Electrochemical impedance spectroscopy (EIS) device to resolve individual membrane electrical properties of epithelial and endothelial tissues

Abstract

Systems, devices and methods are provided for predicting characteristics of a tissue sample. An electrochemical impedance spectroscopy (EIS) device includes at least two electrodes for measuring electrical characteristics of sample, wherein a first electrode corresponds to an apical side of the sample, and a second electrode corresponds to a baso-lateral side of the sample; circuitry for converting electrical signals measured by the electrodes into digital information indicating values of the electrical signals; and a processor coupled to a memory storing instructions that, responsive to being executed by the processor, cause the EIS device to: identify a constraint parameter based on a type of the sample and / or one or more device parameters; and calculate, in accordance with an intracellular circuit model including two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the sample based on the values of the electrical signals and the constraint parameter.

Description

LEYDIG REF. 774627HHS REF. E-249-2023-0-PC-01ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY (EIS) DEVICE TO RESOLVE INDIVIDUAL MEMBRANE ELECTRICAL PROPERTIES OF EPITHELIAL AND ENDOTHELIAL TISSUESInventors: Colby LewallenArvydas MaminishkisKapil BhartiRuchi SharmaAssignee: The United States of America, as represented by the Secretary, Department ofHealth and Human ServicesEntity: LargeELECTROCHEMICAL IMPEDANCE SPECTROSCOPY (EIS) DEVICE TO RESOLVE INDIVIDUAL MEMBRANE ELECTRICAL PROPERTIES OF EPITHELIAL ANDENDOTHELIAL TISSUESSTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with Government support under Project Number Z01- HD000261 awarded by the National Institutes of Health. The Government has certain rights in this invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This Patent Application claims priority to U.S. Provisional Patent Application No. 63 / 708,904, entitled “ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY (EIS) DEVICE TO RESOLVE INDIVIDUAL MEMBRANE ELECTRICAL PROPERTIES OF EPITHELIAL AND ENDOTHELIAL TISSUES”, filed October 18, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0003] Epithelial tissues are a protective layer of cells that line cavities and organs throughout the body and are found in many glands. Endothelial tissues are a layer of cells that make up the lining of blood vessels, lymphatic vessels, and the heart. Trans-epithelial / trans- endothelial electrical resistance (TEER) is a measurement used to assess the function of a layer of epithelial and / or endothelial cells. Cultivation of epithelial / endothelial stem cells is commonly performed for various studies and TEER is a common measurement technique for evaluating the viability of the culture.

[0004] There are several techniques for measuring TEER. One common technique is to place two electrodes on each side of a cell layer and pass a low frequency current between the electrodes. The resistance of the barrier layer can be measured in this manner. Although traditional devices capable of measuring membrane-specific properties offer high quality data, the devices are expensive, destructive to cells, low-throughput, and require a highly trained operator. Thus, there is a need for addressing these issues and / or other issues associated with the prior art.SUMMARY

[0005] Embodiments of the present disclosure relate to systems, devices and methods for non-invasively measuring properties of epithelial / endothelial tissue. The embodiments herein provide non-invasive techniques using electrochemical impedance spectroscopy (EIS) that enable the collection of unique, pipette-like data.

[0006] In accordance with an embodiment, an EIS device for predicting characteristics of a tissue sample is provided. The EIS device includes at least two electrodes for measuring electrical characteristics of the tissue sample, wherein a first electrode of the at least two electrodes corresponds to an apical side of the tissue sample, and a second electrode of the at least two electrodes corresponds to a baso-lateral side of the tissue sample, and circuitry for converting electrical signals measured by the at least two electrodes into digital information indicating values of the electrical signals. The EIS device also typically includes a processor coupled to a memory storing instructions that, responsive to being executed by the processor, cause the EIS device to identify a constraint parameter based on a type of the tissue sample and / or one or more device parameters, and to calculate, in accordance with an intracellular circuit model that comprises two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the tissue sample based on the values of the electrical signals and the constraint parameter.

[0007] According to certain aspects, the constraint parameter defines a ratio of paracellular resistance to cellular resistance of the tissue sample.

[0008] According to certain aspects, the constraint parameter is based on user input.

[0009] According to certain aspects, the user input comprises a value defining a ratio of paracellular resistance to cellular resistance of the tissue sample.

[0010] According to certain aspects, the device parameters include a distance between the at least two electrodes, a distance between each electrode and a surface (apical and / or baso-lateral surface) of the tissue sample, a dielectric constant of the tissue sample, and a resistivity of a solution or solutions in which the tissue sample is located. For example, the tissue sample may be located in a single solution or culture medium, or the apical side may be located proximal to a solution different from the solution proximal to the baso-lateral side of the tissue sample.

[0011] According to certain aspects, no active electrode is located or positioned within the tissue sample.

[0012] According to certain aspects, the values of electrical signals include resistance and reactance values at each of a plurality of frequencies corresponding to a varying waveform applied to the at least two electrodes.

[0013] According to certain aspects, the constraint parameter is a value derived based on the imaginary component of a sum of the resistance and reactance values at each of the plurality of frequencies of the varying waveform applied to the at least two electrodes. For example, the constraint parameter may be a BCC value or parameter as described herein.

[0014] According to certain aspects, the constraint parameter is derived based on taking the imaginary component of limit w -> infinity (ZR(w)), wherein w is the frequency and ZR(w) is the frequency dependent impedance of the tissue sample

[0015] According to certain aspects, the calculating includes fitting the values of the electrical signals to an impedance equation of the intracellular circuit model for the plurality of frequencies of the applied varying waveform, and constraining the fit based on the constraint parameter to produce a discrete set of two or more combinations of membrane-specific values corresponding to the membrane-specific components of the intracellular circuit model.

[0016] According to certain aspects, the membrane-specific values of the membranespecific components of the intracellular model include two or more of an apical resistance value, a baso-lateral resistance value, an apical capacitance value, a baso-lateral capacitance value, an apical solution resistance value and a baso-lateral solution resistance value.

[0017] According to certain aspects, the EIS device further includes a first ion-selective electrode corresponding to, or positioned proximal to, the apical side of the tissue sample and a second ion-selective electrode corresponding to, or positioned proximal to, the baso-later side of the tissue sample, and the processor may be further configured to generate at least one additionalreference value based on a differential potential between the first and second ion-selective electrodes, and the calculating the at least one membrane-specific value may be further based on the at least one additional reference value.

[0018] According to certain aspects, the at least one membrane-specific value includes a first value corresponding to an apical side of the tissue sample and a second value corresponding to a baso-lateral side of the tissue sample.

[0019] According to certain aspects, the tissue sample comprises epithelial tissue.

[0020] According to certain aspects, the epithelial tissue comprises retinal pigment epithelium (RPE) cells.

[0021] According to certain aspects, the tissue sample comprises endothelial tissue.

[0022] According to certain aspects, the device further includes a high-speed camera configured to capture one or multiple images of the tissue sample, and a communications interface for transmitting one or more images of the tissue sample captured by the camera to the memory.

[0023] According to certain aspects, the processor is further configured to analyze one or more images of the tissue sample captured by the camera to generate at least one additional reference value corresponding to the intracellular circuit model, wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

[0024] According to certain aspects, the camera is configured to capture fluorescence intensity values of the apical side or baso-lateral side of at least a portion of the tissue sample after a) exposure of the tissue to dextran molecules, or other substance, on an opposite side of the tissue sample, or b) introduction of a voltage sensing dye to the tissue sample.

