HK1082002B - Impedance based apparatuses and methods for analyzing cells and particles - Google Patents

Description

Apparatus and method for impedance-based analysis of cells and particles

[0001] This patent application incorporates by reference the following patent applications: U.S. provisional application 60/397,749, filing date 2002, 7/20; united states provisional application 60/435,400, filing date 2002, 12 months, 20 days; U.S. provisional application 60/469,572, filing date 5/9/2003. This patent application also incorporates by reference the following PCT patent applications: PCT application PCT/US2003/022557, entitled "IMPEDANCE BASEDDEVICES AND METHODS FOR USE IN ASSAYS," patent attorney docket number ACEA-0202, filed concurrently with this patent application.

Background

Technical Field

[0002] The present invention relates generally to the field of analysis of cells and particles. In particular, the present invention provides impedance-based devices, microtiter plates, and methods for analyzing cells and particles. The devices, microtiter plates, and methods of the invention can be used to monitor attachment, migration, and invasion of cells or particles. The devices, microtiter plates, and methods of the invention may also be used to identify modulators of cell or particle attachment, migration, and growth.

Background

[0003] Growth and metastasis and pathological migration of cells are fundamental characteristics of malignant cells. Further insight into the molecular mechanisms of pathological cell migration is undoubtedly beneficial in the treatment and prevention of cancer.

[0004] Both in vivo and in vitro models were used to study this mechanism. The in vivo model can monitor the entire course of cancer metastasis in experimental animals, but is not suitable for large-scale evaluation of the characteristics of malignant cells. In vitro models can more quantitatively analyze the growth and migration activity of cancer cells, including cell colonization on single or multiple layers of cultured cells or explant tissue, cell growth through natural and artificial extracellular matrices or cell-like structures, and cell motility in response to chemotactic agents.

[0005] Currently, there are generally 3 methods available for detecting cell growth and/or cell migration in vitro, including:

[0006](1) chemoattractant-induced migration (i.e., chemotaxis, of a chemical agent to a biological cell or organism)Concentration gradient-directed reaction)/invasion and transwell migration analysis (trans-Well migration assay) (Falk, W., "A48 Well MicroChemicals Assembly for Rapid and Accurate Measurement of Leucocyte Migration," Journal of Immunological Methods, Vol.33, p.239-247, 1980; richards k.l. and j.mccullough, "a modified micro chamber Method for chemistry and chemistry," Immunological Communications, volume 13, pages 49-62, 1984): the cells in the insert invade and migrate through an artificial porous membrane (polycarbonate membrane without polyvinylpyrrolidone or polyethylene terephthalate membrane) into the lower compartment containing a given chemoattractant. The migrated cells are usually attached to the other side of the membrane and can then be labelled with a chemical dye (Neuroprobe, inc., see Neuroprobe, inc. www. Neuroprobe. com/protocol/pt — 96a. html) or a fluorescent dye and the number of cells is calculated with a microscope or a fluorescence spectrophotometer (TECAN, Coster, BD Biosciences, (Ilsley, s.r. 1996).Basement Membrane Cell Invasion Chamber.Becton Dickinson Technical Bulletin #422；www.bdbiosciences.com/discovery_labware/technical_resources/techbulletins.html；BD BioCoat^TM FluoroBlok^TMTumor Cell inventory System, www.bdbiosciences.com/discovery _ subnet/Products/drug _ discovery/insert _ systems/fluorooblock _ inventory/; tecan. www.tecan. com/migration _ introl.pdf). For example, U.S. patent 5,284,753 discloses an apparatus and method for multi-site chemotaxis experiments. In one method, the chemokines and controls are placed on a preselected area of the top surface of the base plate, while a membrane filter having pores of a suitable size and on which the cell suspension is placed on the base plate, such that droplets of the chemokines and controls are in direct contact with the filter membrane placed under the location of the cell suspension. Thus, over a period of time, cells that migrate through the membrane filter under the influence of chemokines and controls are counted and determined.

[0007] (2) in situ cell diffusion and migration analysis: cells were spotted on glass slides coated with chemoattractant. The diameter of the spot where the cells are present will increase as the cells migrate and spread from the initial spotting location. The diameter of this spot was measured after the cell culture was completed. Migration is determined based on The size of The cell spots during culture (Berens, M.E., et al, "The role of extracellular matrix in human astrocytomization and proliferation in a micro scanner assay", Clinical and Experimental metals, Vol.12, p.405 & 415, 1994; Creative Scientific methods, w.w.critical-sci.com/process. html.).

[0008] (3) in vitro wound healing assay: the healing process of the scratch was measured under a microscope after scraping the cells on a cell monolayer (Miyata K. et al, "New wind-healing modeling focused healing end cells.1.quantitative healing processes", Jpn J Ophthalmol. Vol.34, p.257-266, 1990).

[0009]Cell in situ diffusion and in vitro wound healing analysis methods are easy to operate and low in cost. However, since neither of these methods can distinguish between cell migration and cell proliferation, the results obtained from these two analytical systems are not very reliable. Transwell invasion and migration assays are the most commonly used and well accepted in vitro methods for analyzing cell invasion and migration. This assay uses a device with an insert that forms upper and lower chambers. The two chambers are separated by a porous membrane (or membrane filter) coated with an artificial extracellular matrix (ECM). Invasive cells in the insert or upper chamber can invade and migrate through the microporous membrane coated with artificial ECM into the lower chamber. The activity of invasion and migration of cells passing through the ECM-coated membrane into the lower chamber can be determined by counting the cells. Obviously, the in vitro analysis system can simulate the invasion and migration process of in vivo cells and is also suitable for large-scale analysis of corresponding genes and intracellular signal transduction paths. However, are currently suitable for such bodiesThe data acquisition methods or techniques of the external system are dye-based or radioisotope-based methods, and in most cases, the acquisition of data requires manual counting. Counting cells that invade and migrate across the membrane to the lower chamber is difficult and time consuming, so the accuracy and throughput of this assay is greatly limited. And when the data acquisition method is used, the problems of sensitivity, repeatability, simplicity and the like are often encountered. Currently, it is known that the analysis of molecules and intracellular signal transduction pathways associated with cell invasion and migration is rapidly increasing, and thus a more effective in vitro analysis system is urgently required. To improve the accuracy and throughput of the assay, Amersham Pharmacia and Becton Dickson (BD) biosciences Inc. propose two new assay systems. The Amersham System used a scintillation microtiter plate (cytostar-T) (Graves, R. et al, "A novel assay for cell introduction cytostra-T scanning microplates", Scientific potter, http:// www1.Amersham biosciences. com/aptrix/up00919. nsf/Content/drug + Scientific + potter) to use¹⁴C]And 2³⁵S]Labeled cells to measure cell invasion and migration. An underlying layer of ECM gel is applied to each well to form a barrier to prevent labeled cells from reaching the scintillant-containing substrate. Labeled cells were then added to the upper ECM gel layer and the entire microtiter plate was incubated overnight. Only cells that invade the underlying ECM gel layer and are close to the scintillant will produce a signal in the assay. This system can be used to automatically and real-time monitor cells that penetrate ECM gels. However, the use of radioisotope labels limits the application of this system. The set of systems developed by BD-Biosciences used a light-shielding Fluoroblok PET film that was specially designed to block the transmission of light at 490-700 nm (BD BioCoat)^TM FluoroBlok^TMTumor Cell Invasion System: http:// www.bdbiosciences.com/discovery _ labware/Products/drug _ discovery/insert _ systems/fluorooblock _ innovation /). The experimental cells were stained with a fluorescent dye and placed on a light-tight Fluoroblok PET membrane insert. The invaded cells reaching the back of the insert can be monitored in real time by a fluorescence detectorThe analysis was not disrupted. Using this assay system, the productivity and throughput of invasive assays can be significantly increased. However, the use of this system is greatly limited because not all cell types can be uniformly labeled with fluorescent dyes, and labeling cells can in some cases alter cell invasion and migration.

[0010] Bioelectronics is a developing interdisciplinary field of research involving the integration of biological materials and electronics. The use of electronic methods in cell manipulation and cell analysis has attracted increasing attention.

[0011] Cell-substrate impedance (cell-substrate impedance) measurement is an electronic method for cell monitoring and sensing. Adherent cells are cultured on the surface of the microelectrode structure on the solid substrate. The presence or absence of cells attached to the electrode surface sensitively affects the electron and ion conduction between the cell culture medium and the electrode structure (see, for example, Giaever I. and Keese C.R., "Monitoring fiber glass viewer in tissue culture with an applied cross-section field", Proc. Natl. Acad. Sci. (USA), 1984, Vol. 81, p. 3761-3764). Thus, interrogating the electrode impedance value can provide important information on the biological state of the cells present on the electrode. U.S. Pat. No. 5,187,096 discloses a cell-substrate electrical impedance sensor with a multi-electrode array. Each electrode pair on the impedance sensor for measuring the impedance of the cell substrate comprises a small electrode (measurement electrode) and a large electrode (reference electrode), which are located in two different layers. The difference in size of the two electrodes ensures that the change in the measured impedance value relative to the impedance value when no cells are present on the electrodes is directly related to the number and size of the cells, typically 20-50 cells, or even a single cell attached or growing on the measuring electrode. Some applications of such cell sensors include monitoring conditions in bioreactors, monitoring conditions in cell cultures, detecting cytotoxicity of compounds, studying cell biology to probe cell motility, metabolic activity, cell attachment and diffusion, and the like. However, the impedance sensor including the double-layer structure is complicated because the measuring electrode is in one layer and the reference electrode is in another layer. Furthermore, the electrode area selected for the small electrodes limits the maximum of 50 cells to be monitored at a time.

[0012] U.S. patent No. 4,686,190 discloses a device for studying cell migration through a monolayer of epithelial cells in vitro, which simultaneously measures the transepithelial electrical resistance of the epithelial cell layer.

[0013] WO 02/42766a2 discloses a device and method for studying the effect of chemicals and other factors on cell activity. In this device, cells migrate in an agarose environment and their position can be monitored by a system that measures the impedance and other system electrical parameters of the target on which the assembly is lithographed on a substrate as the cells reach the target.

[0014] Other current data collection methods or techniques for in vitro cell migration analysis are generally dye-based and in most cases require manual counting. Thus, sensitivity, reproducibility and simplicity problems are often encountered when using dye-based methods.

[0015] To overcome the drawbacks of the prior art, the object of the present invention is to expand the use and application of the electrical field and other electronic methods for measuring and analyzing cells, non-cellular particles and biological, physiological and pathological conditions of the cells or non-cellular particles. To this end, the present invention provides an innovative cell migration analysis apparatus using microelectronics technologies.

Summary of The Invention

[0016] In one aspect, the invention provides a device for monitoring the migration of biological particles (e.g., the migration of cells). The device includes an upper chamber for receiving and holding a cell sample, a lower chamber comprising at least two electrodes, and a biocompatible porous membrane having a porosity sufficient to allow migration of cells therethrough. The film is placed in the apparatus to separate the upper and lower chambers. Migration of cells that migrate through the porous membrane allows the migrated cells to contact one or more electrodes located within the lower chamber. This contact causes a measurable change in impedance between or among the electrodes.

[0017] In a preferred embodiment, the device comprises one or more capture reagents immobilized on the surface of at least two electrodes. These capture reagents have the ability to bind to target cells and/or particles.

[0018]The electrodes may be mounted on the surface of the membrane located in the lower chamber. In one embodiment, the lower chamber has a bottom area large enough for attaching a cell population selected from the group consisting of 1-10, 10-100, 100-300, 300-700, 100-1,000, 700-1,000, 1,000-3,000, 3,000-6,000, 6,000-10,000 and 1,000-10,000 cells. In another embodiment, at least 5% of the surface of the membrane located within the lower chamber is covered by the electrode. In another embodiment, the surface area of the lower chamber is less than 1mm²。

[0019] In a preferred embodiment, the device further comprises an impedance analyzer in electrical communication with the electrode.

[0020] The biocompatible porous membrane may comprise an insulating material. The insulating material may be glass, sapphire, silicon dioxide on silicon, or one or more polymers. In a preferred embodiment, the thickness of the biocompatible porous membrane is from 2 to 500 microns.

[0021] In addition, the biocompatible porous membrane may also contain a coating to facilitate attachment of one or more cells.

[0022] In another embodiment, the apparatus further comprises a conductive trace (trace) extending from and in electrical communication with at least one electrode pair, and means for establishing an electrical connection between the conductive trace and the impedance analyzer.

[0023] In another aspect, the invention also includes a method of monitoring cell migration. The method includes introducing cells into the upper chamber of the device and determining whether there is a change in impedance between or among the electrodes. If there is a change in impedance during the experiment, it is an indication that there is cell migration into or through the biocompatible porous membrane.

[0024] The method further comprises adding a known or suspected cell migration modulator to the lower chamber of the device.

[0025] In another embodiment, the method further comprises adding a known or suspected cell migration modulator to the upper chamber of the device.

[0026] The cell can be a mammalian cell. In one embodiment, the mammalian cell is a cell suspected of being malignant. In another embodiment, the mammalian cell is a neuronal cell.

[0027] The cells may also include one or more microorganisms.

Brief Description of Drawings

[0028] FIG. 1 is a schematic diagram of a cell migration measurement apparatus comprising an upper chamber 110, a transmembrane pore 120, and a lower chamber 130 having an impedance-based counter electrode 150. An impedance analyzer may be connected to the electrodes 150 to monitor cell migration/invasion. In operation, cells capable of migration/invasion migrate from upper chamber 110 to the lower chamber through the pores in the transmembrane. Some or all of the cells that migrate from the upper chamber to the lower chamber may be attached to the bottom surface of the lower chamber 130 where the electrodes 150 are located, such that the cells may contact and adhere to the electrodes 150 to cause a change in impedance at the electrodes 150. This change in impedance can be used to monitor the number of cells migrating from the upper chamber.

[0029] FIG. 2 is a schematic illustration of cell migration determination by directly monitoring the change in impedance between upper chamber 210 and lower chamber 220 as the cells pass through the pores in membrane 230. The change in impedance is monitored by the impedance analyzer 240.

