Abstract

1. Introduction

Endothelial cells provide a nonthrombogenic monolayer surface that lines the lumen of blood vessels and functions as a cellular interface between blood and tissue.¹ Epithelial cells line and provide a protective layer for both the outside and the inside cavities and lumen of the body.² Epithelial and endothelial cells are connected to each other via intercellular junctions that differ in their morphological appearance, composition and function. The tight junction or zona occludens is the intercellular junction that regulates diffusion³ and allows both of these cell layers to form selectively permeable cellular barriers that separate apical (luminal) and basolateral (abluminal) sides in the body, thereby controlling the transport processes to maintain homeostasis. Barrier integrity is vital for the physiological activities of the tissue. To successfully treat certain diseases of organs protected by physiological barriers, it is necessary to develop methods that can enable the transport of therapeutic drugs across these barriers in order to reach the target tissue.

Organs-on-chips⁴ or body-on-a-chip ^5-9 systems are microengineered biomimetic devices containing microfluidic channels and chambers populated by living cells, which replicate key functional units of living organs to reconstitute integrated organ-level pathophysiology in vitro. In recent years, organs-on-chips have garnered increasing attention due to both ethical and scientific reasons. In the European Union, in vitro methods will play a major role¹⁰ in future legislation on testing chemicals and also in relation to the seventh amendment to the Cosmetic Directive. Both of these policies call for broad replacement, reduction and refinement of animal experiments. Therefore, an extensive interest has been shown by many pharmaceutical, food and cosmetic industries in applying these body-on-a-chip systems for studying drug, nutrient and xenobiotic absorption and possible toxic effects.

The development of chip-based systems with in vitro cell barrier models can be used to study parameters that control permeability and predict drug transport across these barriers in the early stages of drug discovery. The growing interest in body-on-a-chip systems is due to their potential for providing a high throughput, cost-effective and reliable method for predicting drug interactions in humans including transport phenomena. These cell culture models also have an advantage of precisely controlling important transport parameters and experimental conditions. To perform permeability assessments on the cellular barriers, the complexity¹¹ of the in vitro models in these systems should reflect the variety of membrane transport systems, metabolic pathways involved and include a polarized cell layer. The in vitro models should also incorporate apical as well as basolateral compartments with appropriate composition of the aqueous medium on each side of the cell membrane. It may not be possible to develop a single in vitro system that can simulate all the in vivo conditions, but use of various in vitro systems with more than one type of cell (co-culture) as decision making tools in early drug discovery¹² is a common practice.

Numerous barrier systems^13-14 for predicting drug permeability, typically including cell cultures grown on permeable membranes, have been reported. The configuration in these systems is designed to allow access to both apical and basolateral compartments. These models primarily include cells that grow in a monolayer when seeded on permeable membranes, and have physiologic characteristics similar to the barrier physiology and functionality. The successful application of a system to predict drug absorption depends on how closely the in vitro model can mimic the characteristics of the in vivo barrier integrity. These in vitro models can be based on primary cells¹⁵ or cell lines.¹⁶

To perform reliable in vitro experiments, qualitative and quantitative techniques have been developed to first confirm and quantify the barrier integrity before proceeding with drug testing. A freeze-fracture electron microscopy of transmembrane fibrils and immunostaining for proteins characteristic of tight junctions (occluding, ZO-1 and ZO-2) can provide qualitative insights into the barrier integrity of an endothelial or epithelial monolayer. A simple assay that has been widely used is based on the permeability of the barrier to paracellular tracer compounds of various molecular weights. The first use of sucrose (molecular weight: 342 Da) labeled with carbon-14 for flux measurement on a brain endothelial monolayer has been reported.¹⁷ Radiolabeled markers provide the required sensitivity, but they need special safety precautions for handling and storage. Radiolabeled markers have a short half-life and are not suitable for long term storage. Some of the other frequently used paracellular tracers include inulin (molecular weight: 5 kDa) and mannitol (molecular weight: 182 Da). Albumin (molecular weight: 67 kDa) is another transendothelial permeability marker and has been observed in endothelial vesicles.¹⁸ Evans blue dye–albumin complex,¹⁹ albumin,²⁰ or biotin–albumin²¹ are used for quantification of serum protein permeability. An alternative approach that has been used is based on non-radioactive fluorescence labeled marker proteins such as fluorescein isothiocyanate (FITC)-labeled dextrans.²² However, solutes labeled with nonradioactive fluorophore compounds do not provide the required sensitivity to reflect the subtle changes in monolayer permeability that are due to poor specific activity (fluorescence/mg protein) or fluorophore instability.²³ Enzymatic markers such as horseradish peroxidase (HRP) have also been used to study macromolecule diffusion across endothelial monolayers by tracking supernatant HRP activity.²³ However, the activity of the enzymatic markers can be affected by factors such as pH, temperature and serum constituents, thereby limiting their application. The permeability with these above methods is quantified by the endothelial permeability coefficient^24-25 which is calculated based on the measured flux of the selected tracer across monolayers and cell-free inserts.