[0025] According to certain aspects, the processor is configured to analyze one or more images of the tissue sample captured by the camera to determine fluorescence intensity values in the one or more images.

[0026] According to certain aspects, the tissue sample is disposed in a sample holder and submerged in a culture medium.

[0027] According to certain aspects, the sample holder includes a plurality of electrodes integrated into the culture medium.

[0028] According to certain aspects, the membrane-specific components, or circuit elements, of the intracellular model include two or more of an apical resistance, a baso-lateralresistance, an apical capacitance, a baso-lateral capacitance, an apical solution resistance and a baso-lateral solution resistance.

[0029] According to an embodiment, a method of predicting characteristics of a tissue sample is provided. The method may utilize any electrochemical impedance spectroscopy (EIS) device embodiment as described herein. The method typically includes positioning a first electrode proximal to an apical side of a tissue sample, the apical side being located in a first solution, and positioning a second electrode proximal to a baso-lateral side of the tissue sample, the baso-lateral side being located in a second solution. The method also typically includes measuring electrical signals across the first and second electrodes, and calculating, in accordance with an intracellular circuit model that comprises two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the tissue sample based on values of the electrical signals and a constraint parameter, wherein the constraint parameter is based on a type of the tissue sample and / or one or more device parameters.

[0030] According to certain aspects, the measuring electrical signals includes applying a varying waveform to the first and second electrodes and measuring resistance and reactance values at each of a plurality of frequencies of the applied varying waveform.

[0031] According to certain aspects, the varying waveform is an alternating current waveform or an alternating voltage waveform.

[0032] According to certain aspects, the membrane-specific components of the intracellular model include two or more of an apical resistance, a baso-lateral resistance, an apical capacitance, a baso-lateral capacitance, an apical solution resistance and a baso-lateral solution resistance.

[0033] According to certain aspects, the constraint parameter defines a ratio of paracellular resistance to cellular resistance of the tissue sample.

[0034] According to certain aspects, the method further includes receiving user input including the constraint parameter. According to certain aspects, the user input constraint parameter comprises a value defining a ratio of paracellular resistance to cellular resistance of the tissue sample.

[0035] According to certain aspects, the device parameters include one or more of a distance between the at least two electrodes, a distance between the first electrode and the apical side of the tissue sample, a distance between the second electrode and the baso-lateral side of thetissue sample, a dielectric constant of the tissue sample, and a resistivity of a solution or solutions in which the tissue sample is located.

[0036] According to certain aspects, no active electrode is located or positioned within the tissue sample.

[0037] According to certain aspects, the measuring electrical signals includes receiving input specifying waveform parameters and applying a varying waveform to the first and second electrodes according to the specified waveform parameters, and wherein the values of the electrical signals include resistance and reactance values at each of a plurality of frequencies corresponding to the varying waveform applied to the first and second electrodes.

[0038] According to certain aspects, the constraint parameter is a value derived based on the imaginary component of a sum of the resistance and reactance values at each of the plurality of frequencies of the varying waveform applied to the first and second electrodes.

[0039] According to certain aspects, the calculating includes fitting the values of the electrical signals to an impedance equation of the intracellular circuit model for the plurality of frequencies of the applied varying waveform, and constraining the fit based on the constraint parameter to produce a discrete set of two or more combinations of membrane-specific values corresponding to the membrane-specific components of the intracellular circuit model.

[0040] According to certain aspects, the membrane-specific values of the membranespecific components of the intracellular model include two or more of an apical resistance value, a baso-lateral resistance value, an apical capacitance value, a baso-lateral capacitance value, an apical solution resistance value and a baso-lateral solution resistance value.

[0041] According to certain aspects, the EIS device includes a first ion-selective electrode corresponding to, or positioned proximal to, the apical side of the tissue sample and a second ion- selective electrode corresponding to, or positioned proximal to, the baso-later side of the tissue sample, wherein the method further includes generating at least one additional reference value based on a differential potential between the first and second ion-selective electrodes, and wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

[0042] According to certain aspects, the at least one membrane-specific value includes a first value corresponding to an apical side of the tissue sample and a second value corresponding to a baso-lateral side of the tissue sample.

[0043] According to certain aspects, the tissue sample comprises epithelial tissue. According to certain aspects, the epithelial tissue comprises retinal pigment epithelium (RPE) cells.

[0044] According to certain aspects, the tissue sample comprises endothelial tissue.

[0045] According to certain aspects, the EIS device further includes a memory, a camera configured to capture images of the tissue sample, and a communications interface, and the method further includes capturing one or multiple images of the tissue sample using the camera, and transmitting one or more images of the tissue sample captured by the camera to the memory. The images transmitted may include all or a subset of the images captured.

[0046] According to certain aspects, the method further includes analyzing the one or more images to generate at least one additional reference value corresponding to the intracellular circuit model, and wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

[0047] According to certain aspects, the method further includes exposing the tissue to dextran molecules on one side of the tissue sample, or introducing a voltage sensing dye, and wherein the camera is configured to capture fluorescence intensity values of the apical side or baso-lateral side of at least a portion of the tissue sample after a) the exposing of the tissue to dextran molecules on an opposite side of the tissue sample, or b) the introducing of the voltage sensing dye.

[0048] According to certain aspects, the method further includes analyzing the one or more images to determine fluorescence intensity values in the one or more images.

[0049] According to certain aspects, the method further includes disposing or positioning the tissue sample in a sample holder and submerging the tissue sample in a culture medium.

[0050] According to certain aspects, the sample holder includes a plurality of electrodes integrated into the culture medium.

[0051] According to certain aspects, the membrane-specific components of the intracellular model include two or more of an apical resistance, a baso-lateral resistance, an apical capacitance, a baso-lateral capacitance, an apical solution resistance and a baso-lateral solution resistance.

[0052] According to certain aspects, the method further includes providing an indication of acceptance or rejection of the tissue sample based on the at least one membrane specific value.For example, the indication may include an output presented to a user, e.g., on a monitor or display device, enabling the user to accept or reject the tissue sample, or may be used to control an automated process to reject or accept a tissue sample.

[0053] According to certain aspects, constraining any one or a combination of circuit components / elements during the fitting process provides a distinct solution, yielding insights into membrane-specific epithelial electrophysiology.

[0054] According to certain aspects, the calculating includes constraining the baso-lateral capacitance to a fixed value during the fitting of the values of the electrical signals to the impedance equation of the intracellular circuit model.

[0055] According to certain aspects, the calculating includes constraining the ratio of transcellular resistance to paracellular resistance of the tissue sample to a fixed value during the fitting process.

[0056] According to certain aspects, the calculating includes constraining the imaginary component of the ratio of apical impedance to baso-lateral impedance to a fixed value during the fitting process.

[0057] According to certain aspects, making independent readouts of general electrical states provides the additional constraint required for a unique solution in the calculation of membrane-specific values.

[0058] According to certain aspects, the underlying solution for obtaining complete membrane-specific insights requires one additional independent assumption or constraint applied during the fitting process.

[0059] According to certain aspects, the baso-lateral capacitance is constrained because it does not change significantly during a typical experiment, making it a convenient method for achieving a unique solution.