[0030] Figure 3 illustrates the resistance and capacitive reactance of 8 different types of electrodes with NIH3T3 cells attached and without NIH3T3 cells attached. The impedance shown on the Y-axis in fig. 3-7, 10, 11 is a resistance and the capacitance shown on the Y-axis in fig. 16 to 29 is a capacitive reactance. Both the resistance and the capacitive reactance are in ohms.

[0031] FIG. 4 illustrates quantitative measurement of cells using a 3B electrode.

[0032] FIG. 5 illustrates the real-time monitoring of the proliferation of NIH3T3 cells and PAE cells using 3C and 3B electrodes.

[0033] Fig. 6 illustrates impedance comparisons of 4 different cell types measured with 3C electrodes.

[0034] Fig. 7 illustrates the repeatability of the impedance measurements.

[0035] FIG. 8 is a diagram of an electronic cell chip design. This figure shows 5 representative electronic cell chip designs. Gold electrodes of different shapes and sizes are located in the center of the glass substrate. The size of the glass substrate is 1cm × 1 cm. The gold electrode is connected to an electron detection interface (electro-deposition interface) via a connection electrode pad (connection electrode pads) on the side of the glass substrate.

[0036] FIG. 9 illustrates the mechanism of cell detection by the electrode.

[0037] FIG. 10 illustrates the real-time monitoring of PAE cell proliferation on a test device. Cells were seeded at different densities (8,000 and 1,000 cells) on the coated electrodes. The resistance (shown as impedance) and reactance (not shown) were measured at different time intervals as shown to monitor cell proliferation. "t 0" is shown as measured immediately after seeding the cells. At both cell seeding densities, the impedance (i.e., resistance) values increased with increasing culture time, indicating that the cells were in a proliferative state. The proliferation rate of cells with high seeding density is obviously higher than that of cells with low seeding density. Sd: and (4) inoculating density.

[0038] FIG. 11 illustrates the quantitative measurement of cells on a test device and using the MTT assay. Serial dilutions of NIH3T3 cells (10,000, 5,000, 2,500, 1,250 and 625 cells) were added to fibronectin-coated assay devices or 96-well plates. For the analysis using the device, the impedance (in this figure, resistance) was measured 16 hours after inoculation. For the MTT assay, cells were stained with MTT dye 16 hours after inoculation and read at 540nm on an ELISA plate reader. As shown, the device can quantitatively measure changes in cell number. The results obtained for both methods were almost identical. Further, current chip designs have the ability to detect less than 600 cells, which is comparable to the MTT assay.

[0039] FIG. 12 shows a 16-unit electronic cell chip for migration analysis.

[0040] FIG. 13 illustrates an exemplary apparatus for migration analysis. (A) A test cell is shown: comprises a chamber containing an electronic cell chip (lower chamber), and an insert with a microporous membrane. Cells were seeded into the insert and cultured. The cells capable of invasion pass through the membrane in a manner to facilitate invasion and migration and are attached to the electrodes of the chip, thereby being detected by the impedance analyzer. (B) The cross-section of the display device has 8 cells per row. (C) A 16-cell device is shown and connected to an impedance analyzer.

[0041] Fig. 14 is a schematic diagram of a cell migration/invasion measurement device comprising an upper chamber 10, a trans-pore membrane 20 with an impedance-based counter electrode or electrode array 25, and a lower chamber 30. An electrode array 25 is located on the bottom surface of the cross-pore membrane 20, facing the lower chamber 30. An impedance analyzer or impedance measuring circuit 60 is operatively connected to the electrodes 25 by a specific electrical connection cable/wire or other means for monitoring the number of cells on the electrodes. The change in electrode impedance is caused by cell attachment from the upper chamber, reflecting the number of cells migrating from the upper chamber.

[0042] Fig. 15 is a schematic view of the shape of the electrode for monitoring cell migration/invasion shown in fig. 14. 15(a) an array of interleaved parallel wire-like electrodes, the electrodes having a width that may be greater than, equal to, or less than an electrode gap (gap); 15(B) a staggered electrode structure of a tooth shape; 15(C) electrode structure of adding disk electrode on the line electrode; 15(D) a straight electrode structure of a tooth type; 15(E) a sinusoidal electrode structure; 15(F) concentric electrode configuration. The characteristic dimension of the electrodes may be as small as less than 10 microns and as large as more than several hundred microns. The entire active electrode area may have different shapes, such as a regular shape, such as a rectangle, (fig. 15(a), 15(B), 15(E)) or similar circle (15(C), 32(D)) or other regular or irregular shape. Preferably, the area of the entire electrode area (including the area of the electrode and the electrode gap) may cover substantially the entire bottom surface of the upper chamber. The electrode structure is connected to an impedance measuring circuit (e.g., an impedance analyzer) via a connection pad (as shown in fig. 15(a) and 15 (B)), and the connection pad may be directly connected to the electrode part (as shown in fig. 15(a), 15(C), and 15(E)) or connected to the electrode part via another electrical connection (as shown in fig. 15(B) and 15 (D)). In FIGS. 15(A), (C) and (E), the connection pads are also electrically conductive connection paths in the electrode structure to which the electrode components are connected.

[0043] FIG. 16 is a schematic view of an apparatus comprising a plurality of upper chambers (1610) and lower chambers (1630). In this case, a plurality of upper chambers (1610) are connected to a membrane (1620) with pores sized for cell migration or invasion. Each upper chamber (e.g., 1610a) has a corresponding lower chamber (e.g., 1630 a). For each upper chamber, there is an electrode structure on the bottom surface of the membrane facing the lower chamber. In operation, the electrode structure on the membrane may be connected to the impedance measuring device via different methods. For example, the electrode structures are connected to connection pads at the edges of the membrane via conductive traces or vias on the membrane. The connection pads may be connected to the impedance measuring device by different methods. One method is as follows: wires operatively connected to the impedance measuring device or circuit may be soldered or bonded to the connection pads by conductive connections. Various embodiments also exist for the upper chamber, in one embodiment, multiple upper chambers are individually separated and only connected together when connected to the same membrane (1620). In another embodiment, the upper chambers are interconnected and assembled together (e.g., the upper chambers may be plastic and made by injection molding). One of these upper chambers is then bonded to the membrane.

[0044] FIG. 17 is a schematic of an apparatus containing multi-site cell migration and invasion measurements. The floor (1730) includes a plurality of lower chambers. The top plate (1710) includes a plurality of insertion holes (1715). The bottom surface of each insertion hole is a membrane (1720) having an electrode structure on its bottom surface facing the lower chamber. In operation, the top plate (1710) rests on the bottom plate (1730). The electrode structure located at the bottom surface of each insertion hole may be connected to the impedance measuring apparatus in various ways. For example, an electrode structure located on the bottom surface of the membrane may be connected to a connection point (1790) at the outer edge of the insertion hole via a conductive via (1780) located outside the insertion hole. This connection point (1790) can further be connected to a connection pad (1795) on the base plate when the insertion hole is inserted into the base plate. The connection pad (1795) on the base plate is operatively connected to the impedance measuring circuit. In another example, the electrode structures on the bottom surface of the membrane may be connected to connection pads on the membrane (not shown). When the insertion hole is inserted into the base plate, the connection pad is contactingly connected to a needle-like or other-shaped connection point (1750) provided on the lower chamber.

[0045]FIG. 18 is a schematic cross-sectional view of the cell migration/invasion device shown in FIG. 17, illustrating the electrode structures on the membrane of each insertion hole before the cells migrate through the holes in the membrane (upper panel, impedance Z)₀) And after invasion/migration of cells through the pores in the membrane (lower panel, impedance Z)_cell) Impedance measurement of (2).

[0046] Fig. 19 is a schematic of an apparatus comprising a plurality of upper chambers (1910), a trans-orifice membrane 1920, and a lower chamber (1930). Wherein a plurality of upper chambers (1910) are connected to a membrane (1920) with an aperture sized for cell migration or invasion. Each upper chamber (e.g., 1910a) has a corresponding lower chamber (e.g., 1930 a). For each lower chamber, there is an electrode structure on the bottom surface of the lower chamber, facing the upper chamber. In operation, the electrode structure in the lower chamber can be connected to an impedance measurement circuit or device via various methods to measure cells attached and/or adhered to the electrodes. The monitored impedance may reflect the number of cells migrating from the upper chamber to the lower chamber.

[0047] FIG. 20 is a schematic of an apparatus with multi-site cell migration and invasion measurements. The bottom plate (2030) includes a plurality of lower chambers. The electrode structure is disposed on or integrated into the bottom surface of the lower chamber, facing the upper chamber. The top plate (2010) includes a plurality of insertion holes (2015). The bottom surface of each insertion hole is a membrane. In operation, the top plate (2010) rests on the bottom plate (2030). The electrode structures on the bottom surface of each lower chamber may be connected to the impedance measuring device by various methods.

[0048]FIG. 21 is a schematic cross-sectional view of the cell migration/invasion device of FIG. 20, illustrating the electrode structure located on the lower chamber before cell migration through the pores in the membrane (upper panel, impedance Z)₀) And impedance measurement after invasion/migration of cells through the pores in the membrane (lower panel, impedance is Z)_cell)。

[0049] FIG. 22 is a comparison of the invasion and migration activities of non-invasive NIH3T3 cells and invasive HT1080 cells in a cell migration device with the electrode structure integrated into the bottom surface of the lower chamber, as shown in FIG. 1.

[0050] FIG. 23(A) is a comparison of the invasion and migration activities of non-invasive NIH3T3 cells and invasive HT1080 cells in a cell migration device (FIG. 23(B)) with the electrode configuration on the microporous membrane of the insert (similar to the configuration shown in FIG. 16).

[0051] Fig. 24 shows the results of monitoring the inhibitory effect of doxycycline on cancer cell invasion and migration in real time with a cell migration device, in which the electrode structure is located on the porous membrane of the insert (similar to the structure shown in fig. 16). (A) A time and dose dependent inhibition of HT1080 cell invasion and migration caused by doxycycline. (B) Dynamic inhibition of HT1080 cell invasion and migration by doxycycline was monitored in real time using fully automated equipment with software-controlled impedance measurement data acquisition. This migration process was continuously monitored every 15 minutes as shown in fig. 24 (B).

[0052]FIG. 25(A) shows typical spectra of measured resistance of a "circle-on-line" electrode structure mounted on a glass substrate under two conditions: (a) hollow tags, tissue culture medium containing HT1080 cells was added to the wells containing electrode structures on the bottom surfaces of the wells shortly after (within 10 minutes, cells were not attached to the electrodes and substrate surfaces); (b) solid tags, media containing HT1080 cells were added to the bottom of the wells2 hours and 40 minutes after the electrode structure in the wells (cells have attached to the electrode and substrate surface). Within this 2 hour 40 minutes, the wells were placed at a temperature of 37 ℃ and 5% CO₂Horizontal tissue culture chambers. The electrode structure was designed as shown in fig. 3B with a line width of 30 microns, a line gap of 80 microns, and a diameter of a continuous circle on the line of 90 microns. The total area covering the electrodes and the electrode gap is in this case approximately equal to a circle of 3mm diameter. The electrode structure on the glass substrate formed the bottom surface of a conical hole with a top surface diameter of 6.5mm and a bottom surface diameter of about 5 mm. In the experiment, a total volume of 100 microliters of tissue culture medium containing about 7,000 HT1080 cells was added to wells containing electrode structures on the bottom surface of the wells.

[0053] Fig. 25(B) shows a reactance spectrum measured under the same conditions as in fig. 25(a) in the same electrode configuration. Note that the absolute magnitude of the reactance is plotted as a logarithmic value (in the same manner as the curve in fig. 16). Except for the high frequencies of 1MHz and 580kHz, the reactance of the electrode structure measured shortly after (within 10 minutes) the tissue culture medium containing HT1080 cells was added to the wells containing the electrode structure was negative (capacitive reactance). The reactance measured at 2 hours and 40 minutes after the cell suspension was added to the wells containing the electrode structures was negative throughout the frequency range of 100Hz to 1MHz of the measurement.

[0054] Fig. 25(C) shows a spectrum of the ratio of the resistance value when cells are attached to the electrode surface to the resistance value when no cells are attached, based on the result shown in fig. 25 (a).

[0055] Fig. 25(D) shows a spectrum of the ratio of the reactance value when a cell is attached to the electrode surface to the reactance value when no cell is attached, based on the result shown in fig. 25 (a). Note that the reactance polarity (i.e., capacitive and inductive reactance) has been taken into account when calculating the reactance ratio.

[0056]FIG. 26(A) shows typical spectra of measured resistance of "round on line" electrode structures mounted on a glass substrate under two conditions: (a) hollow tag, tissue culture medium containing HT1080 cells was added shortly after the bottom surface of the well containing the electrode structure(within 10 minutes, the cells have not attached to the electrodes and substrate surface); (b) solid label, media containing HT1080 cells was added 2 hours 40 minutes after the electrode structures were included on the bottom surface of the wells (cells had attached to the electrode and substrate surfaces). The wells were placed at 37 ℃ and 5% CO in 2 hours and 40 minutes₂Horizontal tissue culture chamber. The electrode structure was designed as shown in fig. 3B with a line width of 30 microns, a line gap of 80 microns, and a continuous circle diameter of 90 microns on the line. In this case the total area covering the electrodes and the electrode gap is approximately equal to a circle of 3mm diameter. The electrode structure on the glass substrate formed the bottom surface of a conical hole with a top surface diameter of 6.5mm and a bottom surface diameter of about 5 mm. For the experiments, a total volume of 100 microliters of tissue culture medium containing about 3,200 HT1080 cells was added to wells containing electrode structures on the bottom surface of the wells.

[0057] Fig. 26(B) shows a reactance spectrum measured under the same conditions as in fig. 26(a) in the same electrode configuration. Note that the absolute magnitude of the reactance is plotted as a logarithmic value (in the same manner as the curve in fig. 16). Except for the high frequencies of 1MHz and 580kHz, the electrode reactance measured shortly after (within 10 minutes) tissue culture medium containing HT1080 cells was added to the wells containing the electrode structure was negative (capacitive reactance). The reactance measured at 2 hours and 40 minutes after the cell suspension was added to the wells containing the electrode structures was negative throughout the measurement frequency range 100Hz to 1 MHz.

