid contact between the pipette tip and the fluid on top of the chip before inserting the tip into an inlet. This can be facilitated by adding a small drop at the in/outlet prior to pipette insertion.</li>
		<li>Also add pipette tip reservoirs in the outlets to prevent the channels from drying.</li>
		<li>Incubate the chips at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
</ol>
3. On-chip TEER measurements
<ol>
	<li>Set up the TEER measurement apparatus, consisting of a lock-in amplifier and a probe cable circuit as was specified in Van der Helm et al.16 Alternatively, potentiostats or impedance analyzers are also suitable for these impedance-based TEER measurements.</li>
	<li>Take the chip from the incubator and allow it to reach room temperature for at least 10 min. Remove any fluid from the plastic dish around the electrodes to prevent electrode bridging outside of the chip.</li>
	<li>Take the impedance spectrum from 200 Hz to 1 MHz for each combination of two electrodes, resulting in 6 impedance spectra per chip in only 5-10 min. Check if the impedance spectra have the expected magnitudes and shapes to validate the TEER measurement, as is described in the results section.</li>
	<li>Put the chip back in the incubator at 37 &#176;C and 5% CO2 after finishing the measurement.</li>
	<li>Process the obtained impedance spectra to arrive at a normalized TEER value.
	<ol>
		<li>Get the measured resistance <img alt="Equation 1" src="/files/ftp_upload/56334/56334eq1.jpg" /> between electrodes <img alt="Equation 2" src="/files/ftp_upload/56334/56334eq2.jpg" /> and <img alt="Equation 3" src="/files/ftp_upload/56334/56334eq3.jpg" />, as denoted in Figure 2B and D, from the resistive plateau at 10 kHz in the corresponding impedance spectra.</li>
		<li>Calculate the TEER using the six measured resistances corresponding to one chip at one time point by using the equation of Figure 2G16: 
		<img alt="Equation 20" src="/files/ftp_upload/56334/56334eq20.jpg" /> 
		In this equation, <img alt="Equation 4" src="/files/ftp_upload/56334/56334eq4.jpg" /> is the culture area through which the resistance was measured, <img alt="Equation 5" src="/files/ftp_upload/56334/56334eq5.jpg" /> is the resistance of the cellular barrier and the membrane, <img alt="Equation 6" src="/files/ftp_upload/56334/56334eq6.jpg" />, <img alt="Equation 7" src="/files/ftp_upload/56334/56334eq7.jpg" />, <img alt="Equation 8" src="/files/ftp_upload/56334/56334eq8.jpg" /> and <img alt="Equation 9" src="/files/ftp_upload/56334/56334eq9.jpg" /> are the resistance values measured through the cellular barrier and <img alt="Equation 10" src="/files/ftp_upload/56334/56334eq10.jpg" /> and <img alt="Equation 11" src="/files/ftp_upload/56334/56334eq11.jpg" /> are the resistance values measured in the straight channels.16 The equation is derived from the equivalent circuit illustrated in Figure 2C.</li>
	</ol>
	</li>
	<li>Subtract the blank TEER from the TEER at all time points to obtain the development of the TEER in time.</li>
</ol>

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The authors have nothing to disclose.