[0060] According to certain aspects, the tissue sample includes non-epithelial or non- endothelial tissues without a clear apical and baso-lateral orientation.

[0061] According to certain aspects, the EIS device includes more than two electrodes configured in alternative arrangements, such as ring electrodes or microelectrodes, to accommodate different tissue architectures.

[0062] According to certain aspects, the electrodes are arranged in a three-dimensional configuration to measure tissues grown in 3D cultures or organoids.

[0063] According to certain aspects, the calculating includes utilizing (trained) machine learning algorithms or advanced statistical models to interpret the electrical signals and predict characteristics of the tissue sample.

[0064] According to certain aspects, the constraint parameter is automatically determined using predefined algorithms or databases without the need for user input.

[0065] According to certain aspects, the camera captures dynamic images to observe realtime changes in the tissue sample during electrical stimulation or exposure to compounds.

[0066] According to certain aspects, the EIS device is used for applications in drug testing and screening to assess drug efficacy or toxicity on the tissue sample.

[0067] According to certain aspects, the EIS device is employed in disease modeling to detect pathological changes in the tissue sample or early disease markers.

[0068] According to certain aspects, the EIS device is utilized in regenerative medicine to monitor tissue engineering processes or stem cell differentiation.

[0069] According to certain aspects, the EIS device includes an enhanced user interface providing interactive features for parameter input and data visualization.

[0070] According to certain aspects, the EIS device implements automated decisionmaking algorithms that automatically accept or reject tissue samples based on predefined criteria.

[0071] According to certain aspects, the tissue sample is cultured under variable media conditions, including different nutrient levels or hypoxic environments, and the device accounts for these conditions in its measurements.

[0072] According to certain aspects, the EIS device includes features to prevent or detect contamination in the culture medium, ensuring sterility and reliability of measurements.

[0073] According to certain aspects, the method includes calibration procedures to ensure accuracy and reproducibility of the EIS device measurements.

[0074] According to certain aspects, the EIS device complies with medical device regulations and standards, making it suitable for clinical use.

[0075] Reference to the remaining portions of the specification, including the drawings and claims, will realize other features and advantages of the present invention. Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with respect to theaccompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] The present systems and methods for classifying intracellular dynamic activity of one or more cells in a tissue sample are described in detail below with reference to the attached drawing figures, wherein:

[0077] FIG. 1 A illustrates a physical model of epithelial tissue showing three pathways for ion transport, in accordance with the prior art;

[0078] FIG. IB illustrates an extracellular circuit model, in accordance with the prior art;

[0079] FIG. 1C illustrates an intracellular circuit model, in accordance with the prior art;

[0080] FIG. 2 is an example of a measured response of RPE to a chemical stimulus, measured in accordance with an approach in the prior art;

[0081] FIG. 3 is an example of a measured response of RPE to the same chemical stimulus, measured in accordance with the assumption-based approach using a ratio of paracellular resistance to cellular resistance, in accordance with at least one embodiment;

[0082] FIG. 4 shows a pipette inserted into the cytoplasm of a single RPE for other experiments, and also the mathematical model for electrical transport across epithelial cells derived from the equivalent circuit, in accordance with at least one embodiment;

[0083] FIG. 5 shows an experimental setup used to measure cells for validation;

[0084] FIG. 6A shows intracellular and transepithelial electrode potential traces;

[0085] FIG. 6B shows a comparison of the electrical response of 3 iRPE samples between the invasive pipette method and the non-invasive BCC method according to an embodiment;

[0086] FIG. 7 illustrates a system for measuring electrical characteristics of a tissue sample using voltage sensing dyes, in accordance with at least one embodiment;

[0087] FIG. 8 is an example of the use of voltage sensing dyes being used in mammary epithelial cells to track the propagation of an electrical signal across the epithelial cells, in accordance with at least one embodiment;

[0088] FIG. 9 is a flowchart of a method for measuring electrochemical response of a tissue sample, in accordance with some embodiments; and

[0089] FIG. 10 illustrates an exemplary computer system, in accordance with some embodiments.DETAILED DESCRIPTION

[0090] New techniques are described herein that overcome the limitations of conventional electrophysical measurements of epithelial and / or endothelial tissues. Prior art techniques required invasive devices that used intracellular microelectrodes that lead to end-point assays to provide insight into the membrane-specific electrical properties of these tissues. This measurement technique would often be accompanied by complex measurements that used a sequence of drugs / compounds to target membrane-specific responses. Although traditional devices capable of these measurements would offer high-quality data, they are expensive and complex to administer, as well as destructive to the measured tissue, offer low-throughput, and require a high amount of training to be deployed in the field.

[0091] A non-invasive measurement of trans-epithelial resistance (TER), though offering a less expensive and higher throughput alternative to the above, results in low resolution data because it merges all the membrane-specific resistances into a single parameter - TER. For example, TER is measured by utilizing a single low-frequency measurement of the trans-epithelial electric response (TEER) to a stimulus. The input stimulus and output signal of the epithelial tissue is applied to Ohm’s law (7 = / ■ / ?) to calculate the effective resistance of the epithelium. One shortcoming of this technique is that it is particularly susceptible to “shunting” of electrical current due to non-biological effect, such as the attachment of the perimeter of the tissue sample to the culture dish. Furthermore, shunting of the electrical current in TER leads to misleading conclusions about the integration of the tight-junctions of the cells resulting in an incorrect estimation of quality of the cells. For example, shunting too much electrical current will result in lower TER estimates and lead to the erroneous conclusion that the tissue being measured is of lower quality. TER is currently an important release assay in a stem cell derived retinal pigment epithelium (RPE) manufacturing pipeline. For example, TER may be used in a phase I clinical trial to transplant induced pluripotent stem cell derived RPE into the back of the eye of patients suffering from dryform age-related macular degeneration. Incorrect TER measurements can lead to false rejections of cell colonies.

[0092] Systems, devices and methods are disclosed for measuring properties of epithelial and / or endothelial tissues. In at least one embodiment, a system includes an electrochemical impedance spectroscopy (EIS) device is used for determining or predicting characteristics of atissue sample. The device includes at least two electrodes for measuring electrical characteristics of the tissue sample, wherein a first electrode of the at least two electrodes corresponds to an apical side of the tissue sample, and a second electrode of the at least two electrodes corresponds to a baso-lateral side of the tissue sample. The device also includes readout circuitry for converting electrical signals measured by the at least two electrodes into digital information indicating values of the electrical signals. The system includes a processor or multiple processors coupled to a memory storing instructions that, responsive to being executed by the processor(s), cause the system to identify a constraint parameter based on a type of the tissue sample and / or one or more device parameters, and to calculate, in accordance with an intracellular circuit model that comprises two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the tissue sample based on the values of the electrical signals and the constraint parameter.