[0058] Fig. 26(C) shows a spectrum of the ratio of the resistance value when cells are attached to the electrode surface to the resistance value when no cells are attached, based on the result shown in fig. 26 (a).

[0059] Fig. 26(D) is a graph showing a spectrum of the ratio of the reactance value when a cell is attached to the electrode surface to the reactance value when no cell is attached, based on the result shown in fig. 26 (a). Note that: in calculating the reactance ratio, the polarity of the reactance (i.e., the capacitive and inductive reactances) has been taken into account.

[0060]FIG. 27(A) shows a typical measured resistance of a "round on line" electrode structure mounted on a glass substrate under two conditionsType spectrum: (a) hollow tags, tissue culture fluid containing HT1080 cells was added to wells containing electrode structures on the bottom surface of the wells shortly (within 10 minutes, cells were not attached to the electrode and substrate surfaces); (b) solid label, media containing HT1080 cells was added 2 hours 40 minutes after the electrode structures were included on the bottom surface of the wells (cells had attached to the electrode and substrate surfaces). The culture wells were placed at 37 ℃ and 5% CO for 2 hours and 40 minutes₂Horizontal tissue culture chamber. The electrode structure was designed as shown in fig. 3B with a line width of 30 microns, a line gap of 80 microns, and a continuous circle diameter of 90 microns on the line. In this case the total area covering the electrodes and the electrode gap is approximately equal to a circle of 3mm diameter. The electrode structure on the glass substrate formed the bottom surface of a conical hole with a top surface diameter of 6.5mm and a bottom surface diameter of about 5 mm. For the experiments, a total volume of 100. mu.l of tissue culture medium containing about 500 HT1080 cells was added to wells containing electrode structures on the bottom surface of the wells.

[0061] Fig. 27(B) shows a reactance spectrum measured under the same conditions as in fig. 27(a) in the same electrode configuration. Note that the absolute magnitude of the reactance is plotted as a logarithmic value (in the same manner as the curve in fig. 16). The reactance of the electrode structure measured under both conditions was negative (capacitive reactance) except for 1MHz and 580kHz at high frequencies.

[0062] Fig. 27(C) shows a spectrum of the ratio of the resistance value when cells are attached to the electrode surface to the resistance value when no cells are attached, based on the result shown in fig. 27 (a).

[0063] Fig. 27(D) is a graph showing a spectrum of the ratio of the reactance value when a cell is attached to the electrode surface to the reactance value when no cell is attached, based on the result shown in fig. 27 (a). Note that: in calculating the reactance ratio, the polarity of the reactance (i.e., the capacitive and inductive reactances) has been taken into account.

[0064] FIG. 28(A) is a spectrum of the ratio of the resistances of different numbers of cells added to wells with the same type of "round on line" electrode configuration (electrode shape 3B). Fig. 28(a) is a comprehensive illustration of the frequency spectrums shown in fig. 25(C), 26(C) and 27 (C). One method of calculating the "cell number index" is based on the frequency spectrum of these resistance ratios, which is first to determine the maximum value of the resistance ratio and then to subtract this by one to obtain the "cell number index". The cell number indices obtained by this method were 5.17, 1.82 and 0.17 when different cell numbers of 7,000, 3,200 and 500 were added, respectively, it is clear that the larger the cell number, the higher the cell number index.

[0065] FIG. 28(B) is a spectrum of the ratio of the reactance of different numbers of cells added to a well with the same type of "round on line" electrode configuration (electrode shape 3B). Fig. 28(a) is a comprehensive illustration of the frequency spectrums shown in fig. 25(D), 26(D) and 27 (D).

Modes for carrying out the invention

[0066] The following detailed description of the invention is to be taken in a number of sections for clarity of disclosure and not for purposes of limiting the invention.

[0067]Cell-based analysis with impedance sensing using the devices and systems disclosed in this patent application can provide noninvasive measurements so that cells can be monitored continuously in real time and cells can continue to be used for other cell-based or molecule-based analyses after impedance-based sensing. The disclosed devices and systems can provide dynamic information on cell number and cell biological status (including adhesion status, viability, etc.). Furthermore, the measurement process does not require any labeling of reagents, thereby saving the cost of labeling reagents and the associated handling and manpower required to add those labels. Further, the entire measurement process can be computer controlled and fully automated. The researcher need only inoculate the cells for the desired analytical procedure and add the appropriate compounds and media. Finally, the measurement system is accurate and has a high degree of reproducibility and repeatability. The measurement system is also very sensitive, expressed as the number of cells per unit area (e.g. about 5 cells/mm)²)。

[0068] The effect of most of the cells added to the wells in the disclosed embodiments on the change in impedance between the electrodes on the device of the present application is monitored. This is achieved by a large electrode width and electrode gap ratio and, in one embodiment of the device of the invention, by a sensing electrode which covers the entire area of the aperture in the plate. Embodiments in which more cells are monitored for the effect of changes in impedance between the electrodes thus have the advantage that experimental variation between wells in the same plate or between wells in different plates can be reduced. This also improves the accuracy and sensitivity of measuring small numbers of cells in the assay.

[0069] The selection of the electrode width, gap width and electrode component distribution ensures that the electric field distribution is relatively uniform. For example, the design of the electrodes ensures that for any two adjacent electrode components on different electrode structures, the sum of the electrical resistances from each of these two components to their respective connection pads is almost constant for any given two adjacent electrolytic components. Thus, the effect of any two adjacent components on the impedance is nearly the same. So that their effect on the impedance change is the same wherever the cells are located.

[0070] A convenient method of inter-electrode connection can ensure that multiple sensors can be easily added to the same slide.

[0071] In certain embodiments of the electrode for impedance sensing, there is a non-uniform electric field distribution. This has been demonstrated and demonstrated in many references to dielectrophoresis, wherein under certain electrical and cell suspension conditions, cells are subjected to so-called dielectrophoretic forces and move towards and away from the edges of the electrode assembly. The following documents, for example, clearly demonstrate this effect occurring for various electrodes: "selective electrophoretic compositions of biophotonic in potential energy wells", by Wang X-B et al, J.Phys.D.: appl.phys. volume 26: page 1278-1285, 1993; "Positive and negative dielectrically reflecting of colloidal particles using intercalated particulate microorganisms", by Pethig R. et al, J.Phys.D.: appl.phys. volume 25: page 881-888, 1992. In addition, the following theoretical papers provide additional information on the non-uniform electric field distribution of the interleaved electrodes: "A the organic method of electrical field analysis using Greens's the item", by Wangx-J et al, J.Phys.D.: appl.phys. volume 29: pages 1649 and 1660, 1996.

[0072]Table 1 compares the MTT assay results with the cell impedance sensing assay results. Glass slides with 16 electrode units (each electrode unit having the behavior shown in 3B, see table 2 below) were used in this experiment. A 16-hole plastic strip with a bottom diameter of 5mm was mounted on the glass slide with liquid adhesive. Different numbers of HT1080 cells were seeded in different wells of a 16 x device. The corresponding cell number index can be derived by calculating the maximum relative change in the measured series resistance of each electrode unit. Table 1 shows the cell number index at 2.5 hours after cell inoculation. The well surfaces (including electrode surfaces) were pre-coated with fibronectin by soaking the wells in a fibronectin solution at a concentration of 50 μ g/ml (micrograms per ml) for one hour. The experiment was ended 2.5 hours after the start of the impedance measurement. Cells in the same device were simultaneously analyzed using MTT for comparison. For MTT assay, 10. mu.l of MTT reagent solution was added to each well. After the reaction was incubated at 37 ℃ for 4 hours, 100. mu.l of stop solution (stop solution) was added to each well. After addition of the stop solution, the reaction was further incubated at 37 ℃ for 12 hours to dissolve insoluble purple formazan formed during the MTT reaction(formalzan) crystal. Color density in the Device was measured on a 96 × plate reader Molecular Device with a wavelength set at 550 nm. As a reference absorbance (at a wavelength of 650 nm) for measuring non-specific readings.

TABLE 1

Number of holes	Number of cells	Index of cell number	MTT reading
				B1	1.54E+04	4.74E+00	1.43E+00
C1	1.19E+04	4.71E+00	1.43E+00
				D1	9.13E+03	3.73E+00	1.18E+00
E1	7.03E+03	5.80E+00	1.01E+00
				F1	5.41E+03	4.41E+00	9.40E-01
G1	4.17E+03	2.79E+00	7.62E-01
				H1	3.21E+03	1.59E+00	6.86E-01
H2	2.47E+03	7.99E-01	5.99E-01
				G2	1.90E+03	6.94E-01	5.39E-01
F2	1.47E+03	7.67E-01	4.29E-01
				E2	1.13E+03	5.89E-01	3.83E-01
D2	8.69E+02	3.61E-01	3.30E-01
				C2	6.69E+02	2.62E-01	3.14E-01
B2	5.15E+02	1.39E-01	2.48E-01
				A2	3.97E+02	1.51E-01	2.54E-01
A1	0.00E+00	9.38E-02	2.38E-01

A. Definition of

[0073] The following detailed description of the invention is to be taken in a number of sections for clarity of disclosure and not for purposes of limiting the invention.

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, publications and other publications cited herein are incorporated by reference in their entirety. If a definition set forth in this section is contrary to or contrary to a definition set forth in a patent, application, published publication or other publication that is incorporated herein by reference, the definition set forth in this section applies and is not incorporated herein by reference.

[0075] As used herein, "a" or "an" means "at least one" or "one or more".

[0076] As used herein, "at least two electrodes are mounted on the same side of the substrate" means that if the insulating substrate is a multilayer, at least two electrodes are mounted on the same layer of the substrate.

[0077] As used herein, "film" refers to a sheet of material.

[0078] As used herein, "biocompatible membrane" refers to a membrane that has no deleterious effects on cells, including having no deleterious effects on viability, adhesion, diffusion, motility, growth, cell division, and the like.

[0079] When a suspension containing live, undamaged, epithelial or endothelial cells is added to a vessel, the surface of the vessel is "suitable for cell attachment" meaning that a significant proportion of the cells attach to the vessel surface within 12 hours. Preferably, at least 50% of the cells are attached to the surface of the vessel within 12 hours. More preferably, a surface suitable for cell attachment has surface properties such that at least 70% of the cells are attached to the surface within 12 hours of plating (i.e., adding cells to the vessel). Or more preferably, the surface properties of the surface suitable for cell attachment are such that at least 90% of the cells are attached to the surface within 12 hours of plating. Most preferably, the surface properties of the surface suitable for cell attachment are such that at least 90% of the cells are attached to the surface within 8, 6, 4, 2 hours of plating. In order to have the surface properties required for cell attachment, the surface is subjected to chemical treatment (e.g.treatment with acids and/or bases) and/or physical treatment (e.g.treatment with plasma), and/or biochemical treatment (e.g.coating with one or more molecules or biomolecules that facilitate cell attachment). In this invention, a biocompatible surface (e.g., membrane) is preferably adapted to allow the attachment of the cell type used in the assay for the biocompatible surface (membrane), and more preferably to allow at least 90% of the cells in contact with the biocompatible surface to attach during the assay.

[0080] "biomolecule coating" refers to a coating on a surface comprising a natural biomolecule or biochemical molecule, or a biochemical molecule derived from or based on a natural biomolecule or biochemical molecule. For example, the biomolecule coating may comprise extracellular matrix components (e.g., fibronectin, collagen), or derivatives thereof, and may also comprise biochemical molecules such as polylysine or polyornithine, which are polymers based on naturally occurring biochemical molecules lysine and ornithine. Naturally occurring biochemical molecules, such as amino acids, based polymer molecules, isomers and enantiomers of the naturally occurring biochemical molecules may be utilized.

[0081] "extracellular matrix component" refers to a molecule present in the extracellular matrix of an animal. It may be an extracellular matrix component derived from any species and any tissue type. Non-limiting examples of extracellular matrix components include laminin, collagen fibronectin, other glycoproteins, peptides, glycosaminoglycans, proteoglycans, and the like. The extracellular matrix component also includes growth factors.

[0082] "electrode" refers to a structure having a high electrical conductivity, which is much higher than the surrounding material.

[0083] As used herein, "electrode structure" refers to a single electrode, particularly an electrode having a complex structure (e.g., a spiral-type electrode structure), or a combination of at least two electrode components in electrical communication. All of the electrode components in an "electrode configuration" are in electrical communication.

[0084] As used herein, "electrode component" refers to a single configuration in an electrode structure, for example, a finger-projection structure in an interleaved electrode structure.

[0085] As used herein, an "electrode structure unit" refers to a structure formed by two or more electrode structures having a size and spacing that, when connected to a signal source, acts as a unit to generate an electric field in the space around the electrode structures. The preferred electrode building blocks of the present invention measure the change in impedance as the cell attaches to the electrode surface. Non-limiting examples of electrode structural elements include interleaved electrode structural elements and concentric electrode structural elements.

[0086] An "electrode trace" refers to a conductive path from an electrode or electrode component or electrode structure to one end or periphery of a device or apparatus to connect the electrode, electrode component or electrode structure to a signal source. The ends or perimeters of the devices may correspond to connection pads on the devices or apparatuses.

[0087] As used herein, "at least two electrodes (electrode arrays) have substantially the same surface area" means that there is no large difference in the surface area of any two electrodes (electrode arrays), such that the change in impedance caused by cell attachment or growth on a large electrode (electrode array) contributes to the total detectable change in impedance to the same or similar degree as the change in impedance caused by cell attachment or growth on a small electrode (electrode array). In other words, either large electrodes (electrode arrays) or small electrodes (electrode arrays) contribute to the overall impedance change when a cell is attached or grown thereon. In general, the ratio of the surface area between the largest electrode (electrode array) and the smallest electrode (electrode array) is less than 10. Preferably, the ratio of the surface area between the largest electrode (electrode array) and the smallest electrode (electrode array) is less than 5,4, 3,2, 1.5, 1.2 or 1.1. More preferably, at least two electrodes (electrode arrays) have approximately equal or equal surface areas.