In this manuscript, the engineering of an organ-on-chip device and the direct determination of the transendothelial electrical resistance (TEER) of a cellular barrier cultured in the device were presented. The presented method of integrating electrodes without cleanroom equipment and the direct TEER determination using four electrodes is applicable to any organ-on-chip device with two microfluidic compartments. The chip layout and geometry can be adapted to fit the requirements of the envisioned experiments, as long as the four electrodes are separated in two compartments. The four electrodes can even be conveniently inserted in the inlets of existing chips, provided that they are fixated in place for the duration of the six measurements. The leakage-free bonding method can be optimized for different membranes and channel geometries by changing the PDMS/toluene ratio. A higher toluene content results in a thinner spin-coated layer of mortar26 and may be more suitable for shallower and narrower channels in the PDMS parts. A lower toluene content results in a thicker mortar layer26 and may be more suitable to enclose thicker membranes between the PDMS parts.
As can be seen in the schematic impedance spectra in Figure 2A and the experimental spectra in Figure 2E and 2F, the impedance measurements are influenced by the double layer capacitance at the electrode-medium interface. Due to the small size of the electrodes inside the microchannels, the double layer capacitance can dominate over the resistive plateau of the cellular barrier in impedance spectra, complicating quantification of the TEER. To overcome this, the electrodes can be inserted further into the culture channels before fixation. This will increase the surface area of the electrode exposed to the culture medium and with that the double layer capacitance will increase as well. This results in a shift of the capacitive slope to lower frequencies, so that the resistive plateau of the cellular barrier can be more easily recognized and quantified. Although the resistance of the measured path between two electrodes will become smaller, this will not influence the TEER quantification following the presented method.
While measuring through the cellular barrier, it is possible that the extra resistive plateau cannot be recognized. This can be the result of a fluidic connection between the two channels, for example if the membrane is poorly enclosed by the mortar, leading to a measured path around the cellular barrier. In addition, there can be electrical bridging outside the chip if the electrodes are connected by a droplet of culture medium. This is generally combined with a lower measured impedance and can be solved by removing this bridge of culture medium. Lastly, if there is no resistive plateau and the measured impedance is orders of magnitude higher than expected, there may be a loose connection in the electrical wiring or at the power source.
In the future, the physiological relevance of the current BBB-on-chip can be increased by exposing the endothelial cells to shear stress at physiological levels, which is reported to promote BBB differentiation and increase barrier tightness and is hard to achieve in conventional in vitro models.29 In addition, the bottom channel of the presented device provides a suitable compartment for brain-derived cells to be co-cultured with the endothelium. This is also expected to increase the barrier function and also enables the study of the complex interactions between relevant cell types under pathological conditions.29
In conclusion, the organ-on-chip device described in this publication can be fabricated using standard laboratory equipment and is shown to provide direct TEER measurements using four integrated electrodes that do not impede visual inspection of the studied cell layer. The applicability of this device in the organs-on-chips field was demonstrated by mimicking the BBB in this chip and monitoring the TEER during the culture period. A host of other (barrier) organs can be mimicked by including other relevant cell types into this chip. In addition, the method for measuring TEER can be easily applied in other two compartment-based organ-on-chip devices to arrive at reproducible and meaningful TEER values that can be compared across devices and systems.

