Electrical simulations and experimental results of 4-electrode AC transepithelial electrical resistance measurements in gut-on-a-chip

ELECTRICAL SIMULATIONS AND EXPERIMENTAL RESULTS

OF 4-ELECTRODE AC TRANSEPITHELIAL ELECTRICAL RESISTANCE

MEASUREMENTS IN GUT-ON-A-CHIP

Marinke van der Helm

_{, Olivier Henry}

_{, Tiama Hamkins-Indik}

_{, Michael Cronce}

_,

William Leineweber

_{, Mathieu Odijk}

_{, Andries van der Meer}

_{, Jan Eijkel}

_{, Donald Ingber}

_,

Albert van den Berg

_{, and Loes Segerink}

1

_{BIOS Lab on a Chip group, University of Twente, NETHERLANDS,}

2

_{Wyss Institute for Biologically Inspired Engineering, Harvard University, USA and}

_{Applied Stem Cell Technologies, University of Twente, NETHERLANDS}

Transepithelial electrical resistance (TEER) measurements are often used in organs-on-chips to monitor the barrier tightness of e.g. gut epithelium. Here, we present a chip with four integrated electrodes and a four-terminal alternating current (AC) measurement protocol to perform TEER measurements. The resulting impedance spectra are interpreted using electrical simulations, which include the chip with microfluidic channels, the four electrodes, the AC measurement protocol and the intestinal barrier cultured inside the device, which is modelled as a flat monolayer or as tissue with villi. Eventually, these simulations will be used to quantify TEER in ŸāFP2_{to enable}

comparison among different platforms.

KEYWORDS: Transepithelial electrical resistance, gut-on-a-chip, impedance spectroscopy, electrical simulations INTRODUCTION

In organs-on-chips, transepithelial electrical resistance (TEER) is often used to non-invasively monitor the formation and maturation of cellular barriers. Here, we show four-terminal alternating current (AC) TEER measurements in a gut-on-a-chip with integrated electrodes, accompanied by electrical simulations for understanding and interpretation. We previously demonstrated the value of electrical simulations to quantify direct current (DC) TEER measurements in a gut-on-a-chip [1], while Yeste et al. have recently simulated four-terminal DC TEER measurements for various integrated electrode configurations [2]. Here we progress on these works by performing four-terminal AC TEER and barrier capacitance measurements and interpreting the results by simulations. Additionally, wrinkled cell layers were included in the simulations, as gut epithelial cells differentiate towards complex 3D villi when cultured “dynamically” (i.e., under flow) [3]. Thus, we compare simulated with experimental impedance spectra for both epithelial monolayers (“static” culture) and 3D villi-structured tissue (“dynamic”) in a gut-on-a-chip.

EXPERIMENTAL

The chip, that was recently published [4], comprises two polydimethylsiloxane (PDMS) parts with channels, separated by a porous membrane and sandwiched between polycarbonate (PC) substrates with four deposited semi-transparent gold electrodes (Figure 1A-C). Caco-2 gut epithelium was cultured inside the device either statically (medium refreshed daily) or dynamically (30 μL/hr medium perfusion). For TEER measurements, electrochemical impedance spectroscopy was performed daily using a four-terminal setup (two excitation and two readout electrodes) in galvanostatic mode.

For electrical simulations the volume inside the chip was represented by a network of nodes with electrical properties (resistance and/or capacitance) reflecting the corresponding system structures (Figure 1D). An AC current was applied to the excitation electrodes, after which the resulting potential in each node was determined using Kirchhoff’s and Ohm’s laws. From the input current and the potential difference between the readout electrodes, the impedance was determined.

978-0-692-94183-6/µTAS 2017/$20©17CBMS-0001 1114 21st International Conference on Miniaturized Systems for Chemistry and Life Sciences October 22-26, 2017, Savannah, Georgia, USA

Figure 1: A. Picture of gut-on-a-chip device, reprinted from [4]. B. Exploded view. Channel width is 1 mm. C. Schematic cross section. Bare membrane (before seeding cells), Caco-2 monolayer (when cultured statically) and 3D villi (when cultured dynamically) are all schematically illustrated. To measure TEER, an AC current is applied between the excitation electrodes and the resulting potential is measured between the readout electrodes. D. Build-up of electrical model of the chip and meas-urements.