[0093] FIGS. 1A-1C illustrate known models of epithelial cell equivalent electrical circuit models. FIG. 1A illustrates a physical model 102 of epithelial tissue showing three pathways for ion transport, in accordance with the prior art. As shown in FIG. 1A, a layer of epithelial tissue includes a number of epithelial cells. The apical side of the layer of tissue faces the lumen (i.e., inside of a hollow organ) or external environment, and the basolateral side of the layer of tissue faces or is adjacent to the underlying tissue of the organ. Epithelial cells are held together into a layer of tissue by tight junctions, which hold together individual cells and form tissue spaces to regulate movement of solutes (e.g., water) across the barrier. Ions (e.g., Na+, K+, Cl") and / or water (i.e., H2O) may pass into or out of the epithelial cell via channels or transporters in the apical and / or basolateral cell membranes. Water and / or ions may also pass between cells, through the tight junctions, via a paracellular pathway. Finally, water and / or ions may pass through the cell via a transcellular pathway, in which the water and / or ions are absorbed on one side (e.g., apical or basolateral) of the cell membrane and secreted on the other side (e.g., basolateral or apical) of the cell membrane. Each side of the cell membrane may refer to a continuous portion of the cell membrane between the tight junctions between cells.

[0094] FIG. IB illustrates an extracellular circuit model 104 that models the epithelial cell as a simple RC circuit in order to estimate TER. This model is widely used in the field despite its low fidelity and can be measured with commercial instruments. The model measures current between two nodes (labeled 1 and 2 in FIG. IB) on opposite sides of the epithelial tissue. The cellis modeled as a resistor and capacitor in parallel, which represent the TER and trans-epithelial capacitance (TEC) of the cell. A second resistor (Rbiank) is coupled in series with the RC circuit to represent the resistance of the surrounding media and growth substrate. It is clear from looking at this representation that model 104 is extremely simplified in that it doesn’t account for different electrical properties of the intracellular structures. In addition, it is difficult to measure or estimate Rbiank for a given physical measurement setup independent of the epithelial tissue.

[0095] FIG. 1C illustrates an intracellular circuit model 106 that models the epithelial cell as a more complex RC circuit to account for the different electrical properties of the different transport pathways. As shown, the components of the electrical model 106 include resistors: Rs - paracellular (shunt) pathway resistance; Ra - apical pathway resistance; Rb - basolateral pathway resistance; and RSOIA and RsoiB, which represent resistance of surrounding media and growth substrates in the apical and basolateral pathways, respectively. The components of the electrical model 106 also include capacitors: Ca - apical pathway capacitance; and Cb - basolateral pathway capacitance. As also shown in FIG. 1C, there is an added third node (node 3) between the two sets of RC circuits representing the apical and basolateral pathways, which is necessary to measure the differences between the TER / TEC of these two pathways.

[0096] Electrochemical Impedance Spectroscopy (ElS)-based measurements rely on the known intracellular circuit model 106 of FIG. 1C. Due to the complexity of model 106, and specifically the need to measure current and / or voltage at node 3, to resolve each of the components of model 106 would normally require (1) use of an intracellular reference electrode; or (2) external chemical permutations with complementary assumptions about the cellular response to said chemicals. The use of (1) is complex as placement of an electrode within the cell requires a high degree of skill. The use of (2) relies on certain assumptions that degrade the quality of the data if the assumptions are not accurate.

[0097] Instead, embodiments herein advantageously allow for omission of these steps and offer a non-invasive assessment of tissue, e.g., RPE, physiology.

[0098] In an embodiment, a consistent solution to the equations (e.g., model fit) is calculated using a paracellular resistance vs cellular resistance ratio as a constraint, which ratio is a known property of each type of epithelial tissue. For example, the three major types of epithelia may be defined as: (1) tight; (2) leaky; or (3) intermediate. These properties are maintained over the life of mature epithelia and serve as one of the functional definitions of the tissue. A contributortothis definition is the tight junction (TJ) resistance. For example, for RPE used in a phase I clinical trials, a range of an intermediate resistance ratio of 1 / 5-1 / 10 may be used, based on empirical measurements, literature of epithelial tissues, and chemical properties of phospholipid membranes.

[0099] When asserting the paracellular resistance vs cellular resistance ratio as a constraint, the absolute values of the components of the model may not exactly match the values calculated using the microelectrode technique. However, the changes of direction of the values measured for each membrane during chemical stimulation are the same. The discrepancy between absolute values can be at least partially explained by the difference in measuring values for a single cell (dependent on placement of the electrode) and measurements of an entire cell colony of an epithelial sheet using the assumption-based method disclosed herein.

[0100] FIG. 2 is an example of a measured response of RPE to a chemical stimulus, measured in accordance with an approach in the prior art using an internal electrode. The sample points in the charts are collected over a period of time (e.g., 60 seconds), and each data point represents a single measurement collected using an alternative approach to the techniques disclosed herein. The shaded portion of the graph (located between 20 and 40 seconds, represents the administration of a chemical stimulus to the tissue sample (e.g., epithelial or RPE cells). The chemical stimulant used here was a solution of 100 pM (micromole) of ATP (Adenosine triphosphate). In this case, the chemical stimulant was applied to the apical membrane of the epithelial tissue.

[0101] FIG. 3 is an example of a measured response of RPE to the same chemical stimulus, measured in accordance with an assumption-based approach using a ratio of paracellular resistance to cellular resistance, according to embodiments herein. The vertical lines between 20 and 40 seconds represent the start time and end time of the same 100 pM ATP stimulus. The charts provide three different measurements calculated according to different assumed ratios (0.1, 0.2, and 0.5) based on the type of epithelial tissue. The key insight from the charts in FIG. 3 is that the response calculated using the techniques disclosed herein are similar in magnitude and direction to the response seen in FIG. 2 using the prior art technique of inserting a microelectrode into a single epithelial cell.

[0102] In another embodiment, a mathematical model with a constraint function is used, where the constraint function is derived based on the high frequency asymptote of the imaginary (i.e., i = V”-!) component of the membrane ratio (Zr). This constant is termed the bio-chamberconstant (BCC). Validation of the constraint method was performed using an invasive pipette method that measures the distinct resistance and capacitance values of each transport barrier. The response of RPE to the apical application of 100 pM adenosine triphosphate (ATP) was simultaneously recorded using the new BCC method and the previously validated, pipette method.

[0103] The model was tested using induced pluripotent stem cell (iPSC)-derived retinal RPE (iRPE) tissues. The tissues were stimulated using ATP because the pathway is well studied and the response at the apical and basolateral membranes are opposite. For example, in a typical ATP response, the apical membrane resistance should increase due to potassium channel closure. The basolateral membrane resistance should decrease due to calcium-activated chloride channel opening. This pathway can provide unique insights into the functional state of the apical and basolateral membranes of intact epithelia.

[0104] A pipette was inserted into the cytoplasm of a single RPE for other experiments as depicted in FIG. 4. The currents ( / ) were controlled by external computer hardware to perform galvanostatic E1S measurements. Voltages in the apical bath (Fa), basolateral bath (7b), and RPE cytoplasm (VPiP) were continuously recorded. The mathematical model for electrical transport across epithelial cells was derived from the equivalent circuit depicted in FIG. 4. RPE membranes can be modeled as a single, frequency (m) dependent, impedance (Z) that is comprised of the membrane resistance ( / ?) and membrane capacitance (C). The apical and basolateral membrane impedance (Za(m) and Zb(w), respectively) are shown below and are inserted into the total epithelial impedance equation Z(m).

[0105] The experimental setup used to measure the cells for validation is depicted in FIG.5. A modified Ussing chamber was used to mount iRPE between two, continuously perfusing media baths (one for apical and one for basolateral). After inserting the electrode and allowing for approximately 15 minutes of baseline electrical readings, ATP was added to the apical perfusing media. After about 10 minutes of treatment, the apical media was returned to control media for an additional 30 minutes.