Organs-on-chips are rapidly emerging as a new and promising class of in vitro tissue models.1 In these models, cells are cultured inside microfluidic devices that are engineered in such a way that they mimic the physiological microenvironment of those cells.1,2 This results in more realistic physiological or pathological behavior of those cells than can be expected from conventional in vitro models of simple design and basic function.3,5,6 In addition, organs-on-chips provide a better controllable environment than in vivo models and can incorporate both healthy and diseased tissue from human origin to faithfully replicate both human physiology and pathology. The recently summarized advances in the development of blood-brain barriers on chips (BBBs-on-chips) show that the field is quickly moving forward.7
Another advantage of organs-on-chips is that they enable real-time and continuous monitoring of the tissue cultured inside the device by microscopy, on-line biochemical analyses and integrated sensors.1,2 For example, measuring transendothelial or transepithelial electrical resistance (TEER) is a powerful method for monitoring non-invasively the development and disruption of barrier-forming tissues.8,9,10 TEER is the electrical resistance across a cellular barrier and is therefore indicative of the barrier integrity and permeability.10 In organs-on-chips, cellular barriers are generally cultured on a membrane that separates two fluidic channels, representing the apical and basolateral compartments of that barrier tissue. In such chips, TEER measurements can be conveniently conducted with electrodes inserted into the inlets and outlets of the two channels.3,4,11,12,13,14,15 However, manual insertion and reinsertion of the electrodes can easily result in placement errors and thus in variations in the measured resistance as e.g. the differences in resistance of longer or shorter paths through microchannels are significant compared to the cell barrier resistance.16 To eliminate reinsertion errors, devices with integrated electrodes have been proposed. However, most of these integrated electrodes block the view when inspecting the tissue culture17,18,19,20,21 and/or require specialized cleanroom processes for fabrication.17,22
The organ-on-chip device described in this publication, first applied in an earlier publication,16 combines the stability of integrated electrodes with visibility on the measured cell layer and easy fabrication. The design and fabrication of this chip is depicted in Figure 1. In short, this device consists of two polydimethylsiloxane (PDMS) parts with channel imprints that are bonded together leakage-free with a polycarbonate membrane with 0.4 &#181;m pores in between. Four platinum wire electrodes are inserted and fixed into place with a photocurable adhesive well outside of the culture area. All of these fabrication steps can be conducted with general laboratory equipment, without the need for a cleanroom environment. On top of this, six impedance measurements can be done using these four electrodes, thereby allowing direct isolation of the measured TEER, independent of the resistance of the microchannels leading up to the cross section and thus minimizing the influence of non-biological variations in the system such as (re)insertion errors.16
To show the applicability of this device and the direct TEER measurements, the blood-brain barrier (BBB) was replicated in this chip. This biological barrier consists of specialized endothelial cells and regulates transport between blood and brain to provide brain homeostasis.23,24 To mimic the BBB, the top channel of the microfluidic device was lined with human cerebral microvascular endothelial cells from the hCMEC/D3 cell line (kindly provided by Dr. P.-O. Couraud, INSERM, Paris, France).25 The presented method is, however, more generally applicable to any organ-on-chip device with two compartments, enabling direct TEER determination using easily integrated electrodes.
In this manuscript, first the fabrication process of the organ-on-chip device with integrated electrodes is described. Next, the seeding procedure and culture of brain endothelial cells inside the device is explained, as well as the on-chip TEER measurements. In the results section, representative TEER measurements are shown and data processing is clarified. Lastly, the barrier function of the BBB-on-chip, monitored during 3 days, is presented, showing the applicability of the presented device and methods to monitor TEER.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Polydimethylsiloxane (PDMS) base agent and curing agent: Sylgard 184 Silicone elastomer kit</td><td>Dow Corning</td><td>1673921</td><td></td></tr><tr><td>Scotch Magic tape</td><td>3M</td><td></td><td></td></tr><tr><td>Biopsy punch, 1.0 mm diameter</td><td>Integra Miltex</td><td>33-31AA-P/25</td><td></td></tr><tr><td>Polycarbonate membrane, 0.4 &amp;micro;m pore size</td><td>Corning</td><td>3401</td><td>Cut from Transwell culture inserts</td></tr><tr><td>Toluene</td><td>Sigma-Aldrich</td><td>244511</td><td></td></tr><tr><td>Platinum wire, 200 &amp;micro;m diameter</td><td>Alfa Aesar</td><td>10287</td><td></td></tr><tr><td>UV-curable adhesive: Norland Optical Adhesive 81</td><td>Norland Products</td><td>NOA 81</td><td></td></tr><tr><td>Epoxy adhesive: Loctite M-31 CL Hysol</td><td>Henkel</td><td>30673</td><td></td></tr><tr><td>hCMEC/D3 cells</td><td></td><td></td><td>Human cerebral microvascular endothelial cell line, kindly provided by Dr. P.-O. Couraud, INSERM, Paris, France</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma</td><td>P4417</td><td></td></tr><tr><td>Human plasma fibronectin, 20 &amp;micro;g/ml</td><td>Gibco</td><td>33016015</td><td></td></tr><tr><td>Endothelial growth medium-2 (EGM-2)</td><td>Lonza</td><td>CC-3162</td><td>Endothelial basal medium-2 (EBM-2) supplemented with EGM-2 SingleQuots</td></tr><tr><td>Trypsin-EDTA, 0.05%</td><td>Gibco</td><td>15400-054</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>26140-079</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Oven</td><td>Binder</td><td>9010-0190</td><td></td></tr><tr><td>Spin coater: Spin 150</td><td>Polos</td><td>SPIN150-NPP</td><td></td></tr><tr><td>UV light source, 365 nm for 5 s at 350 mW/cm&lt;sup&gt;2&lt;/sup&gt;</td><td>Manufactured in-house</td><td></td><td></td></tr><tr><td>Centrifuge: Allegra X-12R Centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Incubator</td><td>Binder</td><td>CB E2 150</td><td></td></tr><tr><td>Boxense</td><td>LocSense</td><td></td><td>Lock-in amplifier with probe cable circuit, specialized for on-chip TEER measurements</td></tr></tbody></table>

fabrication and validation of an organ-on-chip system with integrated electrodes to directly quantify transendothelial electrical resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