[0088] "staggered" means that protruding structures from one direction are staggered with protruding structures from the other direction in the manner of a finger or a gripping hand (with the proviso that the staggered electrode parts preferably do not touch each other).

[0089] As used herein, "a device having a surface adapted for cell attachment or growth" means that the electrode and non-electrode areas of the device have suitable physical, chemical, or biological properties to allow the cells required for the experiment to attach to their surface and remain attached during growth on the surface of the device. However, the device or device surface does not necessarily have to carry the necessary substances to supply cell viability or growth. These essential substances, such as nutrients or growth factors, can be supplied by the culture medium. Preferably, when a suspension containing live, undamaged, epithelial or endothelial cells is added to a "surface suitable for cell attachment", at least 50% of the cells attach to the surface within 12 hours. More preferably, a surface suitable for cell attachment has surface properties such that at least 70% of the cells are attached to the surface within 12 hours of plating (i.e., adding cells to a chamber or well containing the device described above). Even more preferably, the surface properties of the surface suitable for cell attachment are such that at least 90% of the cells are attached to the surface within 12 hours of plating. Most preferably, the surface properties of the surface suitable for cell attachment are such that at least 90% of the cells are attached to the surface within 8, 6, 4, 2 hours of plating.

[0090] As used herein, "a measurable change in impedance between electrodes" means that a cell attached or growing to the surface of the device will result in a significant change in impedance between the electrodes, which can be measured by an impedance analyzer or an impedance measuring circuit. The change in impedance refers to the difference between the impedance when a cell is attached or growing on the surface of the device and the impedance when no cell is attached or growing on the surface of the device. Generally, a change in impedance of more than 0.1% can be measured. Preferably, the measurable change in impedance value is more than 1%, 2%, 5%, 8%. More preferably, the measurable resistance value changes by more than 10%. The impedance between the electrodes is generally a function of the frequency of the electric field applied for measurement. "measurable changes in impedance between electrodes" does not require measurable changes in impedance at all frequencies, but only changes in impedance at any single or multiple frequencies. Further, the impedance consists of two parts, resistance and reactance (reactance can be divided into two categories, i.e. capacitive reactance and inductive reactance). "a measurable change in impedance between electrodes" requires that a change in one of resistance and reactance be measured at any single or multiple frequencies.

[0091] As used herein, "at least two electrodes have substantially different surface areas" means that the surface areas of either electrode are dissimilar to each other, and thus the impedance change caused when a cell is attached or grown to a large electrode does not contribute to the total measurable impedance to the same or similar extent as the impedance change caused when a cell is attached or grown to a small electrode. Preferably, the change in impedance caused by the attachment or growth of a cell to the large electrode is significantly less than the change in impedance caused by the attachment or growth of a cell to the small electrode. In general, the ratio of the maximum electrode to the minimum electrode surface area should exceed 10. Preferably, the ratio of the maximum electrode to the minimum electrode surface area exceeds 20, 30, 40, 50, or 100.

[0092] As used herein, "highly likely to contact the electrode means" means that if the cell is placed randomly in the sensor area of the device or apparatus of the present invention, the likelihood that the cell (or particle) will contact the electrode means is calculated from the average diameter of the cell, the size of the electrode means, and the size of the gap of the electrode means used on or in the device or apparatus of the present invention, and should be greater than about 50%, more preferably greater than about 60%, more preferably greater than about 70%, even more preferably greater than about 80%, greater than about 90%, or greater than about 95%.

[0093] An "insert tray" is a structure containing one or more depressions or holes (hereinafter referred to as inserts) that can fit into a liquid container; alternatively, if the insert tray includes a plurality of inserts, they may fit into a plurality of liquid containers or multi-well plates, such that one or more of the inserts inserted into the tray defines a chamber in a liquid container or a well in a multi-well plate. In certain aspects of the invention, an insert tray is provided with at least one device of the invention, forming the bottom of at least one insert, such that when the insert is placed in a liquid container, the chamber formed thereby has a non-conductive substrate with at least one electrode on its bottom surface.

[0094] As used herein, a "dose-response curve" refers to the dependence of a cell on the response of a test compound to a dose concentration. The response of a cell is measured by a number of different parameters. For example, a test compound is suspected of being cytotoxic and causing cell death. The response of the cells can be expressed as the percentage of non-viable (or viable) cells after treatment of the cells with the test compound.

[0095] As used herein, "sample" refers to any substance containing a portion that is separated, manipulated, measured, quantified, detected, or analyzed using the device, microtiter plate, or method of the present application. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. Biological tissue is a structural material that generally has a collection of cells of a particular type together with their intercellular material that can form a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelial, muscle, and neural tissue. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cells. The biological sample may also further comprise a cell suspension, a solution comprising biomolecules (e.g., proteins, enzymes, nucleic acids, carbohydrates, chemical molecules that bind to biomolecules).

[0096] As used herein, a "liquid (fluid) sample" is a sample that naturally exists in a liquid or fluid form, such as a biological fluid. "liquid sample" may also refer to a sample that is naturally present in a non-liquid state, e.g., a solid or gas, but is made into a liquid, fluid, solution, or suspension containing the solid or gas sample material. For example, the liquid sample can be a liquid, fluid, solution, or suspension containing biological tissue.

B. Device and method for detecting cell migration

[0097] In one aspect, the invention is a device for monitoring cell migration or invasion, comprising: a) an upper chamber for housing migrating or invading cells or cells suspected of migrating or invading cells; b) a lower chamber including two electrodes; c) a biocompatible membrane having a plurality of pores therein, the pores being sized to allow passage of said migrated or invaded cells, the membrane being connected between and separating the upper and lower chambers, wherein said migrated or invaded cells pass through the pores of said membrane and contact and/or attach to the electrodes of said lower chamber, causing a change in impedance between the electrodes, said change being useful for monitoring migration or invasion of cells.

In those aspects of the invention that include an upper chamber and a lower chamber separated by a porous membrane through which migrating or invading cells can pass, wherein the lower chamber includes at least two electrodes, the at least two electrodes in the lower chamber are preferably located on the top surface of the bottom of the lower chamber. In some embodiments of these aspects, the lower surface of the membrane has one or more portions to prevent cell attachment. Non-limiting examples of such moieties are certain formulations of polyethylene glycol. In other embodiments, the membrane may be chemically treated (e.g., acid treatment), or physically treated (e.g., some radiation such as plasma radiation), or biologically treated (e.g., some biomolecule coating) such that the lower surface of the membrane prevents cell attachment with minimal cell attachment. In these aspects, attachment of the membrane-mobilized cells to the bottom of the lower chamber causes a change in impedance at the bottom electrode of the lower chamber.

Fig. 19 and 20 show two exemplary embodiments of devices comprising multiple sites for cell migration and invasion. The device in fig. 19 comprises a plurality of upper chambers (1910), a cross-porous membrane (1920), and a plurality of lower chambers (1930). A plurality of upper chambers (1910) are attached to a membrane (1920) having an aperture sized for cell migration or invasion. Each upper chamber (e.g., 1910a) has a corresponding lower chamber (e.g., 1930 a). The bottom surface of each lower chamber has an electrode structure facing the upper chamber. The lower chamber containing the electrode structure is constructed as described in section B of U.S. provisional application 60/435,400 filed 12/20/2002, and the apparatus for monitoring and/or measuring cell-substrate impedance is the same or similar. Thus, the description of the device, multiwell plate, the method of assembling the multiwell plate with a substrate containing electrodes, the method of connecting the electrode structures to an external impedance measurement circuit, and the description of the method of assembling the multiwell plate with the substrate, in section B of U.S. provisional application 60/435,400, filed on 12/20/2002, are also applicable to the lower chamber used herein for monitoring cell migration/invasion. In operation, the electrode structure in the lower chamber is connected to an impedance measuring circuit or instrument via various methods to measure cells that have migrated from the upper chamber and have attached and/or adhered to the electrode structure. The monitored impedance can be used to calculate a cell number index (or cell migration index), which is an indication (or reflection) of the number of cells that have migrated from the upper chamber to the lower chamber.

[0098] The device shown in fig. 20 comprises a top plate and a bottom plate. The top plate (2010) includes a plurality of insertion holes (2015). The bottom surface of each insertion hole is a membrane. The base plate (2030) includes a plurality of lower chambers. An electrode structure is fitted or integrated on the bottom surface of each lower chamber, facing the insertion hole. Construction of the lower base with electrode Structure the apparatus for monitoring and/or measuring cell-substrate impedance described in section B above is the same or similar. The descriptions in section B above regarding the device, the porous plate, the method of assembling the porous plate with the substrate containing the electrode, and the method of connecting the electrode structure to the external impedance measurement circuit are also applicable to the lower chamber and the bottom plate used herein for monitoring cell migration/invasion. In operation, a top plate (2010) placed in an appropriate buffer or culture medium loaded with cells for migration/invasion analysis is placed in a bottom plate (2030) loaded with a buffer or culture medium containing appropriate reagents. The electrode structures disposed on the bottom surface of each lower chamber are connected to an impedance measuring circuit or device via various methods.

[0099] In another aspect, the invention relates to a device for monitoring cell migration or invasion, comprising: a) an upper chamber for housing migrating or invading cells, or cells suspected of migrating or invading cells, said upper chamber containing an electrode; b) a lower chamber containing an electrode; c) a biocompatible membrane comprising at least one pore sized to allow migration or invasion of cells therethrough, the membrane being connected to and separating the upper and lower chambers, wherein movement of migrated or invaded cells through the pore of the membrane causes a change in impedance between the electrodes of the upper and lower chambers, which change can be used to monitor migration or invasion of cells.

[00100] In another aspect of the invention, the device of the invention comprises a) an upper chamber for receiving cells; b) a lower chamber; c) a biocompatible polymer membrane comprising a plurality of pores sized to allow passage of all or part of the cells therethrough, wherein the membrane separates the upper and lower chambers from each other; and d) at least two electrodes are mounted on the membrane in the lower chamber such that the active surfaces of the electrodes face the lower chamber.

[00101] The biocompatible membrane used in the present device may comprise any suitable material. Non-limiting examples of such materials include glass (e.g., quartz glass, lead glass, borosilicate glass), silicon dioxide on silicon, silicon-on-insulator (SOI) wafers, sapphire, plastic, and polymers. Some preferred polymers are polyimides (such as Kapton, polyimide film available from DuPont), polystyrene, polycarbonate, polyvinyl chloride, polyester, polyethylene terephthalate (PET), polypropylene and urea resins. Polymers such as polycarbonates, polyesters, polyethylene terephthalate (PET) are particularly preferred. The biocompatible membrane can be of various thicknesses, ranging from as thin as about 2 microns to as thick as about 500 microns. Preferably, the biocompatible membrane of the device of the present invention has a thickness of about 5 to about 50 microns, more preferably about 8 to about 25 microns.

[00102] Any non-conductive substrate surface exposed to cells is preferably biocompatible when the device or apparatus of the invention is in use. Preferably, at least one surface of the non-conductive substrates is adapted for cell attachment or growth. Substrate materials that are not biocompatible or suitable for cell attachment or growth may be coated with other substances such as: polymer or biomolecule coatings suitable to allow cell growth attachment and growth thereon. Thus, the biofilm surface of the device of the invention may comprise a substance, such as a plastic, which is suitable for the attachment and growth of cells, or, alternatively or additionally, a coating to attach cells to the biofilm surface.

[00103] The biocompatible membrane optionally comprises a coating that facilitates the attachment of one or more cells. The coating may be a polymer, such as a plastic film, or one or more biomolecules or one or more derivatives of one or more biomolecules, such as, but not limited to, polymers such as polyornithine or polylysine, skin or proteins, or extracellular matrix (or derivatives or analogs thereof) including, but not limited to, gelatin, fibronectin, laminin, collagen, glycosaminoglycans, peptidoglycans, and the like. These coatings preferably, but optionally, cover the entire surface of the substrate, including the electrode surfaces, that the device is capable of contacting or being exposed to cells during use.

[00104]The coating may be a semi-solid or gel, and may optionally include other additional components, such as, but not limited to, growth factors. The coating may also be a simple or complex mixture of biomolecules, and may also mimic or replicate the native extracellular matrix (ECM). For example, Matrigel^TMBasement membrane matrix (BD Bioscience), a solubilized basement membrane preparation extracted from the sarcoma of Engelbreth-Holm-swarm (rehs) mice, which contains abundant extracellular matrix proteins. Its main components are laminin, followed by collagen IV, heparan sulfate, proteoglycans, entactin and tactile proteins. It also contains TGF- β, fibroblast growth factor, tissue plasminogen activator, and other growth factors naturally present in EHS tumors.

[00105]Coating mimicking or replicating ECM can promote cell attachment. Also in some aspects of the invention, a barrier is formed to prevent cell migration/invasion. Cells are passed through an ECM-like barrier (such as, but not limited to, Matrigel)^TM) The permeability of (a) can be detected by an "invasion test" experiment using the device or apparatus of the invention.

[00106] The biocompatible membrane of the present device may have one or more (preferably a plurality of) pores of any suitable size suitable for passage of the cells whose migration or invasion behaviour is being studied. The biocompatible membrane may comprise a plurality of pores having the same size. Alternatively, the biocompatible membrane may comprise a plurality of pores having different sizes. It is preferred that the size of the well should limit the number of cells passing through one well at a time to less than 3, more preferably even 1 cell. For example, the membrane may contain a plurality of pores of the same size, the pores having a diameter of about 1 to about 30 microns, more preferably about 2 to about 10 microns, for mammalian cancer cells, epithelial cells or endothelial cells.

[00107] In other preferred embodiments of the invention, the diameter of one or more pores of the biocompatible membrane does not allow cells to pass through the pores that are used to measure the reactance, resistance, or capacitance of the cell/substrate interface. For example, the one or more pores may be less than about 5 microns, or preferably less than about 1 micron. In some aspects of these embodiments, one or more electrodes are mounted on the membrane for measuring changes in impedance caused by the destruction of a layer of cells growing on the membrane by invading cells. In other aspects of these embodiments, the growth, attachment, detachment, morphology, motility of the cells on the membrane can also be monitored.