The use of tracer compounds can interfere with the transport process under study and can also affect the barrier integrity. Also, the use of chemical dyes renders the tested cells unusable for further experiments. Therefore, non-invasive techniques are best suited to continuously monitor the barrier integrity. Transepithelial/transendothelial electrical resistance (TEER) is the measurement of electrical resistance across a cellular monolayer and is a very sensitive and reliable method to confirm the integrity and permeability of the monolayer. Although the measurement of TEER and of trans-epithelial passage of marker molecules are both indicators of the integrity of the tight junctions and of the cell monolayer, they determine different experimental parameters.¹⁰ TEER reflects the ionic conductance of the paracellular pathway in the epithelial monolayer, whereas the flux of non-electrolyte tracers (expressed as permeability coefficient) indicates the paracellular water flow, as well as the pore size of the tight junctions.¹⁰ The advantages and wide use of the TEER method is because it is non-invasive and can be applied to monitor live cells during their various stages of growth and differentiation. This paper discusses some of the theoretical aspects of different TEER measurement techniques, examples of commercially available TEER measurement systems and their applications, a survey of TEER measurement for in vitro models and microfluidic implementations of a few widely studied cellular barriers, and various factors that affect TEER measurements.

2. TEER Measurement Methods

2.1 Ohm's Law Method

The electrical resistance of a cellular monolayer, measured in ohms, is a quantitative measure of the barrier integrity.²⁶ The classical setup for measurement of TEER, as shown in Figure 1, consists of a cellular monolayer cultured on a semipermeable filter insert which defines a partition for apical (or upper) and basolateral (or lower) compartments. For electrical measurements, two electrodes are used, with one electrode placed in the upper compartment and the other in the lower compartment and the electrodes are separated by the cellular monolayer. In theory, the ohmic resistance can be determined by applying a direct current (DC) voltage to the electrodes and measuring the resulting current. The ohmic resistance is calculated based on Ohm's law as the ratio of the voltage and current. However, DC currents can damage both the cells and the electrodes. To overcome this issue, an alternating current (AC) voltage signal with a square waveform is applied. In the widely used and commercially available TEER measurement system known as an Epithelial Voltohmmeter (EVOM),²⁷ an AC square wave at a frequency of 12.5 Hz is used to avoid any charging effects on the electrodes and the cell layer. The EVOM system has a measurement range of 1-9,999 Ω with a 1Ω resolution and uses a pair of electrodes popularly known as a STX2/“chopstick” electrode pair. Each stick of the electrode pair (4 mm wide and 1 mm thick) contains a silver/silver chloride pellet for measuring voltage and a silver electrode for passing current. The measurement procedure includes measuring the blank resistance (R_BLANK) of the semipermeable membrane only (without cells) and measuring the resistance across the cell layer on the semipermeable membrane (R_TOTAL) The cell specific resistance (R_TISSUE) in units of Ω, can be obtained as:

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Figure 1

TEER measurement with chopstick electrodes. The total electrical resistance includes the ohmic resistance of the cell layer R_TEER, the cell culture medium R_M, the semipermeable membrane insert R_I and the electrode medium interface R_EMI.

$$R_{\mathit{\text{TISSUE}}}(\Omega )=R_{\mathit{\text{TOTAL}}}-R_{\mathit{\text{BLANK}}}$$

Where resistance is inversely proportional²⁸ to the effective area of the semipermeable membrane (M_AREA) which is reported in units of cm².

$$R_{\mathit{\text{TISSUE}}}(\Omega )\propto \frac{1}{M_{\mathit{\text{AREA}}}({\mathit{\text{cm}}}^2)}$$