The schematic results of electrical impedance spectroscopy through a chip without cells (solid line) and through a cellular barrier (dashed line) are shown in Figure 2A. Four main regions can be identified, each dominated by a specific electrical component. Below approximately 1 kHz, the double layer capacitance at the electrode-culture medium interface dominates, characterized by a negative slope for impedance magnitude and a phase shift approaching -90&#176;. The frequency at which the double layer capacitance dominates, depends on the electrode area exposed to culture medium. The resistive plateau above 1 kHz (without cells) or 100 kHz (with cells) with a phase shift close to 0&#176;, corresponds to the resistance of the culture medium inside the microfluidic channels, depending on channel length and cross-sectional area, and inside the membrane, depending on the porosity and thickness. When measuring through a cellular barrier, an extra resistive plateau between 1 and 10 kHz is seen as well as a local maximum in the phase diagram. This region is of critical importance for the determination of the TEER as a clear increase in impedance results when cells are present in the measured path, and is therefore termed the &#34;region of interest&#34;. The extra slope between 10 and 100 kHz corresponds to the cell barrier capacitance, which arises from the electrically insulating lipid bilayer membranes and is dependent on the total area of the cell layer.27 The boundaries of these regions as well as the impedance magnitudes depend on the system being studied and change with, among other things, channel dimensions, culture medium conductivity, electrode position and cell type. For further reading about the theory and practice of electrical impedance spectroscopy on barrier-forming tissues the review article by Benson et al. is recommended.28
Representative data of the TEER measurements is shown for both a blank chip and a chip with a hCMEC/D3 brain endothelial cell layer in Figure 2E and 2F, respectively. In short, impedance spectroscopy was performed using six measurements with four electrodes: two measurements through cell culture medium-filled channels (solid lines) and four measurements through the channels as well as the membrane and - if present - the cellular barrier (dashed lines). These six measurement paths can be identified in the equivalent resistive circuit of Figure 2B, which is derived from the schematic cross section (Figure 2C) and top view (Figure 2D). The blank chip measurements (Figure 2E) show the typical shape of impedance spectra without cells, as illustrated in Figure 2A. The measurements through the cellular barrier (dashed lines in Figure 2F) resemble the typical impedance spectra with cells in Figure 2A. Note that both the impedance magnitude and the phase shift increase towards 1 MHz. This is the typical response of the measurement setup at high frequencies and is not of experimental origin.
To determine the TEER using these experimental impedance spectra, first the measured chip resistance is determined, which is the total resistance of the cell layer, channels and membrane. To this end, a suitable readout frequency in the region of interest was chosen, which is at the local maximum in the phase plot with cells and close to 0&#176; phase shift without cells: 10 kHz. With the six measured resistances at 10 kHz, as measured between the four electrodes, the TEER is directly calculated using the equation in Figure 2F.