[0106] The previously described parameter, called the membrane ratio (ratio of paracellular resistance vs cellular resistance), that can be used to fully constrain a fit to the mathematical model of RPE is written below and is equivalent to the ratio of the apical to basolateral voltage ( A V-J A 7b) at any measured frequency (to).

[0107]

[0108] The BCC constraint may be derived by taking the imaginary component of lim- (a)— >co) [Z_r (m) . If the surface area ( / I) of the solution resistances (R sol) is equal to the membrane areas, BCC simplifies to a function of membrane dielectric constant (f m), solution resistivity (p sol), and the distance between the electrodes (d e), which is determined for this setup to be:

[0109] Here, deA and deB are the distances between the apical and basolateral electrodes and the apical side of the tissue sample and the basolateral side of the tissue sample, respectively.

[0110] FIG. 6B shows a comparison of the electrical response of 3 iRPE samples (orange, blue, and grey) between invasive pipette method and the non-invasive BCC method. Theintracellular and transepithelial electrode potential traces are provided for reference in FIG. 6A. As shown in FIG. 6B, the magnitudes of each circuit resistance element may not match the pipette- validated values; however, the direction of response appears to be similar. ATP was applied at the first vertical grey bar. ATP was replaced with the original control media at the second vertical grey bar.

[0111] By replacing the need for an invasive pipette-based measurement, the BCC method embodiments offer a higher-content, longitudinal, and non-invasive assessment of RPE physiology. The BCC method embodiments also yield an excellent approximation of membranespecific changes. This methodology will significantly enhance drug screening and iRPE-cell quality control, especially in studies targeting membrane-specific responses. The methods herein are applicable to any single-layer epithelial tissue.

[0112] According to an embodiment, a process for determining characteristics of epithelia tissue samples according to embodiments herein includes first arranging the tissue sample in the EIS system or device. This may include, for example, positioning a first electrode (or multiple first electrodes) proximal to, and external to, an apical side of the tissue sample, the apical side being located in a first solution, positioning a second electrode ( or multiple second electrodes) proximal to, and external to, the baso-lateral side of the tissue sample, the baso-lateral side being located in a second solution. The first and second solutions may be different, or they may be the same, e.g., same culture medium). Measuring electrical signals across the first and second electrodes is then performed using the extracellular electrodes. This may include applying a waveform (AC or AV) with varying frequencies and measuring electrical response signals. Circuitry in the EIS device is provided for converting the electrical signals measured by the at least two electrodes into digital information indicating values of the electrical signals. Measuring typically includes measuring the resistance and reactance of the epithelia using extracellular electrodes; the resulting digital information / data (measured EIS data) will include a list of resistances and reactances at each of a plurality of applied / measured frequences during EIS. An applied current or voltage waveform may include discrete frequencies or continuous frequencies within a desired frequency range.

[0113] The data may then be used to calculate, in accordance with an intracellular circuit model that includes two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the tissue sample based on values of the electrical signalsand a constraint function or parameter, wherein the constraint parameter is based on a type of the tissue sample and / or one or more system or device parameters. For example, the constraint parameter may include the BCC or the ratio parameter described herein. The various system / device parameters may include a distance between the at least two electrodes, a distance between the first electrode and the apical side of the tissue sample, a distance between the second electrode and the baso-lateral side of the tissue sample, a dielectric constant of the tissue sample, and a resistivity of a solution or solutions in which the tissue sample is located. Other device parameters may be used.

[0114] The calculation may include deriving an impedance equation for the intracellular circuit model, or using a derived impedance equation. Examples of intracellular circuit models are provided herein and others will be readily available or derivable to one skilled in the art. The impedance is the complex (i.e., i = sqrt(-l)) sum of the resistance and the reactance at a fixed frequency. For example, in an embodiment, based on the acquired data, the measured EIS data is fit to the theoretical impedance equation based on the known cell model for all (or a subset) of the measured / applied frequencies, and the fit is constrained based on the constraint function or parameter (e.g., ratio: Rs / (Ra+Rb) equal to a constant, or BCC equal to a constant) used to determine a discrete set of two or more combinations of membrane-specific values corresponding to the membrane-specific components of the intracellular circuit model. Membrane specific components of the model may include Ra, Rb, Rs, Ca, and Cb as examples. Others will be apparent to one skilled in the art based on the model used. Simplifying assumptions about the cells may be made in certain embodiments to select from the list of potential values for Ra, Rb, Rs, Ca, and Cb after fitting.

[0115] For the ratio Rs / (Ra+Rb), a few assumptions may be made to chose from the list of many possible answers. For BCC, one simplifying assumption may be particularly advantageous: the apical surface area must be larger than the basolateral (therefore Ca > Cb). Other assumptions may be made.

[0116] Constraining any one or combination of specific circuit components / elements of the circuit model during fitting provides one distinct solution, advantageously yielding insights into membrane-specific epithelial electrophysiology. For example, constraining the basolateral capacitance, the ratio of transcellular to paracellular resistance, or the imaginary component of the ratio of apical to basolateral impedance to a fixed value will provide good results. Alternatively,making independent readouts of general electrical states would also yield similarly good results. The underlying solution for complete membrane-specific insights requires one additional independent assumption or constraint; one method is to constrain the basolateral capacitance as this may not change much during a typical experiment.

[0117] One advantage of the present embodiments is that the measurement using a microelectrode as in the prior art to measure voltage and / or current values at the internal-to-the- cell node 3 is restricted to measuring a single cell in a cell culture, whereas the present embodiments measure voltage at nodes external to the cell structure and, therefore, can measure the response over the entire epithelial cell culture. Since the prior art technique only measures a single cell, it is likely that the assumption that all cells in the culture will respond the same as the measured cell is wrong. Either a cell exhibiting unhealthy response could be an outlier, thereby incorrectly rejecting an entire cell colony, or a cell exhibiting a healthy response could be an outlier, thereby incorrectly accepting an unhealthy cell colony for further study.

[0118] The present embodiments utilize a mathematical framework for calculating the absolute values of each parameter in the epithelial cell model in FIG. 1C. For example, the constraint methods (ratio and BCC methods) show relative changes in each membrane. In most cases, these changes in the electrical membrane parameters are more important than the absolute values determined using the previous microelectrode technique. However, to additionally resolve the absolute value of each parameter with a single measurement, there are a number of techniques that can be utilized along with the ratio method.

[0119] In at least some embodiments, the absolute values of the parameters can be measured using voltage sensing dyes with a high-speed camera. FIG. 7 illustrates a system 700 for measuring electrical characteristics of a tissue sample using voltage sensing dyes, in accordance with at least one embodiment. The system 700 includes a sample holder 730 and highspeed camera 710 that includes a communication interface 702 to a computing device 720. The sample holder 730 holds a tissue sample (e.g., epithelial or endothelial cells) in a culture medium. In some embodiments, the sample holder 730 may be transparent or partially transparent. The sample holder 730 may also include protrusions for securing the tissue sample to the sample holder 730, and / or to create cavities both above and below the tissue sample to allow for the culture medium to flow and / or contact the tissue sample. The sample holder 730 may include a lid that screws or snaps onto the sample holder 730. In an embodiment, the lid may include surfaces thathold the tissue sample in place against one or more protrusions within the sample holder 730. The lid may include a transparent window made of, e.g., acrylic or glass that allows the tissue sample to be visible through the lid.