[00108] Each electrode pair is electrically connected to an impedance analyzer. The impedance may be analyzed or measured in any suitable frequency range, such as a frequency range between about 1Hz to about 100MHz, or between about 10Hz to about 5 MHz.

[00109] Most preferably, at least one electrode of each electrode pair is adapted to bind to a cell that has passed completely or partially through the porous membrane. The electrode used to monitor cell migration should have any suitable surface area. For example, the electrodes between which the impedance is measured to monitor cell migration or adhesion may have substantially the same or significantly different surface areas. In another example, the surface area of each electrode surface is sufficient to attach at least 10 cells.

[00110] In a preferred aspect of the invention, two or more electrodes are mounted on one side of the biocompatible membrane. Preferably, at least two of the two or more electrodes have substantially the same area. The at least two electrodes having substantially the same surface area are preferably part of the same electrode building block.

[00111] The electrodes or electrode components in the electrode structure of the present device may have any suitable shape, such as rectangular, circular on a rectangular line ("circular on a line"), square on a rectangular line or sinusoidal on a line. They may also be other curves such as, but not limited to, spirals or arcs. Some examples of electrodes, electrode structures, or electrode structure elements of the devices of the present invention are shown in fig. 9 and 15.

[00112] In some preferred embodiments of the invention, the electrode structure may be an interleaved electrode structure (IDES) or a concentric electrode structure (CCES) as shown in fig. 15A and 15F. For example, an electrode structure may comprise two or more electrodes forming one or more IDES or one or more CCES. The interdigitated electrode structure (IDES) may be further modified or adapted so that the parallel line electrode means has a large perimeter sub-shape (texture), i.e. the portion added to the line electrode means (which may itself be parallel, curved, looped, angled, etc.) may be branched, outcrop, convex, etc. when viewed from above, so that the line electrode path has a larger perimeter than if its boundary only conformed to the directionality of the electrode means path. Examples of the structure having a large circumference are a diamond-shaped on-line electrode structure, a "round-on-line" electrode structure shown in fig. 15C, and a tooth-shaped electrode structure shown in fig. 15B, 15D. The electrode structure with the large perimeter sub-shape is not limited to that described herein and may be regular 25 or irregular in the periodicity and the profile (curved, angular, circular, rectangular) of the sub-shape.

[00113] Preferably, the electrodes and electrode components are distributed over the entire surface area of the device on which they are mounted, wherein this area will or will come into contact with the cells. In other words, the area that is or will be in contact with the cell is covered by the electrode (or electrode part) and the electrode (or electrode part) gap. In preferred devices of the invention, the sensor area should occupy at least 5%, 10%, 30%, 50%, 70%, 80%, 90%, 95% or even 100% of the total surface area of the device that will or will be exposed to the cells in the assay. In other words, in a preferred device of the invention, at least 5%, 10%, 30%, 50%, 70%, 80%, 90%, 95% or even 100% of the surface area that will or will be exposed to the cells in the assay should cover the electrodes (or electrode means) and the electrode (or electrode means) gaps. Preferably, the distribution of the electrodes or electrode components over the sensor area is uniform or nearly uniform.

[00114] In embodiments where the device comprises at least two electrode structures, the two or more electrode components should preferably be arranged in an electrode structure. When the electrode member is not rectangular, "electrode width or electrode member width" refers to the average dimension of the electrode member in the plane of the substrate from the point of contact with one electrode gap to the point of contact with the other electrode gap opposite thereto (in a direction perpendicular to the long axis of the electrode member). When the electrode gap is not rectangular, "electrode gap" refers to the average dimension of the gap in the plane of the substrate from the point where it meets one electrode member to the point where it meets the opposite electrode member (in a direction perpendicular to the long axis of the gap). For the purposes of the present invention, substrate means the material on which the electrodes are mounted. If the electrodes are mounted on a biocompatible membrane, then the membrane is a substrate.

[00115] In order to monitor the behavior of the cells, it is preferred that the gap between the electrode members should not substantially exceed the size of the cells whose behavior is being monitored by the device (e.g., the width of the cells that diffuse into and adhere to the substrate). This reduces the likelihood that the cell will be in contact with the substrate but not with at least a portion of the electrodes or electrode components. Furthermore, it is preferred that the width of the gap between the electrode elements (or the size of the gap) be substantially no less than the size of the cell being monitored by the device (e.g., the average width of the cell being spread across and attached to the substrate) to reduce the likelihood that the cell will contact an adjacent electrode element, which would produce an impedance signal that is particularly large relative to when the cell is in contact with only one electrode. This is particularly important if the width of the electrodes is much larger (e.g. 10 times) than the size of the cells whose behaviour is to be detected by the device. On the other hand, if the electrode width is comparable to the size of the cell (e.g., the average width of the cell spread and attached to the substrate), the gap width between the electrode components may be slightly less than the cell. Preferably, the electrode component gap size of the electrode structure is in the range of about 0.2 to 3 times the average cell width used in an assay employing the present device, although other gap sizes may be used. Preferably, the width of the gap between the electrodes or electrode parts of the device of the invention for monitoring eukaryotic cells, such as mammalian cells (e.g. cancer cells, endothelial cells or epithelial cells), should be from about 3 to 80 microns, more preferably from about 5 to 50 microns, most preferably from about 8 to 30 microns.

[00116] Preferably, the width of the electrode part cannot be too narrow because the resistance thereof increases when the width of the electrode part decreases. The increased resistance along the electrode means results in a large potential difference at different points on the electrode means, so that the impedance signal is different when the cell is in or attached to different areas of the electrode means. Preferably, any area of the cell that is on or attached to the substrate surface should have a similar impedance signature. Thus, for an electrode assembly that is either an interleaved electrode configuration or a concentric electrode configuration, when the device is used to measure eukaryotic cells, such as mammalian cells (e.g., cancer cells, endothelial cells, or epithelial cells), the electrode width should preferably be greater than about 3 microns, and more preferably greater than about 10 microns. The electrode width is also limited on the basis that if the electrode member is wide, a small impedance signal is caused when the cell is located in the central part of this wide electrode, compared to the cell being located at the edge of the electrode where the field strength is significantly higher. Preferably, the width of the electrode means should be from about 0.5 to about 10 times the size of the cells used in the assay in which the device is used (e.g., the average cell width when spread and affixed to a substrate). Preferably, for an electrode member belonging to IDES or CCES, when the device is used to measure eukaryotic cells, such as mammalian cells (e.g., cancer cells, endothelial cells or epithelial cells), the electrode or electrode member should be less than about 500 microns wide, more preferably less than about 250 microns. In some preferred embodiments of the invention, the electrode features should be between about 20 and about 250 microns wide.

[00117] In this patent application, it is preferable that the electrode gap of the electrode part is designed in consideration of the electrode width. Many electrode member width to gap ratios are suitably available, but preferred electrode member width to gap ratios are between about 1: 3 and 20: 1. Preferably, the electrode part width is 1.5 to 15 times the gap width, more preferably 2 to 6 times the gap width; for example, if the electrode width of the widest point of each electrode is 90 microns, the width of the widest point of the gap between adjacent electrodes is about 20 microns. In the present application, the width of the electrodes ranges from less than 5 microns to greater than 10 millimeters. It is preferred that the width of the electrodes is in the range of 10 microns to 1 mm. More preferably the electrode width is between 20 microns and 500 microns.

[00118] Examples of non-limiting materials for the electrodes or electrode parts may be Indium Tin Oxide (ITO), chromium, gold, copper, nickel, platinum, silver, steel and aluminum. The electrodes may comprise a variety of materials. The choice of a suitable material for the electrodes depends on the following factors: whether the material is sufficiently conductive, whether it is difficult to mount the material on a substrate, and whether the material can be used to reliably perform the molecular detection analysis of the present invention.

[00119] The electrodes or microelectrodes in the present invention may be of any conductive material. For example, gold (Au), platinum (Pt), etc. can be used. When a substrate such as a plastic or polymer film is used, a metal adhesion layer such as Cr and Ti may be used. In order to reduce the electrical impedance of the electrode, the electrode having the conductive thin film layer should have a certain thickness. One non-limiting example is: the electrode can be made as a 300 angstrom thick layer of Cr coated with a2,000 angstrom layer of Au. Such an electrode layer is opaque. Furthermore, light-transmissive electrodes may also be used in the device of the present invention. Examples of the light-transmitting electrode include Indium Tin Oxide (ITO). For an ITO coating with proper thickness, the penetration rate of light passing through the electrode of the ITO layer can reach 98%. In other cases, a sufficiently thin conductive film (i.e., a very thin gold film) may also optionally serve as the light-transmitting electrode. The choice of a suitable electrode material depends on the following factors: whether the material is biocompatible and non-cytotoxic, whether the material is sufficiently conductive, and whether the material is readily assembled on a membrane substrate.

[00120]In one aspect of the invention, the electrodes of the device or apparatus of the invention are mounted in a biological organism for monitoring cell migration or invasionOn the film. Various microfabrication methods can be used to assemble the electrodes on the biocompatible membrane. One example is the use of photolithographic methods. Exemplary steps for the photolithography method are as follows: one side of the biocompatible film is first coated with a thin metal film (e.g., about 0.2 μm gold film on a 10nm seed Cr layer) by vapor deposition and/or sputtering, a photoresist (e.g., Shipley photoresist S1830) is spin coated to a certain thickness (e.g., 1 μm) on the gold film, and then exposed to uv light through a mask with the desired electrode array image. The exposed photoresist may be developed with a corresponding developer (e.g., MF351 developer from Shipley), and then the gold and chromium layers are sequentially developed with KI/I, respectively₂And K₃Fe(CN)₆NaOH etching. The mask is commercially produced on an ultra-high resolution flat panel using electron beam writing technology. Due to the "flexible" nature of the thin film, care must be taken when using photolithographic methods to ensure repeatable and precise fabrication of the microelectrode structure on the film. Care is taken, especially during assembly, to ensure that the film remains flat, extended, assembled and in good contact with the screen. In addition, since the membrane has at least one hole of a size suitable for cell migration/invasion, care is taken during assembly not to affect the holes if they are already present before assembling the electrode. Alternatively, the holes may be formed by assembly or micro-machining (e.g., laser drilling) after the electrodes are assembled. Those skilled in the art of lithographic assembly and other microfabrication and micromachining methods can readily select and apply appropriate materials and processes to assemble the microelectrodes and microwells.

Another method of micro-assembling or patterning electrodes is laser ablation (laser). For the most extensive ablation, one side of the film is first coated with a thin metal film (e.g., about 0.1 μm gold film on a 10nm seed Cr layer) by vapor deposition and/or sputtering. This thin metal film is then exposed to a laser of suitable intensity (e.g., 248 or 351nm excimer laser) through a mask bearing the desired electrode array image. In the areas of the mask that are "transparent" to the laser light, the laser light impinges on the metal film and interacts with it to ablate it from the film. Because the interaction of a film (e.g., a polymer film) with a laser is different from the interaction of a metal film with a laser, the laser conditions (wavelength, intensity, pulse width) can be selected to be suitable so that the laser ablates the metal film with little or no effect on the film. In the areas where the laser light is "blocked" on the mask, the metal film remains on the film. The mask is commercially produced on an ultra-high resolution flat panel using electron beam writing technology. Due to the "flexible" nature of the film, extra care is required when using laser ablation to ensure repeatable and precise fabrication of the microelectrode structure on the film. Care is especially taken to ensure that the polymer film remains flat and extended during mask-based laser ablation. Those skilled in the art of laser ablation and the use of laser ablation to create the corresponding pattern in the film will readily select the appropriate method and laser wavelength, intensity, mask to create the electrodes on the polymer film.

[00121] If the assembly method of assembling the well and the electrode or electrode structure on the biocompatible membrane allows, it may be possible to ensure that the well for cell migration/invasion is located in the area corresponding to the electrode surface, while avoiding gaps in the electrode or electrode components. While not necessary or limiting in this disclosure, these are still preferred embodiments of the apparatus according to aspects of the invention. For example, in one aspect of the invention, a biocompatible membrane is attached to the bottom surface of the top chamber (or upper chamber) and the membrane has electrodes on the surface facing the bottom chamber (or lower chamber). The details and applications of this structure will be described in the following section "apparatus comprising upper and lower chambers" (see below). In operation, cells are placed in the apical chamber in a suitable culture medium and the migration behavior of the cells through the biocompatible membrane is monitored by measuring changes in impedance as a result of the electrode structures that the cells migrate to the basal chamber and adhere to the membrane. An advantage of locating the pores that will allow cell migration/invasion in the region of the membrane corresponding to the electrode area is that as cells migrate through the pores, they can contact and adhere to the electrode surface as they leave the pores in the membrane. Linking the attachment of the electrode surface and migration through the pores by fitting the pores at the electrode site ensures that all cells migrating through the pores in the membrane contribute to the measured impedance change.

[00122]The electrode components, electrodes, electrode structures and electrode building blocks of the devices of the invention may have any suitable configuration, surface area or surface modification. For example, at least one electrode structure may have at least two electrode members. In another example, the surface area of the electrode or electrode structure may be modified with moieties that promote cell adhesion. Any suitable moiety that promotes cell adhesion may be used in the device of the present invention, such as a self-assembled monolayer (SAM) layer (e.g., thiolane (alkanethiolate) on gold, alkylsiloxane on SiO₂Or SiO_xAbove), proteins (e.g., fibronectin, gelatin, collagen, laminin, proteins that promote attachment of specific or non-specific cells to an electrode or electrode array surface region), peptides (e.g., poly-L-lysine), polymer layers and a charged group.