TEER values are typically^28-29 reported (TEER_REPORTED) in units of Ω.cm² and calculated as:

$${\mathit{\text{TEER}}}_{\mathit{\text{REPORTED}}}=R_{\mathit{\text{TISSUE}}}(\Omega )\times M_{\mathit{\text{AREA}}}({\mathit{\text{cm}}}^2)$$

The TEER readings with EVOM2 are highly dependent on the electrode positions and a careful handling of the electrodes is required while introducing them into the well under test to avoid any disturbance to the cells. The uniformity of the current density generated by the electrodes across the cell layer has a significant effect on the TEER measurements. The STX2/chopstick electrode cannot deliver a uniform current density over a relatively large membrane,²⁷ such as the ones used in 24 mm diameter tissue culture inserts, and leads to an overestimation of the TEER value. As an alternative to STX2/chopstick electrodes, an EndOhm chamber³⁰ which allows the cups from culture wells to be inserted can be used. In an EndOhm chamber, both the chamber and the cap contain a pair of concentric electrodes: a voltage-sensing silver/silver chloride pellet in the center plus an annular current electrode. The symmetrical arrangement of circular disc electrodes on both sides of the membrane in an EndOhm chamber generates a more uniform current density across the membrane when compared to STX2/chopstick electrodes. Also, with Endohm's fixed electrode geometry, variation of measurements on a given sample is reduced³⁰ to 1-2 Ω when compared to 10-30 Ω with STX2 electrodes. Apart from the EVOM/STX2/EndOhm systems, a few of the other commercial systems available for TEER measurements include Electric Cell-Substrate Impedance Sensing (ECIS)³¹ (Applied BioPhysics Inc., Troy, NY), REMS AutoSampler (World Precision Instruments, Sarasota, FL), Millicell-ERS system^{10, 32} (Millipore Corp., Bedford, USA) and Ussing Chamber Systems³³ (Warner Instruments, Hamden, CT).

2.2 Impedance Spectroscopy

Impedance spectroscopy when combined with a fitting algorithm provides a more accurate representation of TEER values than traditional DC/single frequency AC measurement systems.³⁴ Impedance spectroscopy is performed by applying a small amplitude AC excitation signal with a frequency sweep and measuring the amplitude and phase response of the resulting current. Figure 2 (a) indicates a schematic which illustrates the concept of impedance measurement.

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Figure 2

(a) TEER measurement concept based on impedance spectroscopy (b) Components of Impedance.

Electrical impedance (Z) is the ratio of the voltage-time function V (t) and the resulting current-time function I (t):

$$\mathrm{Z}=\frac{\mathrm{V}\left(\mathrm{t}\right)}{\mathrm{I}\left(\mathrm{t}\right)}=\frac{{\mathrm{V}}_{\mathrm{o}}sin\mathrm{\theta }}{{\mathrm{I}}_{\mathrm{o}}sin(2\mathrm{\pi }\text{ft}+Ø)}=\frac{1}{\mathrm{Y}}$$

$$\mathrm{Z}=Z_R+j{Z}_I$$

where Vo and Io are the peak voltage and current, f is the frequency, t is the time, Φ is the phase shift between the voltage-time and current-time functions, and Y is the complex conductance or admittance. Z is a complex function and can be described by the modulus |Z| and the phase shift Φ or by the real part Z_R and the imaginary part Z_I, as illustrated in the Figure 2(b). An in depth analysis of impedance spectroscopy has been published.³⁵