[0120] In operation, the sample holder including the tissue sample is placed under the highspeed camera 710. In an embodiment, the computing device 720 includes an application that provides a graphical user interface for operating the high-speed camera 710 and capturing a number of images of the tissue sample. The computing device 720 may be triggered by the application via a signal sent over the communication interface 702, which may be a wired or wireless interface such as WiFi, Bluetooth, or the like. The high-speed camera 710 will then capture a series of images of the tissue sample over a period of time. For example, the high-speed camera may capture 10-60 images per second over a number of seconds. In an embodiment, the images may be stored in a memory in the camera and then transmitted to the application on the computing device 720 via the communication interface 702 at a later time. In another embodiment, each image is transmitted to the application on the computing device 720 via the communication interface 702 as it is captured in real time and stored in a memory of the computing device 720.

[0121] In an embodiment, the high-speed camera 710 includes an illumination device (e.g., light source, flash, etc.) used to illuminate the tissue sample. In another embodiment, the system 700 includes a separate light source 712 used to illuminate the sample. For example, a light source 712 may be located under the sample holder 730 to illuminate the tissue sample from below. In such embodiments, the sample holder 730 should be either transparent or translucent to allow light to pass through the tissue sample.

[0122] Voltage sensing dyes change fluorescence intensities of cells or cell structures depending on existent electrical charge. These voltage sensing dyes can be used as a substitute in place of the microelectrodes and provide missing reference information to precisely calculate absolute values for components of the intracellular circuit model. This also allows for the subpopulation measurements of tissues with multiple epithelial phenotypes, which have been shown in a laboratory setting to be an important physiological feature of RPE cells, for example. Furthermore, the use of voltage sensing dyes can be exceptionally powerful for quality control (QC) of epithelial tissues as an acceptance criteria in surgical studies and would also be extremely important in pharmaceutical applications to evaluate large sets of epithelial cell response in drug discovery assays.

[0123] FIG. 8 is an example of the use of voltage sensing dyes being used in mammary epithelial cells to track the propagation of an electrical signal across the epithelial cells. The image on the left shows cellular ROIs and, adjacent to the image is color-corresponding R / R0time series data for a subset of cells in the image. The chart in the upper right shows a plot of distance of the first cell as a function of time at maximum hyperpolarization. The chart in the lower right shows a plot of the peak transient amplitude (%A7? / Ro) as a function of distance from the first cell showing the transient hyperpolarization.

[0124] In some embodiments, the setup using the high-speed camera may be utilized with a genetically modified cell line, where the genetic modification encodes a voltage indicator within the cell. The use of genetic modification may be inappropriate for QC or pharmaceutical applications. However, the genetic modification would allow for study of the same parameters over a longer-term in a non-cytotoxic manner, which can be useful in certain basic research or drug discovery applications.

[0125] In yet another embodiment, paracellular flux can be measured using Dextran molecules. Dextran molecules are primarily transported through the tight junctions of epithelial cells. By tracking the fluorescent intensity of Dextran molecules that have passed through the tight junction, relative to a concentration of Dextran molecules in the application bath, a more accurate estimate of the paracellular resistance to intracellular resistance ratio is possible. By more accurately measuring the ratio, the calculation of the membrane specific properties can be provided, which in turn provides a more useful estimation of the physiological state of the epithelial cells.

[0126] In yet another embodiment, paracellular flux can be measured using an ion sensitive electrode. In this application, two ion sensitive electrodes can be placed in the apical and basolateral baths (e.g., reservoirs for culture medium in contact with one of the two sides of the tissue sample). By monitoring the difference in potential between the two baths, it is possible to monitor the concentration of a specific type of ion that is passing through the tissue sample. The electrode should be sensitive to a particular ion that is likely to only pass through the tight junction, thereby providing a more accurate estimate of paracellular flux and paracellular resistance.

[0127] In yet another embodiment, growth substrate can be built in a sample holder with integrated electrodes in a pattern that enables local electrical response of the epithelial tissue atmultiple locations across the tissue sample. By measuring a drop in the electrical field between electrodes, an estimation of the paracellular resistance may be made.

[0128] Appendix A shows exemplary EIS device prototypes according to embodiments.

[0129] FIG. 9 is a flowchart of a method 900 for measuring electrochemical response of a tissue sample, in accordance with some embodiments. Although the method is described in conjunction with the system 400, it will be appreciated that the method 900 can be performed using a number of different optical imaging devices capable of imaging cellular structures of epithelial cells in conjunction with a high-speed camera. Furthermore, the steps described herein may be performed, at least in part, using one or more processors of a computing device 420. The steps may be implemented by executing instructions in any combination of hardware, firmware, and / or software.

[0130] At 902, a constraint parameter (e.g., ratio or BCC) is identified for the tissue sample and device setup. In an embodiment, a correspondence between tissue type and a pre-set ratio may be provided, e.g., through literature and / or experimental results. Given a type of tissue sample secured in the sample holder, a ratio can be retrieved from a lookup table based on the selected type of tissue sample. In other embodiments, the ratio can be measured using, e.g., fluorescent voltage sensing dyes.

[0131] At 904, measurements of the tissue sample may be collected via one or more extracellular electrodes. Electrodes located in the culture medium associated with an apical and basolateral side of the tissue sample may be used to measure electrical properties of the tissue sample. A number of data points may be collected over a period of time. A microelectrode to measure electrical properties in the interior of a cell is not used.

[0132] At 906, a number of components of an intracellular circuit model are calculated based on the measurements and the constraint parameter. Membrane-specific values (e.g., TER and / or TEC) for the apical and basolateral membranes of the epithelial cells may be calculated separately. In some embodiments, the calculated values may represent the average values for a colony of cells.

[0133] At 908, the component values are utilized to analyze the characteristics of the cells in the tissue sample. For example, membrane-specific values for TER may be utilize as an acceptance criteria for a particular set of RPE cells being measured.

[0134] An example system suitable for use in implementing some embodiments of the present disclosure is set forth below. It should be understood that this and other arrangements described herein are set forth only as examples. Other arrangements and elements (e.g., machines, interfaces, functions, orders, groupings of functions, etc.) may be used in addition to or instead of those shown, and some elements may be omitted altogether. Further, many of the elements described herein are functional entities that may be implemented as discrete or distributed components or in conjunction with other components, and in any suitable combination and location. Various functions described herein as being performed by entities may be carried out by hardware, firmware, and / or software. For instance, various functions may be carried out by a processor executing instructions stored in memory. Furthermore, persons of ordinary skill in the art will understand that any system that performs the operations of the method is within the scope and spirit of embodiments of the present disclosure.

[0135] FIG. 10 illustrates an exemplary computer system 1000, in accordance with some embodiments. The computer system 1000 includes a processor 1002, a non-volatile memory 1004, and a network interface controller (NIC) 1020. The processor 1002 can execute instructions that cause the computer system 1000 to implement the functionality various elements of the system 400 described above. For example, the computing device 420 and / or the high-speed camera 410 can each take the form of the computer system 1000.