To show that the presented device is suitable for determining TEER in organs-on-chips, the BBB was replicated inside the chip and its TEER was monitored during 3 days of culture. In Figure 3A the average TEER &#177; standard error of the mean (SEM) is shown for four BBBs-on-chips, resulting in a plateau of 22 &#177; 1.3 &#8486; cm2, which is comparable to the TEER of this cell line as reported in literature.29 Furthermore, after termination of the experiment and fluorescence staining of the nuclei, it can be seen that the brain endothelium formed a continuous monolayer inside the device (Figure 3B). Immunofluorescence staining of tight junction protein Zonula Occludens-1 (ZO-1) showed that the brain endothelial cells maintained their BBB-specific phenotype and formed tight junctional complexes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56334/56334fig1.jpg" /> 
Figure 1: Design and assembly of the organ-on-chip device with integrated electrodes. 
(A) Exploded view of the microfluidic chip, consisting of a top PDMS part with the top channel (TC), a membrane (M) and a bottom PDMS part with the bottom channel (BC). Four platinum wire electrodes (E1-4) are inserted and fixed in the side channels. In the top channel and on top of the membrane, hCMEC/D3 brain endothelial cells were cultured to replicate the BBB. (B) Assembled chip, fixed to a plastic dish. Reprinted and adapted with permission from Elsevier.16 <a href="https://cloudflare.jove.com/files/ftp_upload/56334/56334fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56334/56334fig2.jpg" /> 
Figure 2: Representative impedance data and determination of TEER. 
(A) Schematic impedance spectrum showing impedance magnitude (&#8486;) and phase shift (&#176;) versus frequency (Hz), typical for electrical impedance spectroscopy on chips without cells (solid line) and with cells (dashed line). There are four main regions, each dominated by: the double layer capacitance at the electrodes, the culture medium resistance, the cell barrier resistance and the cell membrane capacitance. The &#34;region of interest&#34; indicates where the contribution of the cell layer can be quantified (red arrow). (B) Equivalent resistive circuit of the chip, showing the top channel resistors R1 and R3, the bottom channel resistors R2 and R4 and the membrane and EC barrier resistor Rm. (C) Schematic cross section showing the endothelial cells (EC) cultured in the top channel. (D) Schematic top view of the BBB chip showing the configuration of the electrodes and the culture area of 0.25 mm2 through which the impedance is measured. (E) Representative impedance spectra of a blank chip filled with cell culture medium. (F) Representative impedance spectra of a chip in which hCMEC/D3 brain endothelial cells were culture for 3 days. (G) Formula to calculate TEER from the measured resistances between all six combinations of four electrodes. Reprinted and adapted with permission from Elsevier.16 <a href="https://cloudflare.jove.com/files/ftp_upload/56334/56334fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56334/56334fig3.jpg" /> 
Figure 3: Representative TEER development of BBB-on-chip. 
(A) Average TEER &#177; standard error of the mean (SEM) of four BBBs-on-chips during a culture period of three days, reaching a plateau at 22 &#177; 1.3 &#8486; cm2 (average &#177; SEM). For comparison, data of blank chips is included, showing marginal variation and deviation from 0 &#8486; cm2 in the same period compared to the variation and TEER value of chips with cells. (B) Fluorescence microscopy of stained nuclei revealed a continuous monolayer of endothelium, both on PDMS and the membrane at the location indicated in the inset. (C) Immunofluorescence revealed the presence of tight junction protein Zonula Occludens-1, indicating that BBB-specific tight junctions between the cells give rise to the measured TEER. Reprinted and adapted with permission from Elsevier.16 <a href="https://cloudflare.jove.com/files/ftp_upload/56334/56334fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance at JoVE.com