RESULTS AND DISCUSSION

The experimental impedance spectra of the static culture (Figure 2A) show increasing barrier resistance (at ±10 Hz) at constant capacitance (slope at 100 Hz - 10 kHz) after day 3, indicating formation of a cellular barrier. 8QIRUWXQDWHO\TXDQWLILFDWLRQRIWKH7((5ŸāFP2_{) is not trivial as the effectively measured culture area is not}

known. Furthermore, four-terminal impedance spectra need careful interpretation.

Simulated impedance spectra of the static culture (increasing input TEER and constant capacitance) are similar to the experimental spectra (Figure 2B). Potential distributions (Figure 2C) show that the potential difference between the readout electrodes is smaller than between the excitation electrodes, underestimating the measured impedance. Additionally, the barrier contributes non-uniformly to the total measured resistance, especially at lower TEER values. We therefore plan to use simulated spectra to analyze experimental data and quantify corresponding TEER and barrier capacitance values.

In contrast, experimental spectra of the dynamic culture (Figure 2D) show a decreasing barrier resistance and increasing capacitance after day 5. This is most likely due to differentiation towards 3D villi, as evidenced microscopically. Our simulations confirm this hypothesis: simulated spectra show that for fixed input TEER and capacitance the apparent barrier resistance decreases and the apparent capacitance increases with increasing villi height (Figure 2E). Thus, our electrical simulations aid correct interpretation of these measured spectra.

Figure 2: A. Experimental impedance spectra of developing Caco-2 barrier (static). B. Corresponding simulated imped-ance spectra with increasing input TEER and fixed capacitimped-ance. C. Potential distributions at 10 Hz for three different input TEERs. D. Experimental impedance spectra of developing Caco-2 barrier (dynamic), forming villi (inset: confocal sideview after day 9; blue = nuclei, green = actin). E. Corresponding simulated impedance spectra with increasing villi height at fixed TEER (750 ŸÂFP2_{). Villi are modeled sinusoidally. F. Potential distributions at 10 Hz for three different villi heights.}

CONCLUSION

In conclusion, the presented electrical simulations are a useful tool for correct interpretation of AC TEER measurements in a gut-on-a-chip with four integrated electrodes. Most strikingly, the electrical simulations correctly predicted the influence of villi-formation on the measured impedance spectra. Eventually, we will use the electrical simulations to quantify the 7((5LQŸāFP2_{to enable comparison among different platforms.}

ACKNOWLEDGEMENTS

Funding sources: SRO Biomedical Microdevices (L.I. Segerink) and SRO Organs-on-chips (A.D. van der Meer) by MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente; VESCEL, ERC Advanced Grant to A. van den Berg (Grant no. 669768); Wyss Institute for Biologically Inspired Engineering at Harvard University; and the Defense Advanced Research Projects Agency (Cooperative Agreement Number W911NF-12-2-0036).

REFERENCES

[1] M. Odijk, A.D. van der Meer, D. Levner, H.J. Kim, M.W. van der Helm, L.I. Segerink, J.-P. Frimat, G.A. Hamilton, D.E. Ingber, and A. van den Berg, “Measuring direct current trans-epithelial electrical resistance in organ-on-a-chip microsystems,” Lab Chip, 15(3), 745-752.

[2] J. Yeste, X. Illa, C. Gutiérrez, M. Solé, A. Guimerà, and R. Villa, “Geometric correction factor for transepithelial electrical resistance measurements in transwell and microfluidic cell cultures,” J. Phys. D Appl. Phys., 49(37), 375401.

[3] H.J. Kim, D. Huh, G.A. Hamilton, and D.E. Ingber, “Human gut-on-a-chip inhabited by microbial flora that experiences intestinal peristalsis-like motions and flow,” Lab Chip, 12(12), 2165-2174.

[4] O.Y.F. Henry, R. Villenave, M.J. Cronce, W.D. Leineweber, M.A. Benz, and D.E. Ingber, “Organs-on-chips with integrated electrodes for trans-epithelial electrical resistance (TEER) measurements of human epithelial barrier function,” Lab Chip, 17, 2264-2271.

CONTACT

* M.W. van der Helm; phone: +31 53 489 4514; m.w.vanderhelm@utwente.nl