Impedance measurement across a wide spectrum of frequencies instead of a DC/single frequency AC TEER measurement can provide additional information about the capacitance of the cell layer. An automated measurement system (cellZscope^®, nanoAnalytics GmbH, Germany) has been developed for measuring the transendothelial/epithelial impedance of various barrier-forming cells cultured on permeable membranes of standard cell culture inserts. An equivalent circuit analysis of the measured impedance spectrum is performed to obtain the electrical parameters that can be applied to characterize the cellular barrier properties. Figure 3 (a) (adapted from Benson et al.²⁶) shows a typical equivalent circuit diagram that can be applied to analyze the impedance spectrum of cellular systems.²⁶ In this circuit, the current can flow through the junctions between cells (paracellular route) or through the cell membrane of the cells (transcellular route). The tight junction proteins in the paracellular route contribute to an ohmic resistance (R_TEER) in the equivalent circuit. Each lipid bilayer in the transcellular route contributes to a parallel circuit²⁶ consisting of ohmic resistance (R_membrane) and an electrical capacitance (C_C). In addition to these elements, the resistance of the cell culture medium (R_medium) and the capacitance of the measurement electrodes (C_E) also have to be considered. The high values of R_membrane causes the current to mostly flow across the capacitor and allows an approximation where R_membrane can be ignored²⁶ and the lipid bilayers can be represented with just C_C. Based on this approximation, the equivalent circuit diagram can be further simplified as shown in Figure 3 (b) (adapted from Benson et al.²⁶) and the impedance spectrum observed will have a non-linear frequency dependency as shown in Figure 3 (c) (adapted from Benson et al.²⁶). Typically, there are three distinct frequency regions in the impedance spectrum where the impedance is dominated by certain equivalent circuit elements. In the low frequency range, the impedance signal is dominated by C_E. In the mid frequency range, the impedance signal is dominated by circuit elements related to the cells, namely R_TEER and C_C. In the high frequency range, C_C and C_E provide a more conductive path and the impedance signal is dominated by R_medium. These equivalent circuit parameters can be estimated by fitting the experimental impedance spectrum data to the equivalent circuit model using non-linear least squares fitting techniques to obtain the best fit parameters.

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Figure 3

(a) A typical equivalent circuit diagram that can be applied to analyze the impedance spectrum of cellular systems. (b) Simplified equivalent circuit (c) A typical impedance spectrum with distinct frequency dependent regions. Adapted from Benson et al.²⁶

The following sections describe the advantages of organs-on-chips for TEER measurement, in vitro models of some widely studied cellular barriers, TEER measurements with in vitro models and some microfluidic implementations, and the various factors affecting TEER values.

3. Organs-on-chips

The existing commercial systems for measuring TEER are mostly confined to static and macroscopic cellular environments. The STX/chopstick electrodes are not suitable for integration with body-on-a-chip systems due to the small cell culture area that is not easily accessible to allow electrode placement close to the cell culture area and variation between measurements when electrodes are not firmly secured in the same position. An integration of immobilized TEER electrodes directly within the chip model and in close proximity to the cellular monolayer can not only reduce the contribution of electrical resistance from the cell culture medium but also the signal noise generated by any electrode motion. The size of the electrodes can be scaled relative to the microchannel dimensions within the system. TEER measurements can be achieved with much smaller surface areas in a chip-based system when compared to conventional culture systems. However, with custom designed and embedded TEER electrodes, it is important to ensure that a uniform current density across the cellular monolayer is achieved during measurement. The non-uniformity of current density has proved to be one of the reasons for erratic TEER values.³⁶ The electrode design process for these chips can be combined with electrical simulation/modeling to ensure that a uniform current density is achieved. Organs-on-chips also provide the advantage of enabling the study of cells under physiologically relevant fluid flow conditions that are known to induce mechanotransductive effect on certain cell types. For example, many transport processes in kidney are known to be regulated by fluid flow and shear stress.³⁷ In vitro chips also provide precise control over physiologic stresses,³⁸ chemical signaling³⁹ and degree of cell to cell interaction⁴⁰ while reducing reagent consumption.

4. Blood-Brain Barrier Model

The blood-brain barrier (BBB)⁴¹ is an active interface between the circulatory system and the central nervous system (CNS) which restricts the free movement of different substances between the two compartments and plays a crucial role in maintaining homeostasis in the CNS. Figure 4 (adapted from Wong et al.⁴²) shows a schematic of the various components of a BBB model. Physiologically, the BBB⁴³ consists of an intricate network of vascular endothelial cells which are quite distinct from other endothelium due to the combination of a physical and biochemical barrier. The BBB endothelial cells are surrounded by basal lamina and astrocytic perivascular end-feet with astrocytes providing the cellular link to the neurons. The BBB endothelium⁴³ is characterized by the presence of tight junctions, a lack of fenestrations and minimal pinocytotic vesicles. The tight junction consists of transmembrane proteins (claudin, occludin, and junction adhesion molecules) and cytoplasmic accessory proteins. The details of the principal components of the BBB and their functions have been previously published.^{41, 43-44} The BBB has a dual function of a carrier and a barrier. The barrier function of the BBB is to restrict transport of potentially toxic or harmful substances from the blood to the brain. The carrier function allows for selective transport of nutrients to the brain and removal of metabolites from the cerebral spinal fluid that surrounds CNS tissue. Small lipid soluble compounds (<500 Da) such as alcohol, narcotics and anticonvulsants can pass through the BBB with relative ease. However, the BBB inhibits⁴³ the brain from taking up 100% of large molecule therapeutics (> 1000 Da) that includes genes, recombinant proteins as well as more than 98% of potential neurotherapeutics comprised of proteins and peptides (500-1000 Da). The development of drugs that can cross the BBB is a challenge and consequently belies the importance of developing in vitro models of the BBB that can mimic in vivo barrier properties for evaluation of these drugs.