[0136] Each of the components 1002, 1004, and 1020 can be interconnected, for example, using a system bus to enable communications between the components. The processor 1002 is capable of processing instructions for execution within the system 1000. The processor 1002 can be a single-threaded processor, a multi-threaded processor, a vector processor or parallel processor that implements a single-instruction, multiple data (SIMD) architecture, or the like. The processor 1002 is capable of processing instructions stored in the volatile memory 1004. In some embodiments, the volatile memory 1004 is a dynamic random access memory (DRAM). The instructions can be loaded into the volatile memory 1004 from a non-volatile storage, such as a Hard Disk Drive (HDD) or a solid state drive (not explicitly shown), or received via the network. In an embodiment, the volatile memory 1004 can include instructions for an operating system 1006 as well as one or more applications 1008. It will be appreciated that the application(s) can be configured to provide the functionality of one or more components of the system 200, as described above. The NIC 1020 enables the computer system 1000 to communicate with other devices overa network, including a local area network (LAN) or a wide area network (WAN) such as the Internet.

[0137] It will be appreciated that the computer system 1000 is merely one exemplary computer architecture and that the processing devices implemented in the system 200 can include various modifications such as additional components in lieu of or in addition to the components shown in FIG. 10. For example, in some embodiments, the computer system 1000 can be implemented as a system-on-chip (SoC) that includes a primary integrated circuit die containing one or more CPU cores, one or more GPU cores, a memory management unit, analog domain logic and the like coupled to a volatile memory such as one or more SDRAM integrated circuit dies stacked on top of the primary integrated circuit dies and connected via wire bonds, micro ball arrays, and the like in a single package (e.g., chip). In another embodiment, the computer system 1000 can include a printed circuit board with a number of components soldered thereto, as well as one or more expansion cards coupled to an interface such as a peripheral component interconnect (PCI) express (PCIe), or the like. In yet another embodiment, the computer system 1000 can be implemented as a server device, which can, in some embodiments, execute a hypervisor and one or more virtual machines that share the hardware resources of the server device.

[0138] It is noted that the techniques described herein may be embodied in executable instructions stored in a computer readable medium for use by or in connection with a processorbased instruction execution machine, system, apparatus, or device. It will be appreciated by those skilled in the art that, for some embodiments, various types of computer-readable media can be included for storing data. As used herein, a "computer-readable medium" includes one or more of any suitable media for storing the executable instructions of a computer program such that the instruction execution machine, system, apparatus, or device may read (or fetch) the instructions from the computer-readable medium and execute the instructions for carrying out the described embodiments. Suitable storage formats include one or more of an electronic, magnetic, optical, and electromagnetic format. A non-exhaustive list of conventional exemplary computer-readable medium includes: a portable computer diskette; a random-access memory (RAM); a read-only memory (ROM); an erasable programmable read only memory (EPROM); a flash memory device; and optical storage devices, including a portable compact disc (CD), a portable digital video disc (DVD), and the like.

[0139] It should be understood that the arrangement of components illustrated in the attached Figures are for illustrative purposes and that other arrangements are possible. For example, one or more of the elements described herein may be realized, in whole or in part, as an electronic hardware component. Other elements may be implemented in software, hardware, or a combination of software and hardware. Moreover, some or all of these other elements may be combined, some may be omitted altogether, and additional components may be added while still achieving the functionality described herein. Thus, the subject matter described herein may be embodied in many different variations, and all such variations are contemplated to be within the scope of the claims.

[0140] To facilitate an understanding of the subject matter described herein, many aspects are described in terms of sequences of actions. It will be recognized by those skilled in the art that the various actions may be performed by specialized circuits or circuitry, by program instructions being executed by one or more processors, or by a combination of both. The description herein of any sequence of actions is not intended to imply that the specific order described for performing that sequence must be followed. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0141] The use of the terms "a" and "an" and "the" and similar references in the context of describing the subject matter (particularly in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term “at least one” followed by a list of one or more items (for example, “at least one of A and B”) is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. Furthermore, the foregoing description is for the purpose of illustration only, and not for the purpose of limitation, as the scope of protection sought is defined by the claims as set forth hereinafter together with any equivalents thereof. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illustrate the subject matter and does not pose a limitation on the scope of the subject matter unless otherwise claimed. The use of the term “based on” and other like phrases indicating a condition for bringing about a result, both in the claims and in the written description, is not intended to foreclose any other conditions that bring about that result. Nolanguage in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention as claimed.

Claims

CLAIMS1. An electrochemical impedance spectroscopy (EIS) device for predicting characteristics of a tissue sample, the device comprising: at least two electrodes for measuring electrical characteristics of the tissue sample, wherein a first electrode of the at least two electrodes corresponds to an apical side of the tissue sample, and a second electrode of the at least two electrodes corresponds to a baso-lateral side of the tissue sample; circuitry for converting electrical signals measured by the at least two electrodes into digital information indicating values of the electrical signals; and a processor coupled to a memory storing instructions that, responsive to being executed by the processor, cause the EIS device to: identify a constraint parameter based on a type of the tissue sample and / or one or more device parameters; and calculate, in accordance with an intracellular circuit model that comprises two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the tissue sample based on the values of the electrical signals and the constraint parameter.

2. The EIS device of claim 1, wherein the constraint parameter defines a ratio of paracellular resistance to cellular resistance of the tissue sample.

3. The EIS device of claim 1, wherein the constraint parameter is based on user input.

4. The EIS device of claim 3, wherein the user input comprises a value defining a ratio of paracellular resistance to cellular resistance of the tissue sample.

5. The EIS device of claim 1, wherein the device parameters include a distance between the at least two electrodes, a distance between the first electrode and the apical side of the tissue sample, a distance between the second electrode and the baso-lateral side of the tissue sample, adiel ectric constant of the tissue sample, and a resistivity of a solution or solutions in which the tissue sample is located.

6. The EIS device of claim 1, wherein no active electrode is located or positioned within the tissue sample.

7. The EIS device of claim 1, wherein the values of electrical signals include resistance and reactance values at each of a plurality of frequencies corresponding to a varying waveform applied to the at least two electrodes.

8. The EIS device of claim 7, wherein the constraint parameter is a value derived based on the imaginary component of a sum of the resistance and reactance values at each of the plurality of frequencies of the varying waveform applied to the at least two electrodes.

9. The EIS device of claim 7, wherein the constraint parameter is derived based on taking the imaginary component of limit w -> infinity (ZR(w)), wherein w is the frequency and ZR(w) is the frequency dependent impedance of the tissue sample.

10. The EIS device of claim 7, wherein the calculating includes fitting the values of the electrical signals to an impedance equation of the intracellular circuit model for the plurality of frequencies of the applied varying waveform, and constraining the fit based on the constraint parameter to produce a discrete set of two or more combinations of membrane-specific values corresponding to the membrane-specific components of the intracellular circuit model.

11. The EIS device of claim 10, wherein the membrane-specific values of the membranespecific components of the intracellular model include two or more of an apical resistance value, a baso-lateral resistance value, an apical capacitance value, a baso-lateral capacitance value, an apical solution resistance value and a baso-lateral solution resistance value.