[00123] Preferably, the electrodes, electrode structures, and electrode components are configured such that electrode traces extend from the electrodes on the substrate surface to the edge or end of the substrate (e.g., a biocompatible film) where they may be connected to lines from an impedance measurement circuit or signal source. Where the edge or end of the substrate where the electrode traces terminate corresponds to a connection pad on the substrate. In a preferred aspect of the invention, the tracks on the electrode parts of one electrode structure and the tracks of the electrode parts of the other electrode structure are insulated from each other. In one type of arrangement, the electrode traces are located in spaced apart regions of the substrate so that they do not touch in the path they travel. In another arrangement, the electrode traces need to cross each other and a layer of insulating material can be sandwiched between the two electrode traces. Assembling such devices or apparatuses involves multilayer microfabrication methods.

[00124] The apparatus also includes one or more impedance analyzers connected to the one or more connection pads. The electrodes may be connected directly or indirectly to the connection pads, via which the wires from the impedance analyzer are connected. Preferably, the connection pads are located at the edge or periphery of the device of the invention, but this is not essential to the invention. Optionally, the connection between the electrodes and the connection pads is made by connection vias located at the ends of the substrate. In exemplary embodiments of the invention, the biocompatible membrane on which the electrodes are mounted will be part of, attached to, or located within a plate or fluid container capable of holding a sample liquid. In these embodiments, the connection pads may be located on the fluid container or on a plate with one or more fluid containers.

[00125] The apparatus or device of the present invention may have any suitable dimensions depending on the application. In one example, the apparatus of the present invention may be sized to be compatible with an automated plate handling workstation. In such a case, a plurality of measurement units are mounted on the same board. As shown in fig. 16-21, each device includes 16 measurement units, any of which can be used for cell migration analysis.

[00126]Any suitable surface area may be provided in the lower chamber of the device. For example, the bottom area of the lower chamber can be suitable for the adhesion of about 1-10, 10-100, 100-300, 300-700, 100-1,000, 700-1,000, 1,000-3,000, 3,000-6,000, 6,000-10,000 or 1,000-10,000 cells. In another example, the electrode covers at least 5% of the area of the bottom of the lower chamber. As yet another example, the bottom area of the lower chamber should be less than 10mm²Or less than 3mm²Or less than 1mm²Or less than 300 μm²Or less than 100 μm²。

[00127] The electrodes may be of any suitable shape and placed in any suitable location on the device. For example, the electrode in the lower chamber may be located on the bottom surface of the lower chamber. In preferred embodiments, the sensor area should occupy at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the entire floor of the lower chamber. In another example, the electrodes or electrode structures of the lower chamber may be located on the sidewalls of the lower base. In another example, the electrodes may be needle-shaped within the chamber. In yet another example, the electrode may comprise an electrode structure having at least 2 electrode members. In another example, the electrodes may be rectangular, circular, or rounded on a rectangular line, or sinusoidal.

[00128] In one embodiment, the lower chamber of the device may comprise a modulator of cell migration or invasion, such as: chemoattractants or other agents that stimulate or inhibit cell migration/invasion. Alternatively or additionally, the upper chamber of the device may also contain a modulator of cell migration or invasion.

[00129] A preferred embodiment of the present invention is a system comprising the device of the present invention and further comprising an interface electronics device comprising an impedance measuring circuit and a switch (e.g., an electronic switch) to control and switch the impedance measuring circuit to the electrode structure unit of the device of the present invention. Preferably, the system of the invention further comprises a computer with a software program for measuring or monitoring the impedance between the electrodes or electrode structures of the device of the invention in real time. The measured impedance data may be automatically analyzed and processed to obtain the appropriate parameter (e.g., cell number index or cell migration index) and displayed on a monitor.

[00130] Preferably, the software program has one or more of the following functions: (1) electronically switching the connection of the impedance measuring circuit (or analyzer) to one of a plurality of devices on the inventive device; (2) controlling an impedance measurement circuit (or analyzer) to measure impedance between the electrodes or electrode structures at one or more frequencies; (3) processing the obtained impedance data to obtain an appropriate biologically relevant parameter (e.g., cell number index or cell migration index); (4) displaying the results on a monitor, or storing the results; (5) the functions 1 to 4 described above are automatically performed at regular or irregular time intervals.

Apparatus comprising upper and lower chambers

[00131] In one aspect, the present invention is an apparatus for monitoring cell migration or invasion, comprising a) at least one upper chamber for housing migrated or invaded cells or cells suspected of having the ability to migrate or invade; b) at least one lower chamber; C) a biocompatible membrane having a plurality of pores therein, the pores being sized to allow passage of said migrating or invading cells, the membrane being connected between and separating the upper and lower chambers, the membrane further comprising at least two electrodes positioned on a side of the membrane facing the lower chamber, wherein said migrating or invading cells pass through the pores of the membrane and contact and/or attach to the electrodes positioned on the membrane to cause a change in impedance between the electrodes, thereby providing for monitoring migration or invasion of said cells.

[00132] In general, each fluid container of the cell migration apparatus of the present invention should have a surface area sufficient for attachment or growth of a plurality of cells. In one example, the fluid container in the device of the invention should have a surface area sufficient for at least 10, preferably at least 50, cells to adhere to. In another example, each pair of electrodes or each pair of electrode structures in the device of the invention connected to an impedance analyzer should have a surface area sufficient for at least 10, and preferably at least 50, cells to be attached.

[00133] In one embodiment of the above device, the biocompatible membrane may further be provided with a gel or other coating on the side facing the upper chamber. This gel or coating provides a barrier to cell migration/invasion. The biocompatible membrane may be coated on one or both sides, for example a coating comprising one or more extracellular matrix components on the upper side and polylysine on the lower side.

[00134] In another embodiment of the above device, the bottom compartment may further comprise an agent that promotes or inhibits cell migration or invasion. Alternatively or additionally, the upper chamber may also carry an agent that promotes or inhibits cell migration or invasion.

[00135]The lower chamber of the device of the present invention may be of any shape, such as cylindrical, rectangular, and other shapes. The lower chamber can be made of a variety of substances, including polymers, plastics, or glass. The lower chamber may also be of different volumes and sizes. The lower chamber in the present apparatus may also have any suitable surface area. In one example, the bottom area of the lower chamber may be as small as 1mm²And may be larger than 50mm². In another example, the bottom area of the lower chamber may be less than 10mm²Or less than 3mm²Or less than 1mm²Or less than 300 μm²Or is orLess than 100 μm²。

[00136]The upper chamber of the device of the present invention may be of any shape, such as cylindrical, rectangular, and other shapes. The upper chamber can be made of a variety of substances, including polymers, plastics, or glass. The upper chamber may also be of different volumes and sizes. The bottom surface of the upper chamber may be a biocompatible containment membrane with at least one pore suitable for cell migration/invasion. The membrane may be attached to a wall of the upper chamber. The area of the bottom surface of the upper chamber (i.e., the corresponding area on the membrane) can be any size suitable for performing cell migration/invasion assays. For example, this area may be as small as 1mm²And may be larger than 50mm². In another example, the bottom area of the upper chamber may be less than 10mm²Or less than 3mm²Or less than 1mm²Or less than 300 μm²Or less than 100 μm²。

[00137] The device of the present invention comprising upper and lower chambers separated by the device of the present invention can be made by a variety of suitable methods. For example, a top chamber structure with at least one tube or no bottom hole can be attached to the device of the present invention, and a lower chamber structure (in the form of at least one hole) can also be attached (e.g., reversibly attached) to the top structure with the attached membrane.

[00138] Alternatively, the method may be used on a vessel with a bottom surface (e.g., a well or structure with one or more wells), such as: laser patterning and laser drilling of the mounting electrode to mount the device of the present invention on the bottom surface. In this case, the bottom surface is a biocompatible membrane. The lower chamber structure can then be attached to the upper chamber structure.

[00139] The device of the invention may be part of a system comprising an impedance analyzer or impedance measuring circuit connected to at least two electrodes on the device. Preferably, the electrodes of the device of the invention are connected to an impedance analyzer or an impedance measuring circuit on at least one connection pad. The electrodes may be connected directly or indirectly to at least one connection pad where they are connected to wires from an impedance measurement circuit or impedance analyzer. In one exemplary embodiment, the connection pad may be located in a top chamber structure (top plate) with one or more fluid reservoirs; preferably near or at the border or perimeter of the bottom surface of the device.

[00140] In a preferred embodiment of the invention, a system comprising a device of the invention comprising an upper chamber and a lower chamber separated by a biocompatible membrane also comprises interface electronics comprising impedance measurement circuitry and switches (e.g., electronic switches) to control and switch the impedance measurement circuitry to the various electrode building blocks of the device of the invention. Preferably, the system of the invention also comprises a computer with a software program that can measure or monitor the impedance between the electrodes or electrode structures of the device of the invention in real time. The measured impedance data may be automatically analyzed and processed to obtain the appropriate parameter (e.g., cell number index or cell migration index) and displayed on a monitor. [00141] Preferably, the software program has one or more of the following functions: (1) electronically switching the connection of the impedance measuring circuit (or analyzer) to one of the plurality of units on the device; (2) controlling an impedance measurement circuit (or analyzer) to measure impedance between the electrodes or electrode structures at one or more frequencies; (3) processing the obtained impedance data to obtain an appropriate biologically relevant parameter (e.g., cell number index or cell migration index); (4) displaying the results on a monitor, or storing the results; (5) the functions 1 to 4 described above are automatically performed at regular or irregular time intervals.

Device (membrane) with multiple electrodes or electrode structural units

[00142] In some preferred embodiments of the invention, a device for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface comprises four or more electrodes mounted on the same side of a biocompatible membrane having at least one aperture, wherein at least one surface of the biocompatible membrane is adapted to receive one or more cells. Preferably, the four or more electrodes are arranged in an electrode array (or electrode structure array) comprising two or more IDES or ccs, each IDES or ccs having at least two electrodes.

[00143] Preferably, a device with at least two or more IDES or CCES is part of an apparatus wherein the device is reversibly or irreversibly attached to one or more structures that provide a plurality of separate fluid containers, each of which may contain one or more IDES or CCES, such that such a biocompatible membrane separates the liquid container into an upper chamber and a lower chamber.

[00144] FIG. 16 is an example of a device with multiple top chambers (1610) and multiple bottom chambers (1630). In this example, a plurality of apical chambers (1610) are coupled to a membrane (1620) containing pores sized for cell migration or invasion. Each top chamber (e.g., 1610a) has a corresponding bottom chamber (e.g., 1630 a). Each top chamber has an electrode structure on the bottom surface of the membrane, facing the bottom chamber. In operation, the electrode structures on the membrane are connected to the impedance measuring device via different methods. For example, the electrode structures are connected to connection pads located at the edge of the membrane via conductive traces on the membrane or vias on the membrane. The connection pads may be connected to the impedance measuring device by different methods. One method is as follows: the wires operatively connected to the impedance measuring device or circuit may be soldered or connected to the connection pads by a conductive adhesive. There are also different ways of implementing the top compartment. One is that the top compartments are separate and are only connected together when connected to the same membrane (1620). Another embodiment is where multiple top compartments are interconnected and assembled together (e.g., where the multiple top compartments are plastic and made by injection molding). A portion of these top compartments are then attached to the membrane.

[00145] In making a biocompatible membrane, the area of the individual sensors may cover an area that is larger than the area covered by the well or chamber structure. This ensures that, even in situations where there is no precise alignment, the aperture comprises substantially all of the portion of the membrane that is the sensor area when the upper and lower chamber structures are attached to the membrane.

[00146] However, in a porous structure device, it is not required that all of the pores contain an electrode or electrode structure. For example, in some preferred embodiments, one or more of the pores may include at least a portion of a biocompatible membrane, which portion may be free of electrodes. These one or more wells without electrodes or electrode structures can serve as controls, where the cells under analysis can be observed under a microscope, or used as biochemical assays, such as gene expression, compound detection, or viability assays using optical detection (e.g., absorption or fluorescence), optionally using a plate reader or other detection means.

[00147] In some aspects of the invention, the electrodes are mounted on the upper side of the membrane. In these aspects, the biocompatible membrane has a pore size of less than about 5 microns, preferably less than about 1 micron, and preferably carries a layer of epithelial or endothelial cells on the upper side of the membrane. To establish the epithelial or endothelial cell layer, epithelial or endothelial cells may be added to the apical chamber and the epithelial or endothelial cells may be attached to membrane-mounted electrodes which may have a biomolecular coating to facilitate cell attachment. Attachment of epithelial or endothelial cells, which may or may not be completely assembled confluently at the surface of the membrane, to the membrane-mounted electrodes can be monitored by measuring changes in impedance between the electrodes. Cells capable of migrating or invading through the epithelial or endothelial cell layer, or cells suspected of being capable of migrating or invading through the epithelial or endothelial cell layer, may be added to the apical chamber. Disruption of the epithelial or endothelial cell layer by these cells can produce a change in impedance that can be monitored by the system of the present invention. Optionally, one or more compounds that affect the invasive properties of the cells may be placed in the upper or lower chambers of the device. Non-limiting examples of applications of the device of the present invention include use for measuring cancer cell migration/invasion characteristics and leukocyte migration characteristics.

[00148] For example, leukocyte migration through small blood vessels to target tissues is an essential feature of the inflammatory response. This migration is chemotactic and can be initiated and enhanced in the presence of an external chemical stimulus, known as a chemoattractant. Much research has focused on exploring molecular mechanisms that are intrinsic to migratory leukocytes, as well as discovering anti-inflammatory therapeutic effects that inhibit pathological migration of leukocytes. Cytokines such as IL-1, IL-6, IL-8, IL-12, IL-15, IL-18, GM-CSF, TNF- α, INF- γ, NO, TGF- β, VIP, and somatostatin, cell adhesion molecules such as LFA-1, VLA-4, α -chain, MAC-1, ICAM-1, selectins (L, P, E), chemokines and chemokine receptors such as RANTES, MIP-1, IP-10; the contribution of CCR1, CCR2B and CXCR2 to leukocyte migration.