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Figure 4

A schematic of BBB showing various components, brain capillary endothelial cells (BCECs), basement membrane, pericytes and astrocytes. Adapted from Wong et al.⁴²

Establishing in vitro models of the BBB has been a challenging task due to species differences, low availability of human primary brain endothelial cells, and poor barrier formation by immortalized human brain microvascular endothelial cells. The primary cell cultures widely used are brain endothelial cells from a spectrum of mammals of rat, mouse, pig, bovine or human origin. Some of the well characterized brain endothelial cell lines⁴¹ includes RBE4 (rat origin), GP8 (rat origin) and hCMEC/D3 (human origin). Porcine⁴⁵ and bovine⁴⁶ cell lines are also available but are not very well characterized. Although cerebral endothelial cells are the principal components of the BBB, there is increasing evidence of other cell types such as glial cells, pericytes and neurons playing important regulatory roles in the induction and maintenance of the BBB and can be included to develop more complex in vitro co-culture models of these systems.⁴⁷

TEER has been the most commonly used parameter to evaluate the functionality of the BBB since it was first introduced by Crone et al.⁴⁸ and was used to measure in vivo BBB properties of various species.^49-50 Table 1 indicates a range of TEER values reported for BBB models. More recently, Lippmann and colleagues have developed a better mimetic model by demonstrating the derivation of brain endothelial cells from human induced pluripotent stem cells.⁵¹ In these studies, various methods were used to evaluate the structural and functional properties of the BBB. This included immunolabeling of tight junction proteins, such as ZO-1, Occludin and Clauidin-5, permeability measurements of various types and sizes of molecules through the BBB and measurements of TEER. The TEER values measured in vivo have been reported to be as high as 5900 Ω.cm² which is markedly larger than that achieved by the majority of the currently available in vitro models.⁴⁹ In order to replicate in vivo TEER values in BBB models, endothelial cells have been co-cultured with other brain cell types, exposed to physiological shear stress⁵² or treated with various chemicals.⁵³ The models using cell lines, such as hCMEC/D.3, in co-culture with astrocytes have yielded relatively low TEER values⁵⁴ of 140 Ω.cm², whereas TEER values as high as 300 Ω.cm^{2 55} were achieved utilizing primary endothelial cells from rodents and has been extensively reviewed.⁵⁶ Lippmann et al. recently developed a co-culture model with iPSC-derived endothelial cells and neuronal progenitor cells in conjunction with chemical treatment to promote BBB formation.⁵³ In their model, they showed that retinoic acid treatment can increase the TEER values up to 4000 Ω.cm² and sustain it above 2000 Ω.cm² for four consecutive days. The majority of the studies focused on developing in vitro models using commercially available TEER equipment as previously described in this paper.

Several microfluidic platforms have been recently developed to improve BBB models by more precisely controlling biochemical and mechanical factors in the BBB microenvironment.⁵⁷ Due to design limitations and the small scale of microfluidic systems, it is not suitable to use conventional TEER electrodes. Instead, custom-built microelectrodes are usually integrated or inserted into the system without disrupting the culture conditions. A multi-layered microfluidic device comprising of four PDMS substrates, two glass layers, a porous polycarbonate membrane sandwiched between PDMS layers, and integrated thin-film electrodes has been reported.⁵⁷ The thin-film microelectrodes were fabricated by depositing layers of Cr, Au and Ag on glass, followed by chlorination of the silver surface chemically to generate an electrochemically active AgCl surface. For TEER measurements, these electrodes were connected to a commercial EVOM system that was described in a previous section. By exposing the cells to dynamic conditions which provide shear stress simulation, the TEER of b.End3 (endothelial) cells in co-culture with C8D1A cells (astrocytic) was increased to 250 Ω.cm². A PDMS based BBB microfluidic platform to perform mechanical and biochemical modulation¹⁴ of the BBB that is comprised of the immortalized human brain endothelial cell line hCMEC/D3 has been developed. To perform TEER measurements,¹⁴ an impedance/gain phase analyzer was coupled with two Pt wires (200 μm diameter) that were inserted through the PDMS side walls of the device. An impedance spectrum was recorded by applying the AC signal over the Pt electrodes with an amplitude of 0.2V in a frequency range of 100 Hz to 10 MHz. TEER values were estimated by fitting the measured impedance spectrum to an equivalent electrical circuit by applying a least-square optimization method.