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

fabrication-validation-an-organ-on-chip-system-with-integrated

Research

JoVE Journal

Bioengineering

16.2K Views.  University of Twente. Deze publicatie beschrijft de fabricage van een apparaat van de orgel-on-chip met geïntegreerde elektroden voor directe kwantificering van signaalcomplexen elektrische weerstand (TEER). Voor een validatie, de bloed - hersen barrière werd geïmiteerd binnen dit microfluidic apparaat en zijn functie als barrière werd gecontroleerd. De gepresenteerde methoden voor de integratie van de elektrode en directe kwantificering van de TEER zijn algemeen toepasbaar.

Video: Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Developmental Biology

Scalable Fabrication of Stretchable, Dual Channel, Microfluidic Organ Chips

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human patients. Human Organ-on-a-Chip (Organ Chip) microfluidic cell culture devices, which provide an experimental in vitro platform to assess efficacy, toxicity, and pharmacokinetic (PK) profiles in humans, may be better predictors of therapeutic efficacy and safety in the clinic compared to animal studies. These devices may be used to model the function of virtually any organ type and can be fluidically linked through common endothelium-lined microchannels to perform in vitro studies on human organ-level and whole body-level physiology without having to conduct experiments on people. These Organ Chips consist of two perfused microfluidic channels separated by a permeable elastomeric membrane with organ-specific parenchymal cells on one side and microvascular endothelium on the other, which can be cyclically stretched to provide organ-specific mechanical cues (e.g., breathing motions in lung). This protocol details the fabrication of flexible, dual channel, Organ Chips through casting of parts using 3D printed molds, enabling combination of multiple casting and post-processing steps. Porous poly (dimethyl siloxane) (PDMS) membranes are cast with micrometer sized through-holes using silicon pillar arrays under compression. Fabrication and assembly of Organ Chips involves equipment and steps that can be implemented outside of a traditional cleanroom. This protocol provides researchers with access to Organ Chip technology for in vitro organ- and body-level studies in drug discovery, safety and efficacy testing, as well as mechanistic studies of fundamental biological processes.

Here, we present a protocol that describes the fabrication of stretchable, dual channel, organ chip microfluidic cell culture devices for recapitulating organ-level functionality in vitro.

A significant number of lead compounds fail in the pharmaceutical pipeline because animal studies often fail to predict clinical responses in human ...

The Fabrication and Operation of a Continuous Flow, Micro-Electroporation System with Permeabilization Detection

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is accompanied by high manufacturing costs, which has brought about an interest in using alternative methods for gene delivery. Electroporation is an electro-physical, non-viral approach for the intracellular delivery of genes and other exogenous materials. Upon the application of an electric field, the cell membrane temporarily allows molecular delivery into the cell. Typically, electroporation is performed on the macroscale to process large numbers of cells. However, this approach requires extensive empirical protocol development, which is costly when working with primary and difficult-to-transfect cell types. Lengthy protocol development, coupled with the requirement of large voltages to achieve sufficient electric-field strengths to permeabilize the cells, has led to the development of micro-scale electroporation devices. These micro-electroporation devices are manufactured using common microfabrication techniques and allow for greater experimental control with the potential to maintain high throughput capabilities. This work builds off a microfluidic-electroporation technology capable of detecting the level of cell membrane permeabilization at a single-cell level under continuous flow. However, this technology was limited to 4 cells processed per second, and thus a new approach for increasing the system throughput is proposed and presented here. This new technique, denoted as cell-population-based feedback control, considers the cell permeabilization response to a variety of electroporation pulsing conditions and determines the best-suited electroporation pulse conditions for the cell type under test. A higher-throughput mode is then used, where this 'optimal' pulse is applied to the cell suspension in transit. The steps for fabricating the device, setting up and running the microfluidic experiments, and analyzing the results are presented in detail. Finally, this micro-electroporation technology is demonstrated by delivering a DNA plasmid encoding for green fluorescent protein (GFP) into HEK293 cells.

This protocol describes the microfabrication techniques required to build a lab-on-a-chip, microfluidic electroporation device. The experimental setup performs controlled, single-cell-level transfections in a continuous flow and can be extended to higher throughputs with population-based control. An analysis is provided showcasing the ability to electrically monitor the degree of cell membrane permeabilization in real-time.

Current therapeutic innovations, such as CAR-T cell therapy, are heavily reliant on viral-mediated gene delivery. Although efficient, this technique is ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Reconstituting Cytoarchitecture and Function of Human Epithelial Tissues on an Open-Top Organ-Chip