[00149]An in vitro experimental model called leukocyte transendothelial migration has been well accepted for analyzing molecular mechanisms and for examining inhibitory chemicals or stimuli of migration, where leukocytes migrate through an established monolayer of cells in the presence of chemoattractants. In one example of a routine assay for T cell migration, the carotid artery of a Lewis rat was removed and used to isolate Endothelial Cells (ECs) for leukocyte migration detection. First, the ES cell line MAT-1.3-5X 10 was established⁴Cells/ml were placed in cell culture inserts (polyethylene terephthalate filters, 3 μm pore size, 9.0mm diameter, 24-well format; Becton Dickinson) coated with 0.2% gelatin, cultured for 1-2 days until confluent, and stimulated with recombinant rat IFN-. gamma. (100U/ml, Life technology). Control cells were not treated with IFN- γ. T cells (2X 10 per filter)⁵Cells) were added to the confluent layer of MAT-1 with or without blocking antibody and cultured in a cell incubator. After 3-5 hours, cells that migrated to the bottom well of the plate can be collected and counted under a microscope.

[00150] The device of the present invention may meet the increasing need for a detection system that more efficiently and cost-effectively analyzes leukocyte migration. The device of the present invention enables label-free, and real-time detection of leukocyte transendothelial migration and invasion. The proposed electronic device maintains the analytical model of a commonly used filter-chamber transwell migration device, comprising a microporous membrane insert and a lower chamber, while a microelectronic sensor is mounted on the microporous membrane of the insert to measure the impedance of the cell-substrate. The arrayed sensors can detect cells that are in close contact with the electronic sensor, such as a monolayer of cells and cells that migrate through the monolayer. Thus, in addition to real-time detection of leukocyte transendothelial migration, the device also monitors the integrity of the cell monolayer in a given insert prior to addition of test leukocytes, thereby improving the accuracy and reproducibility of the detection method. The devices, apparatuses, and systems of the invention can also be used to perform similar assays on other cell types (e.g., to detect invasion of cancer cells by ECM or cell layers).

[00151]Another example of the use of the invention includes assays to determine or measure the integrity of a cell monolayer for drug transport or drug permeability assays. For this assay, Caco-2 cells are widely used as an in vitro experimental model to estimate transport of drug candidates across The intestinal epithelial barrier (e.g., "The use of surfactants to enhance The performance of drugs through The intestinal epithelial barrier" (e.g., "The use of drugs to enhance The intestinal epithelial barrier) (Pharm Res., Vol. 13 (4): p. 528. 534, 1996." evaluation of polalized expression systems in Caco-2 cell pa. of modeling cyclosporine a ", AugustifuginePF et al, Biochim. Biophys. Comm. No. 197, Vol. 360, p. 365, Characteris. transmitter a transport, J. 9, J. expression of molecular proteins, J. 9, J. expression of genes. 1989, J. introduction, et al.; The expression of genes, J. 9, J. 3. introduction, J. 9, et al). Generally, the assay is performed on a multi-day culture (e.g., 10 days, 21 days) using cells grown in a multi-well system. Each system comprises a multi-well insert plate (or filter plate, since there is a well-containing membrane at the bottom of each well on the insert plate), and a multi-well plate. The insert plate (or filter plate) is placed on the multi-well plate with each well on the insert plate placed in a corresponding well on the multi-well plate. Caco-2 cells were cultured in individual wells of the filter plate for a certain number of days to achieve a differentiated cell monolayer close to the ridge. The integrity of the monolayer can be detected by various methods, including fluorescent yellow exclusion, and TEER (transepithelial electrical resistance). When the integrity of the monolayer reaches a certain target parameter (e.g., TEER at 250-²In between), then the cell monolayer is formedCan be used to measure the permeability or transport capacity of a drug candidate molecule through this layer.

[00152] The device of the invention can be used to assess the integrity of a cell monolayer (Caco-2 cell layer). In this application, the electrodes are mounted on the upper side of the membrane. In these aspects, the pore size of the biocompatible membrane should be less than 10 microns, preferably less than 3 microns, and even less than 1 micron. For example, the pore size should be about 1 micron or 0.5 micron. This electrode-bearing membrane is then used to replace the microporous membrane of the insert plate (otherwise known as the filter plate) of the conventional Caco measurement system described above. Similar to conventional Caco-2 measurement systems, Caco-2 cells were cultured on membranes inserted into wells contained in plates. The integrity of the cell monolayer can be monitored by measuring the change in impedance between the electrodes on the membrane. The higher the electrical impedance, the more confluent or dense or tight the cell monolayer. Establishing appropriate electrode impedance criteria allows for characterizing the integrity of the cell monolayer and determining whether the cells are sufficiently tight for compound permeability measurements.

[00153] Another application of the invention is in cytotoxicity assays. In this application, the device is constructed similarly to that used in the Caco-2 system described above, except that electrodes are provided at the bottom of the individual wells on a multi-well plate on which the insert plate (or filter plate) is placed. Thus, in this device, the electrodes are mounted on the microporous membrane formed on the bottom of the insert plate, and the electrodes are also mounted on the bottom of the porous plate. For cytotoxicity assays, cells were cultured on membranes inserted into plates, similar to the Caco-2 assay described above. For example, cells that can be used to mimic the metabolism of a pharmaceutical compound in humans in vitro are seeded and cultured on a membrane inserted into a well. The membrane may have electrodes mounted on its upper side, whereby the electrodes may be used to monitor the physiological state of the cells on the membrane. On the other hand, the membrane may not have an electrode. Other methods may also be used to monitor the cell state, such as the integrity of a cell monolayer, and the pharmaceutical compound is added to the insertion hole when the cells reach a certain confluence or tightness on the membrane. These drug compounds are "handled" by cells on the membrane. Certain metabolic molecules of the pharmaceutical compound after treatment by the cells on the membrane are released into solution and into the underlying well, where the insert plate is placed. For such toxicity assays, cells used to detect toxicity of a compound, or cells used to detect toxicity of a compound metabolite after treatment with intestinal cells or other cells (e.g., cultured cells such as hepatocytes, neuronal cells, lung cells, cardiomyocytes or primary cells) are cultured on multi-well plates (the bottom well plates). The physiological state of the cells is monitored or measured by monitoring the impedance or resistance between electrodes mounted on the multi-well plate. Thus, if the drug compound molecules have a toxic effect or their metabolite molecules from the cells on the membrane are toxic to the cells located in the basal compartment, these compound molecules or their metabolite molecules will be transported or released in the lower compartment. Thus, the cells in the lower chamber can be used to measure and monitor these molecules.

[00154] In a variation of the above application, the cells in the bottom well may be specially assembled (e.g., to alter a gene) to increase their sensitivity to the drug candidate molecule or a metabolic molecule from the drug candidate molecule. In this case, the cells located in the bottom wells are used to sense and monitor the metabolite molecules as the drug candidate compound molecules are "processed" by the cells on the membrane. The cells of the bottom hole can be of any cell type, including neuronal cells, cardiac cells (heart cells), hepatocytes, and the like. The electrodes on the bottom hole are shaped to be suitable for measuring electrical impedance, whereby the cell condition can be monitored. The electrodes in the bottom hole may also be shaped to measure other parameters, such as may be used for extracellular recording of excitable cells (e.g., neuronal cells, cardiac cells, etc.). In this case, extracellular recording of such excitable cells in the bottom well can serve as a sensing mechanism or method to monitor metabolic molecules from the well of the insert. Changes in the extracellular recording signal, measured in the time domain and spectral analysis, can be used as an indicator of the type and/or concentration of a particular metabolic molecule after the drug candidate molecule has been metabolized. Some examples of the use of excitable cells for measuring and sensing toxic or non-toxic molecules are described in "Portable cell-based biosensor system for toxin detection", Pancziazo, J.J., et al, Sensorsand Actuators B Chemical, Vol.53, pp.179-185 (1998); genetic associated cell-based biosensors for specific agent classification, DeBusscher B.D., et al, Proceedings of the International Solid-State catalysts and actors Conference-TRANSDUCERS' 99, Sendai, Japan, 6.7-10 days, (1999).

[00155]In other aspects of these embodiments, the electrode is mounted on the underside of the membrane. In this case, the pore size of the biocompatible membrane is greater than about 1 micron, preferably less than about 30 microns, and the upper side of the membrane is preferably provided with at least one substance that facilitates cell adhesion to the underside of the membrane. The upper side of the membrane may carry at least one extracellular matrix component or, preferably, comprise a plurality of extracellular matrix components that form a matrix, e.g., Matrigel^TM. For example, Matrigel^TMBasement membrane matrix (BD Bioscience) is a solubilized basement membrane preparation extracted from Engelbreth-Holm-swarm (rehs) mouse sarcoma, a tumor that is rich in extracellular matrix proteins. Its main components are laminin, followed by collagen IV, heparan sulfate, proteoglycans, entactin and tactile proteins. It contains TGF-. beta.s, fibroblast growth factors, tissue plasminogen activator, and other growth factors naturally present in EHS tumors.

[00156] Alternatively or additionally, the upper side of the membrane may carry an epithelial or endothelial cell layer. Migration/invasion of cells through the epithelial or endothelial cell layer can be monitored by the device of the invention. Also optionally, one or more compounds that affect the invasion/migration behavior of the cells may be placed in the lower chamber of the device. Optionally, one or more compounds that affect the invasion/migration behavior of the cells may be placed in the upper chamber of the device.

[00157] The invention includes a device for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface, wherein a biocompatible membrane comprising two or more electrodes is reversibly or irreversibly attached to a first plate having two or more wells providing a lower chamber of a cell transfer unit, and reversibly or irreversibly attached to a second plate providing a tubular structure providing an upper chamber of the cell transfer unit (e.g., using a bottomless microplate), such that each cell transfer unit comprises an IDES or a CCES. In certain aspects, the membrane may be irreversibly attached to the lower, well-containing plate. In other aspects, the membrane can be irreversibly attached to a tube-containing plate that forms the upper portion of the upper chamber. In the case of a membrane irreversibly attached to the lower plate, some means should be provided for adding the culture medium and optionally other reagents or components for detection to the lower chamber (e.g., the lower chamber may be open at its side walls for addition of culture medium).

[00158] Such devices may be used to detect migration or invasion of one or more cells placed in the upper chamber of the device. In using the device of the invention, the device is part of, or attached to, or within a plate or fluid container in which the cells and culture medium can be placed, such that, in the presence of a suitable cell culture medium, the attachment or detachment of one or more cells on the side of the membrane equipped with at least two electrodes can be measured by the device from changes in impedance, capacitance, resistance.

[00159] The invention also includes a system for measuring the electrical impedance, resistance or capacitance of a cell/substrate interface comprising the apparatus described herein, including a membrane with a plurality of electrode structures surrounded by a plurality of fluid containers, and an impedance analyzer connectable to the plurality of electrodes. An impedance analyzer (or impedance measurement circuit) is also operatively connected to the electrode. The impedance may be analyzed or measured in any suitable frequency range, such as a frequency range between about 1Hz to about 100MHz, or between 10Hz to 5 MHz. The connection between the impedance analyzer and the electrodes in the device may be direct or indirect. For example, the electrodes may extend to the connection pads and be connected to an impedance analyzer at the connection pads. In another example, the electrodes or electrode members are connected to the connection pads via an electrically conductive connection path.

[00160] In a preferred embodiment of the invention, a system comprising the device of the invention also comprises interface electronics including impedance measuring circuitry and switches (e.g., electronic switches) to control and switch the impedance measuring circuitry to the various electrode structure elements of the device of the invention. The system of the present invention also includes a computer with a software program that can measure or monitor the impedance between the electrodes or electrode structures of the device of the present invention in real time. The measured impedance data may be automatically analyzed and processed to obtain the appropriate parameter (e.g., cell number index or cell migration index) and displayed on a monitor.

[00161] Preferably, the software program has one or more of the following functions: (1) electronically switching the connection of the impedance measuring circuit (or analyzer) to one of the plurality of cells in the device of the present invention; (2) controlling an impedance measurement circuit (or analyzer) to measure impedance between the electrodes or electrode structures at one or more frequencies; (3) processing the obtained impedance data to obtain a suitable biologically relevant parameter (e.g., cell number index or cell migration index); (4) the monitor displays the result on the monitor or stores the result; (5) the functions of 1-4 above are automatically performed at regular or irregular intervals.

Multi-cell, two-chamber system with isolation diaphragm

[00162] The invention also includes an apparatus for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface comprising a plate having two or more apertures, each aperture having a membrane with at least two electrodes and one or more small apertures, the membrane dividing the aperture into an upper chamber and a lower chamber, the membrane further having a surface suitable for cell attachment and growth.

[00163] The device can be used to detect migration or invasion of one or more cells when placed in the upper chamber of the device. In the use of the device of the invention, the attachment or detachment of one or more cells on the side provided with at least two electrode films, as a result of the migration or invasion behavior of at least a part of the cells placed in the upper chamber, can be measured by a change in impedance, capacitance or resistance.

[00164] The device of the present invention may be manufactured by any suitable method. For example, a membrane with holes and one or more electrodes mounted thereon may be attached to a bottomless hole structure to form a top chamber, and then attached to a perforated structure that forms a bottom chamber. Alternatively, the device may be attached to a perforated structure which forms the bottom compartment, and then the top portion comprising the tubular structure (without the bottom opening) attached thereto to form the upper compartment. When the bottom plate is irreversibly attached to the membrane and the membrane covers the entire upper surface of the bottom chamber, means (e.g., holes in the side walls of the lower chamber) should be provided for placing the culture medium or optional other reagents into the lower chamber. This can be achieved if the culture medium has been added to the bottom compartment. Another method is to fit a perforated plate (e.g., a perforated plate with a thin polycarbonate film as the bottom) at the bottom of the holes to pattern the electrodes and form the holes, and then attach a perforated bottom structure that forms the bottom compartment.

[00165] The aperture and electrode may be mounted on the membrane by any suitable method depending on the membrane and electrode material. The method of assembling the holes and electrodes may also be used according to the procedure for constructing the top and bottom chambers. For example, the holes may be made by ion bombardment followed by etching, or by laser drilling. Methods of mounting the electrodes on the substrate have been described above, and mounting at the bottom of the hole may include techniques such as laser patterning (or laser ablation). The electrodes may be assembled to the film before it is perforated or, alternatively, the film may be perforated and the electrodes attached to the surface. The electrode and the hole may have a corresponding relationship in position on the membrane, i.e. the hole is located in a region corresponding to the electrode surface.