Microfluidic models of BBB in combination with TEER measurement validation are promising for drug testing studies. The incorporation of recent human iPSC-derived EC models into microfluidic BBB platforms with physiologically relevant TEER values provide more realistic models for these studies.

5. Gastrointestinal Tract Models

Drug administration orally is usually preferred as it reduces any infection or pain associated with other invasive routes, is patient-friendly and doesn't require administration by medical personnel. However, the transport of drugs via the intestinal membrane is a complex and dynamic process that includes the passage of compounds across several pathways in parallel.¹¹ The intestinal mucosa is characterized by the presence of villi that constitutes the anatomical and functional unit for nutrient and drug absorption. The mucosa consists of an epithelial layer, the lamina propria and the muscularis mucosa.¹¹ Figure 5 (adapted from Antonissen et al.⁵⁸) indicates a schematic of the various components of a gastrointestinal (GI) tract model. The epithelial layer is considered to be the limiting hurdle for GI tract drug permeation and is therefore the histological component of most interest for in vitro recapitulation. The bioavailability of nutrients or drugs administered orally depends on the quality of the barrier function of the GI tract and is an important determinant, especially when investigating passive transport of substances through tight junctions. Tight junction complexes restrict passive substance diffusion through the paracellular spaces between epithelial cells, a process that can be compromised in diseased epithelial tissue.^59-60 Active transport mechanisms enable the transport of substances through the cell bodies of epithelial cells. Both active and passive transport can be better investigated when the barrier function of the GI tract tissue is established to mimic either healthy or disease conditions in the human body.

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Figure 5

A schematic of the GI tract model showing various components, intestinal epithelial cells (IEC), goblet cell. Adapted from Antonissen et al.⁵⁹.

At present, there are numerous cell lines and their co-cultures being studied for in vitro models of the GI tract. The most widely used cell line for developing human GI tract in vitro models is the Caco-2 line, derived from a human colorectal adenocarcinoma. These cells can be maintained easily in cell culture for many weeks and are capable of establishing tight junctions in culture. After 2-3 weeks, Caco-2 cells form a densely populated cell layer differentiating spontaneously into polarized enterocytes and a monolayer of columnar cells that are coupled together by tight junction protein complexes.^61-62 The Caco-2 monolayer generates a TEER of 150-400 Ω.cm² that restricts the diffusion of substances across the barrier. Caco-2 cells are often used for studying nutrient and drug transport as they provide the main routes of substance transport from: 1) the paracellular route through tight junction complexes, 2) the passive transcellular route through the bodies of epithelial cells, 3) the active, carrier-mediated paracellular through cells, and 4) transcytosis.⁶³

Caco-2 cells alone do not fully represent the morphology and physiology of GI tract. The GI tract consists of a number of other cell types, particularly goblet cells that do not form tight junctions. Goblet cells are mucus-producing cells that coat the epithelium with a layer of mucus, providing an additional diffusion barrier that plays a role in nutrient uptake.⁶⁴ Peyer patches are formed from aggregated lymphoid cells, allow passage of large peptides and also do not form tight junctions; Peyer patches constitute less than 0.5% of the human intestine. Although other cell types (e.g. paneth and enteroendocrine cells) are present in much smaller numbers,⁶⁵ few groups have begun to include even cells such as goblet cells and lymphocytes into their in vitro models.^66-67 By including these cells, the strength of barrier function of such models is altered, rendering them more permeable and physiological.