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional (3D) structures. One of the main functions of epithelia is the formation of barriers that protect the underlining tissues against physical and chemical insults and infectious agents. In addition, epithelia mediate the transport of nutrients, hormones, and other signaling molecules, often creating biochemical gradients that guide cell positioning and compartmentalization within the organ. Owing to their central role in determining organ-structure and function, epithelia are important therapeutic targets for many human diseases that are not always captured by animal models. Besides the obvious species-to-species differences, conducting research studies on barrier function and transport properties of epithelia in animals is further compounded by the difficulty of accessing these tissues in a living system. While two-dimensional (2D) human cell cultures are useful for answering basic scientific questions, they often yield poor in vivo predictions. To overcome these limitations, in the last decade, a plethora of micro-engineered biomimetic platforms, known as organs-on-a-chip, have emerged as a promising alternative to traditional in vitro and animal testing. Here, we describe an Open-Top Organ-Chip (or Open-Top Chip), a platform designed for modeling organ-specific epithelial tissues, including skin, lungs, and the intestines. This chip offers new opportunities for reconstituting the multicellular architecture and function of epithelial tissues, including the capability to recreate a 3D stromal component by incorporating tissue-specific fibroblasts and endothelial cells within a mechanically active system. This Open-Top Chip provides an unprecedented tool for studying epithelial/mesenchymal and vascular interactions at multiple scales of resolution, from single cells to multi-layer tissue constructs, thus allowing molecular dissection of the intercellular crosstalk of epithelialized organs in health and disease.

The present protocol describes the capabilities and the essential culture modalities of the Open-Top Organ-Chip for the successful establishment and maturation of full-thickness organ-on-chip cultures of primary tissues (skin, alveolus, airway, and intestine), providing the opportunity to investigate different functional aspects of the human epithelial/mesenchymal and vascular niche interface in vitro.

Nearly all human organs are lined with epithelial tissues, comprising one or multiple layers of tightly connected cells organized into three-dimensional ...

TEEI Method for Electroporation Analysis and Delivery Prediction in A431 Cells

Porous Substrate-Based Electroporation with Transepithelial Electrical Impedance Monitoring

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other types of intracellular delivery, PSEP relies heavily on fluorescent markers and fluorescent microscopy to determine successful delivery. To gain insight into the intermediate steps of the electroporation process, a PSEP platform with integrated transepithelial electrical impedance (TEEI) monitoring was developed. Cells are cultured in commercially available inserts with porous membranes. After a 12 h incubation period to allow for the formation of a fully confluent cell monolayer, the inserts are placed in transfection media located in the wells of the PSEP device. The cell monolayers are then subjected to a user-defined waveform, and delivery efficiency is confirmed through fluorescent microscopy. This workflow can be significantly enhanced with TEEI measurements between pulsing and fluorescent microscopy to collect additional data on the PSEP process, and this additional TEEI data is correlated with delivery metrics such as delivery efficiency and viability. This article describes a protocol for performing PSEP with TEEI measurements.

Author Spotlight: Optimizing Porous Substrate Electroporation Through Micro and Nanochannels for Enhanced Monitoring and Intermediate Stage Characterization

Porous substrate electroporation (PSEP) pairs consistent, high throughput delivery with high cell viability. Introduction of transepithelial electrical impedance (TEEI) measurements provides insight into the intermediate processes of PSEP and allows for label-free delivery. This article discusses a method for performing PSEP delivery experiments and TEEI measurement analysis simultaneously.

Porous substrate electroporation (PSEP) is an emerging method of electroporation that provides high throughput and consistent delivery. Like many other ...

A Simple Approach to Perform TEER Measurements Using a Self-Made Volt-Amperemeter with Programmable Output Frequency

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model systems. In most cases, chopstick electrodes are used to determine the electric impedance between the upper and lower compartment of a cell culture filter insert system containing cellular monolayers. The filter membrane allows the cells to adhere, polarize, and interact by building tight junctions. This technique has been described with a variety of different cell lines (e.g., cells of the blood-brain barrier, blood-cerebrospinal fluid barrier, or gastrointestinal and pulmonary tract). TEER measurement devices can be readily obtained from different laboratory equipment suppliers. However, there are more cost-effective and customizable solutions imaginable if an appropriate voltammeter is self-assembled. The overall aim of this publication is to set up a reliable device with programmable output frequency that can be used with commercially available chopstick electrodes for TEER measurement.

Here, we demonstrate how to set up an inexpensive volt-amperemeter with programmable output frequency that can be used with commercially available chopstick electrodes for transepithelial/endothelial electrical resistance measurements.

Transepithelial/endothelial electrical resistance (TEER) has been used since the 1980s to determine confluency and permeability of in vitro barrier model ...

Biology

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...

Medicine

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

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