12. The EIS device of claim 10, further comprising a first ion-selective electrode corresponding to the apical side of the tissue sample and a second ion-selective electrode corresponding to the baso-later side of the tissue sample, wherein the processor is further configured to generate at least one additional reference value based on a differential potential between the first and second ion-selective electrodes, and wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

13. The EIS device of claim 1, wherein the at least one membrane-specific value includes a first value corresponding to an apical side of the tissue sample and a second value corresponding to a baso-lateral side of the tissue sample.

14. The EIS device of claim 1, wherein the tissue sample comprises epithelial tissue.

15. The EIS device of claim 14, wherein the epithelial tissue comprises retinal pigment epithelium (RPE) cells.

16. The EIS device of claim 1, wherein the tissue sample comprises endothelial tissue.

17. The EIS device of claim 1, the device further comprising: a high-speed camera configured to capture images of the tissue sample; and a communications interface for transmitting one or more images of the tissue sample captured by the camera to the memory.

18. The EIS device of claim 17, wherein the processor is further configured to: analyze the one or more images to generate at least one additional reference value corresponding to the intracellular circuit model, wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

19. The EIS device of claim 17, wherein the camera is configured to capture fluorescence intensity values of the apical side or baso-lateral side of at least a portion of the tissue sampleafter a) exposure of the tissue to dextran molecules on an opposite side of the tissue sample, or b) introduction of a voltage sensing dye.

20. The EIS device of claim 19, wherein the processor is configured to analyze the one or more images to determine fluorescence intensity values in the one or more images.

21. The EIS device of claim 1, wherein the tissue sample is disposed in a sample holder and submerged in a culture medium.

22. The EIS device of claim 21, wherein the sample holder includes a plurality of electrodes integrated into the culture medium.

23. The EIS device of claim 1, wherein the membrane-specific components of the intracellular model include two or more of an apical resistance, a baso-lateral resistance, an apical capacitance, a baso-lateral capacitance, an apical solution resistance and a baso-lateral solution resistance.

24. A method of predicting characteristics of a tissue sample using an electrochemical impedance spectroscopy (EIS) device, the method comprising: positioning a first electrode proximal to an apical side of a tissue sample, the apical side being located in a first solution; positioning a second electrode proximal to a baso-lateral side of the tissue sample, the baso-lateral side being located in a second solution; measuring electrical signals across the first and second electrodes; and calculating, in accordance with an intracellular circuit model that comprises two or more membrane-specific components, at least one membrane-specific value that indicates a characteristic of the tissue sample based on values of the electrical signals and a constraint parameter, wherein the constraint parameter is based on a type of the tissue sample and / or one or more device parameters.

25. The method of claim 24, wherein the measuring electrical signals includes applying a varying waveform to the first and second electrodes and measuring resistance and reactance values at each of a plurality of frequencies of the applied varying waveform.

26. The method of claim 25, wherein the varying waveform is an alternating current waveform or an alternating voltage waveform.

27. The method of claim 24, wherein the membrane-specific components of the intracellular model include two or more of an apical resistance, a baso-lateral resistance, an apical capacitance, a baso-lateral capacitance, an apical solution resistance and a baso-lateral solution resistance.

28. The method of claim 24, wherein the constraint parameter defines a ratio of paracellular resistance to cellular resistance of the tissue sample.

29. The method of claim 24, further including receiving user input including the constraint parameter.

30. The method of claim 29, wherein the user input constraint parameter comprises a value defining a ratio of paracellular resistance to cellular resistance of the tissue sample.

31. The method of claim 24, wherein the device parameters include one or more of a distance between the at least two electrodes, a distance between the first electrode and the apical side of the tissue sample, a distance between the second electrode and the baso-lateral side of the tissue sample, a dielectric constant of the tissue sample, and a resistivity of a solution or solutions in which the tissue sample is located.

32. The method of claim 24, wherein no active electrode is located or positioned within the tissue sample.

33. The method of claim 24, wherein the measuring electrical signals includes receiving input specifying waveform parameters and applying a varying waveform to the first and second electrodes according to the specified waveform parameters, and wherein the values of the electrical signals include resistance and reactance values at each of a plurality of frequencies corresponding to the varying waveform applied to the first and second electrodes.

34. The method of claim 33, wherein the constraint parameter is a value derived based on the imaginary component of a sum of the resistance and reactance values at each of the plurality of frequencies of the varying waveform applied to the first and second electrodes.

35. The method of claim 34, wherein the calculating includes fitting the values of the electrical signals to an impedance equation of the intracellular circuit model for the plurality of frequencies of the applied varying waveform, and constraining the fit based on the constraint parameter to produce a discrete set of two or more combinations of membrane-specific values corresponding to the membrane-specific components of the intracellular circuit model.

36. The method of claim 35, wherein the membrane-specific values of the membrane-specific components of the intracellular model include two or more of an apical resistance value, a baso- lateral resistance value, an apical capacitance value, a baso-lateral capacitance value, an apical solution resistance value and a baso-lateral solution resistance value.

37. The method of claim 35, wherein the EIS device includes a first ion-selective electrode corresponding to, or positioned proximal to, the apical side of the tissue sample and a second ion-selective electrode corresponding to, or positioned proximal to, the baso-later side of the tissue sample, wherein the method further includes generating at least one additional reference value based on a differential potential between the first and second ion-selective electrodes, and wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

38. The method of claim 24, wherein the at least one membrane-specific value includes a first value corresponding to an apical side of the tissue sample and a second value corresponding to a baso-lateral side of the tissue sample.

39. The method of claim 24, wherein the tissue sample comprises epithelial tissue.

40. The method of claim 39, wherein the epithelial tissue comprises retinal pigment epithelium (RPE) cells.

41. The method of claim 24, wherein the tissue sample comprises endothelial tissue.

42. The method of claim 24, wherein the EIS device further includes: a memory, a camera configured to capture images of the tissue sample; and a communications interface, and wherein the method further includes: capturing one or multiple images of the tissue sample using the camera; and transmitting one or more images of the tissue sample captured by the camera to the memory.

43. The method of claim 42, further including analyzing the one or more images to generate at least one additional reference value corresponding to the intracellular circuit model, and wherein the calculating the at least one membrane-specific value is further based on the at least one additional reference value.

44. The method of claim 42, wherein the method further includes: a) exposing the tissue to dextran molecules on one side of the tissue sample, or b) introducing a voltage sensing dye, and wherein the camera is configured to capture fluorescence intensity values of the apical side or baso-lateral side of at least a portion of the tissue sample after a) the exposing ofthe tissue to dextran molecules on an opposite side of the tissue sample, or b) the introducing of the voltage sensing dye.

45. The method of claim 44, wherein the method further includes analyzing the one or more images to determine fluorescence intensity values in the one or more images.

46. The method of claim 24, wherein the method further includes disposed the tissue sample in a sample holder and submerging the tissue sample in a culture medium.

47. The method of claim 46, wherein the sample holder includes a plurality of electrodes integrated into the culture medium.

48. The method of claim 24, wherein the membrane-specific components of the intracellular model include two or more of an apical resistance, a baso-lateral resistance, an apical capacitance, a baso-lateral capacitance, an apical solution resistance and a baso-lateral solution resistance.

49. The method of claim 24, further including providing an indication of acceptance or rejection of the tissue sample based on the at least one membrane specific value.

50. The EIS device of claim 1, wherein the processor is further configured to provide an indication of acceptance or rejection of the tissue sample based on the at least one membrane specific value.

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