A careful characterization of transport properties and TEER measurements will help in developing physiological models when utilizing immortalized cells lines. For example, immortalized cell lines such as HT29-MTX cells (goblet-type) have the potential to overgrow compared to Caco-2 cells when introduced into the culture in high numbers, giving rise to different barrier properties. To prevent this alteration in barrier properties, initial seeding ratios can be adjusted and the culture's relevance to physiologic transport can be tested.⁶⁸ In addition, selecting cultures with similar (or a minimum) TEER value before experimentation can be useful in preventing large variations in determination of transport rates due to unintended overgrowth.

IEC-18 cells, a small intestinal crypt-derived rat cell line, have been studied mainly for screening passive transcellular and paracellular transport of hydrophilic molecules.⁶⁹ Also, IEC-18 cells can simulate properties of the small intestine and could also simulate a leakiness/tightness of the paracellular pathway similar to human intestinal tissue.⁶⁹ However, IEC-18 cells are less well differentiated than the Caco-2 cells and not much is known about the carrier-mediated transport systems in these cell lines. TC-7 cells,⁷⁰ which are a subclone of Caco-2 cells, have also been studied for permeability screening and display higher enzyme content when compared with native Caco-2 cells. A co-culture model based on Caco-2 cells and human Raji B lymphocytes which are known to convert enterocytes into M cells (microfold cells) has been developed. The Caco-2/Raji B co-culture model has been applied to study the absorption of large molecules and nanoparticles containing drugs.⁷¹

Human primary GI tract cells harvested from patient samples mimic nutrient and drug transport with the highest similarity to in vivo conditions. These samples can contain all the different cell types of the GI tract to enable the re-establishment of 3D organization in vitro when appropriate 3D scaffolds are provided.⁷² Wang et al. have tested this approach with murine GI tract cells, finding that the cells generated continuous millimeter-scale tissue that resembled the architecture and geometry of the colonic epithelium in vivo.

Depending on the study to be conducted, researchers may choose to use different cells types or cell lines to model nutrient and drug transport, keeping in mind that the cell types used in the in vitro model as well as the cell culture conditions will determine the TEER that will be observed. Table 2 shows a range of some of the TEER values reported for GI tract models. Yee et al. discussed the problem of comparing TEER data across laboratories, suggesting that determining the permeability coefficients for substances may provide a more accurate basis for cross-laboratory comparison.⁷³ In addition, including other cell types into the model, such as mucus-producing goblet cells, has the potential to decrease TEER values which may make the model more physiological. GI epithelia are classified⁷⁴ based on TEER values as “tight” indicated by values of about 2000 Ω.cm², as “intermediate” indicated by values in the range of 300-400 Ω.cm² and as “leaky” indicated by values of 50-100 Ω.cm².

After the inception of microfluidic organs-on-chips, several groups have developed microfluidic models of the GI tract where apical and basolateral sides are accessible via microfluidic streams.^{68, 75-81} Some of these groups have already begun to utilize multiple cell types to develop GI tract models and combine them with models of liver tissue to investigate inter-organ relationships.^{68, 76, 80} The barrier function in microfluidic GI tract models has alternatively been evaluated by determining the permeability coefficients for fluorescently labeled dextran or standard drugs for which coefficients in intact cell cultures are known.⁷⁷

However those microfabricated systems that are designed for TEER measurements can offer TEER data in addition to permeability coefficients.⁷⁸ With the system developed by Kim et al., TEER values of up to 3000 to 4000 Ω.cm² were observed in the microfluidic system, while control cultures that were cultured under static conditions exhibited TEER values between 700 to 800 Ω.cm². The authors have not discussed whether the TEER values obtained from the static and microfluidic system are directly comparable. Nevertheless, such systems may make it possible to investigate the influence shear stress has on the barrier function of in vitro GI tract tissues. Also, these very high values of TEER are certainly not physiologic,⁷⁴ reducing the value of the system to replicate human physiology.

6. Pulmonary Models

The surface of the lung is lined with a continuous epithelium that can be divided into airway and alveolar epithelia. Figures 6 (adapted from Hollenhorst, 2011⁸² shows a schematic of the various cells of the airway and alveolar epithelium respectively. The gold standard culturing technique for obtaining physiologically relevant and functional pulmonary epithelium is to culture cells to confluence in liquid covered condition (LCC) followed by culture at an air-liquid interface (ALI).⁸³ Pulmonary epithelial cells secrete surfactants, develop tight junctions and differentiate into a specific cell type⁸³ when cultured at ALI for longer times. Many conventional and microfluidic in vitro pulmonary models representing pulmonary physiology have been developed by applying this culture technique that are comprised of primary cells, cell-lines and co-culture models.⁸⁴ The recent advances in microfabrication techniques have led to the development of microfluidic platforms for pulmonary models to incorporate and recapitulate complex physiological functions in vitro.^85-86

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Figure 6

A schematic overview of the human lung. Cells of the airway epithelium comprises ciliated, undifferentiated columnar, clara and basal cells. Cells of the alveolar epithelium comprise AT-I and AT-II type cells. Adapted from Hollenhorst, 2011. ⁸²

7. Other in vitro barrier models

Apart from the barrier models described in the previous sections, there are other in vitro barrier models that have been developed for predicting drug transport. These include placental,¹¹⁴ nasal,¹¹⁵ vaginal,¹¹⁶ ocular¹¹⁷ and skin¹¹⁸ based in vitro models. The focus of this paper was restricted to BBB, GI tract and pulmonary models, since they are more widely studied than the other models and so comparisons could be made between different methods and techniques.

8. Factors affecting TEER measurements

9. TEER Measurement – Significance and current challenges

TEER values are accepted as strong indicators of the integrity of the cellular barriers before they are evaluated for transport of drugs or chemicals. Impedance spectroscopy, in combination with equivalent circuit analysis, provides a more accurate representation of TEER values than the DC/single frequency AC measurement approach. The use of cells cultured under static conditions on permeable membranes of standard cell culture inserts with external STX2 electrodes is predominant. In order for these studies to be physiologically relevant, it is necessary to develop in vitro systems that can mimic in vivo conditions. Recently many microfluidic platforms have been developed to more precisely control biochemical and mechanical factors in the cellular microenvironment. Microfluidic platforms can allow the exposure of cells to dynamic conditions which provide shear stress simulation wherever applicable. The integration of microelectrodes within these systems would allow continuous monitoring of cells without disrupting the cell structure. However, there are electrical and microfluidic packaging challenges that would have to be overcome for these microfluidic platforms to be more widely used in labs for long term drug transport studies. TEER measurements can be a good benchmark for barrier integrity when comparing results from different sources if the results of these measurements are correlated with the numerous factors that affect the TEER values. The design of in vitro models should allow reproduction of TEER values close to in vivo conditions since the success of these models for drug development depends on how closely they mimic the complexity of drug absorption of the in vivo barriers.

10. Conclusions

TEER measurement is a valuable non-invasive technique that can be applied to quantify the barrier integrity of cells during their various stages of growth and differentiation. Organs-on-chips or body-on-a-chip systems integrated with TEER measurement capability can be an excellent high-throughput and cost-effective tool for drug toxicity and permeability predictions during the early stages of drug discovery. Numerous in vitro cell models composed of immortalized cells that can be grown on semipermeable supports under controlled conditions have been studied for drug permeability and absorption evaluation. The success of these studies to predict drug absorption depends on how closely the in vitro models can mimic the complexity of the drug absorption processes of the in vivo barriers. TEER measurements based on impedance spectroscopy are more reliable and provide more information about the cells when compared to the Ohm's law method. Even though TEER measurement systems are commercially available, integration of a TEER measurement system within the chip-based devices allows continuous non-invasive monitoring of the cells within an incubator without disrupting the cell culture environment. TEER measurements for various cell types have been reported with commercially available measurement setups and also with custom built microfluidic implementations. Also, for the same cell type, a wide range of TEER values have been reported in the literature. These variations can arise due to various factors affecting values such as the accuracy of the measurement techniques based on selection and usage of electrodes, temperature during measurement, medium formulation, cell culture period and passage number of cells used in the model. It may not always be possible to control every variable, but the aim should be to optimize and simplify conditions whenever needed. TEER values have proven to be simple and rapid indicators of cellular barrier integrity. However, in order to make a meaningful comparison of TEER results reported from different laboratories, it is important to not merely compare the values but also take into consideration all the above factors that affect TEER. Also, when reporting TEER values, laboratories should identify and report the above listed variables and the exact conditions under which TEER measurements were performed. The goal of in vitro models with respect to TEER measurement should be to reproduce the TEER values observed under in vivo conditions. Any use of models with TEER values much higher/lower than in vivo values may lead to incorrect conclusions while evaluating transport of drugs or chemicals.

Acknowledgments

References