[00166] The invention also includes a measurement system for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface comprising a device as described herein having a multi-well plate, each well having a biocompatible membrane comprising at least two electrodes and one or more wells, the device further comprising an impedance analyzer connectable to the plurality of electrodes. An impedance analyzer (or impedance measurement circuit) is also operatively connected to the electrode. The impedance may be analyzed or measured in any suitable frequency range, such as a frequency range between about 1Hz to about 100MHz, or between 10Hz to 5 MHz. The connection between the impedance analyzer and the electrodes on the device may be direct or indirect. For example, the electrodes may extend to connection pads where they are connected to an impedance analyzer. In another example, the electrode or electrode assembly is connected to the connection pad via an electrically conductive connection path.

[00167] A preferred embodiment of the invention is a system comprising a multi-well plate apparatus of the invention, the system further comprising interface electronics including impedance measuring circuitry and switches (e.g., electronic switches) to control and switch the impedance measuring circuitry to different electrode building blocks of the apparatus of the invention. Preferably, the system of the present invention further comprises a computer with a software program for measuring or monitoring in real time the impedance between the electrodes and the electrode structure of the device of the present invention. The measured impedance data may be automatically analyzed and processed to obtain the appropriate parameter (e.g., cell number index or cell migration index) and displayed on a monitor.

[00168] Preferably, the software program has one or more of the following functions: (1) electronically switching the connection of the impedance measuring circuit (or analyzer) to one of the plurality of units in the device; (2) controlling an impedance measurement circuit (or analyzer) to measure impedance between the electrodes or electrode structures at one or more frequencies; (3) processing the obtained impedance data to obtain a suitable biologically relevant parameter (e.g., cell number index or cell migration index); (4) displaying the results on a monitor, or storing the results; (5) the functions of 1-4 above are automatically performed at regular or irregular intervals.

Multi-insertion tray system

[00169] The invention also includes an apparatus for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface, the apparatus comprising an insertion tray having one or more insertion chambers, each chamber comprising a liquid impermeable wall and a porous biocompatible membrane comprising two or more electrodes, the membrane forming the bottom of at least one of the one or more insertion chambers. Preferably, the device further comprises a plate with one or more holes, whereby the insertion tray of the invention is adapted to this plate. Preferably, the insert tray has a plurality of insert chambers and a corresponding plate containing a plurality of apertures, the insert tray being configured such that the insert chambers are aligned with and can be placed into the apertures in the plate. Preferably, each insert structure is placed into a well of a plate such that the well in the plate constitutes a lower chamber and the insert inserted into the tray constitutes an upper chamber of the cell invasion/migration unit.

[00170] The invention also includes a device for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface, wherein the insert tray is provided with a biocompatible membrane forming the bottom of the upper chamber and is reversibly loaded onto a first plate comprising two or more wells providing the lower chambers of the cell transfer unit, whereby preferably such that each cell transfer unit comprises an IDES or a ccs unit.

[00171] Such a device can be used to detect migration or invasion of one or more cells when placed in the upper chamber of the device, attachment or detachment of the cells on the side of the membrane equipped with at least two electrodes upon addition of a suitable cell culture medium, as measured by a change in impedance, capacitance or resistance with the device.

[00172] The invention also includes a system for measuring the electrical impedance, resistance, or capacitance of a cell/substrate interface comprising an insertion tray and a multi-well plate device, and an impedance analyzer connectable to a plurality of electrodes on a membrane insert. Preferably, an impedance analyzer (or impedance measurement circuit) is also operatively connected to the electrode. The impedance may be analyzed or measured in any suitable frequency range, for example: the frequency range is between about 1Hz to about 100MHz, or between 10Hz to 5 MHz. The impedance analyzer and the device electrodes may be connected via connection pads located on the insertion tray.

[00173] Fig. 17 shows a schematic representation of an exemplary embodiment of an apparatus, which includes an insertion tray (top plate 1710) and a perforated bottom plate (1730). The base plate (1730) includes a plurality of base compartments. The top plate (1710) includes a plurality of insertion holes (1715). The bottom surface of each insertion hole is a membrane (1720) having an electrode structure on its bottom surface facing the bottom chamber. In operation, the top plate (1710) is placed onto the bottom plate (1730). The electrode structures located at the bottom surface of each insertion hole are connected to the impedance measuring apparatus in various ways. For example, an electrode structure located on the bottom surface of the membrane may be connected to a connection point (1790) at the outer edge of the insertion hole via a conductive via (1780) located outside the insertion hole. This connection point (1790) may further be connected to a connection pad (1795) of the base plate when the insertion hole is inserted into the base plate. The connection pad (1795) on the base plate is operatively connected to the impedance measuring circuit. In another example, the electrode structures on the bottom surface of the membrane may be connected to connection pads on the membrane (not shown). When the insertion hole is inserted into the bottom plate, the connection pad is contactably connected to a pin-like or other-shaped connection point (1790) of the bottom chamber.

[00174] In a preferred embodiment of the invention, the invention comprises a system of insertion trays and multi-well plate devices, and also interface electronics including impedance measurement circuitry and switches (e.g., electronic switches) to control and switch the impedance measurement circuitry to the different electrode building blocks of the device of the invention.

[00175] The invention also includes a method of monitoring cell migration or invasion, comprising providing a device for cell migration or invasion as described above, placing a cell in the upper chamber of the device, and monitoring a change in impedance between the electrodes to monitor cell migration or invasion.

[00176] In use, the invention relates to a method of monitoring cell migration or invasion, the method comprising: a) providing the above-described device for monitoring cell migration or invasion; b) placing the migrated or invaded cells or cells suspected of migrating or invading into the upper chamber of the device from the upper chamber through the pores of the polymer membrane into the lower chamber; and c) monitoring the change in impedance between the electrodes to monitor cell migration or invasion.

[00177] The methods provided by the present invention can be used to monitor any suitable parameter associated with cell migration or invasion. For example, the method may be based on monitoring impedance for monitoring the amount or number of cells migrating or invading into the lower chamber.

[00178] The methods provided herein can be used to determine whether a test compound modulates, i.e., increases or decreases, cell migration or invasion, or can be used to screen for such modulators. For example, such a method can be performed wherein the migration of cells is monitored in the presence or absence of a test compound, and such a method can be used to determine whether the test compound modulates the migration or invasion of cells. In another example, the method can be performed when migration or invasion of the cell is stimulated by a migration or invasion stimulus, and the method can be used to screen the test compound for antagonists to the agonist.

[00179] The methods of the invention may also be used to monitor normal cell migration or invasion. Alternatively, the methods of the invention may be used to monitor abnormal cell migration or invasion, such as metastasis of a tumor or cancer cell. In a particular embodiment, the method can be used to monitor migration or invasion of tumor cells, endothelial cells, epithelial cells, fibroblasts, myoblasts, neurons, glial cells, and the like.

[00180] An example of the present apparatus and its method of operation is described below. The device for monitoring cell migration/invasion comprises an upper chamber or an insert and a bottom chamber or a lower well separated by a polymer membrane (e.g.a microporous polyethylene terephthalate membrane). The polymer membrane has a plurality of pores sized to permit passage of invading cells therethrough. The microelectrode array on the bottom compartment or lower well carries at least 2 sets of electrode members (i.e. 2 electrode arrays). The electrode arrays are suitably shaped so that when cells are added to and attached to the electrode plane, the impedance between the electrode arrays changes. Examples of electrode arrays include staggered parallel electrode arrays, double parallel spiral electrode arrays, and circular shaped electrode arrays. The device for monitoring cell migration/invasion further comprises an impedance analyzer for determining the impedance between the two sets of microelectrodes.

[00181] In operation, cells to be tested are placed in the apical chamber. Appropriate agents (e.g., cell migration/invasion modulators that stimulate or inhibit cell migration/invasion, or agents suspected of being cell migration/invasion modulators) are added to the apical chamber during or after the cells are placed therein. The bottom compartment contains a suitable buffer or cell culture medium. The buffer or culture medium placed in the basal compartment may carry suitable agents, such as chemoattractants, or other cell migration regulators that stimulate or inhibit cell migration and agents suspected of being cell migration/invasion regulators. When cells enter the bottom compartment from the top compartment through the pores in the trans-compartment membrane, fall onto and attach to the electrode structure located in the bottom compartment, the resulting inter-electrode impedance change will be measured and analyzed. Preferably, the electrode area (i.e. the sensor area) covers a substantial part of the area of the bottom surface of the bottom compartment. Preferably, the surface area of the bottom aperture is reduced somewhat to improve sensitivity. To cover a large dynamic range, a series of bottom chambers of different sizes can be made, so that each area can cover a range of cell numbers, for example: 1-10, 10-100, 100-1,000, 1,000-10,000, etc.

[00182] Another example of the present apparatus and its method of operation is described below. The device comprises an upper chamber and a lower chamber separated by a polymeric membrane (e.g., a microporous polyethylene terephthalate membrane). The polymer membrane has a plurality of pores sized to permit passage of invading cells therethrough. The size and number of pores on the polymer membrane must be precisely controlled to fit different cell numbers and different cell sizes. One electrode in the top chamber and one electrode in the bottom chamber. When a cell invades and passes through the hole in the membrane separating the two chambers, the electrical impedance at the two electrodes will change. Electrodes that can monitor transmembrane impedance can be used to provide information on the migration process.

Impedance spectrum in detecting cells

[00183] FIG. 25(A) shows a typical spectrum of the measured resistance of a "round-in-line" electrode structure mounted on a glass substrate under two conditions: assembly (a) tissue culture medium, hollow labeled HT 1080-containing cells, was added to the wells containing the electrode structures on the bottom surface of the wells shortly (within 10 minutes, cells were not yet attached to the electrodes and substrate surface); (b) solid label, media containing HT1080 cells was added 2 hours 40 minutes after the electrode structures were included on the bottom surface of the wells (cells had attached to the electrode and substrate surfaces). Shortly after the cell-containing medium is added to the wells (within 10 minutes), there is insufficient time for the cells to adhere to the electrodes. This is confirmed by the fact that the impedance (resistance and reactance) measured by the electrode structure in which the cell-containing medium is added to the well is the same or almost the same as the value obtained when the cell-free medium is added to the well. In the case where a cell-free medium is applied to the electrode or where a cell-containing medium is applied to the electrode but the cells do not have sufficient time to adhere to the electrode structure, generally speaking, the impedance (resistance and reactance) at high frequencies (e.g., around 1MHz or above) is determined primarily by the shape of the electrode and the electrical properties (conductivity and permittivity) of the medium in solution introduced into the electrode structure. At low frequencies, there is an "electrode polarization" that produces a resistance and capacitance that varies with frequency (see, e.g., Schwan, H.P., "Linear and nonlinear electrode polarization and biological materials", in Ann. biomed. Eng., Vol. 20, p. 269 & 288, 1992; Jaron, D., Schwan, HP and Geselewitz., "A chemical model for the polarization of the cardiac pa maker electrodes", in Med. biol. Eng., Vol. 6, p. 579 & 594). Under conditions of 2 hours and 40 minutes after the cell-containing medium was placed in the well, and the well was placed in a tissue incubator for 2 hours and 40 minutes, the cells had enough time to attach and spread (cell evidence on the area not covered by the electrodes can be examined under a microscope). Due to the non-conductivity of the cell membrane, the resistance spectrum of the electrode structure changes. Generally, there is an increase at intermediate frequencies (1kHz-100 kHz). There is a small change in the low or high frequency region.

[00184] FIG. 25(B) shows the measured reactance spectra for the same electrode configuration under two conditions as in FIG. 25 (A): (a) hollow marks indicate that tissue culture medium containing HT1080 cells was added to the wells containing the electrode structures on the bottom surface of the wells shortly (within 10 minutes, cells were not attached to the electrode and substrate surfaces); (b) solid label, media containing HT1080 cells was added 2 hours 40 minutes after the electrode structures were included on the bottom surface of the wells (cells had attached to the electrode and substrate surfaces). Shortly after the cell-containing medium is added to the wells (within 10 minutes), there is insufficient time for the cells to adhere to the electrodes. This is confirmed by the fact that the impedance (resistance and reactance) measured by the electrode structure in which the cell-containing medium is added to the well is the same or almost the same as the value obtained when the cell-free medium is added to the well. As described above, in the case where a cell-free medium is applied to the electrode or in the case where a cell-containing medium is applied to the electrode but the cells do not have sufficient time to adhere to the electrode structure, generally speaking, the resistance at high frequencies (e.g., around 1MHz or more) is determined mainly by the shape of the electrode and the conductivity of the solution introduced into the electrode structure. At low frequencies, there is a kind of "electrode polarization" that creates resistance and capacitance that vary with frequency. (see, e.g., Schwan, H.P., "Linear and nonlinear electrode polarization and biological materials", in Ann. biomed. Eng., Vol. 20, p. 269-288, 1992; Jaron, D., Schwan, HP and Geselewitz., "A chemical modification for the polarization estimate of cardiac kinetic maker electrodes", in Med. biol. Eng., Vol. 6, p. 579-594). Under the condition that 2 hours and 40 minutes after the medium containing the cells was put into the well, and the well was placed in a tissue culture chamber for 2 hours and 40 minutes, the cells had enough time to attach and spread (the cell evidence on the area not covered with the electrode could be examined under a microscope). Under such conditions, the reactance spectrum of the electrode structure changes due to the non-conductivity of the cell membrane. Unlike resistance changes, the main relative change in reactance occurs at high frequencies where the total level of reactance increases significantly due to cell attachment to the electrode.

[00185] If the ratio of the resistance measured when the cells are attached to the resistance measured when the cells are not attached (i.e., the relative change in resistance or series resistance) is taken and plotted to show the ratio as a function of frequency, generally, we obtain a peak-like curve (fig. 25 (C)). At low frequencies, there is little or no change in impedance (here, resistance), with a ratio of approximately 1. As the frequency increases, the ratio increases to a peak. The frequency continues to increase and the ratio decreases at high frequencies up to about 1. It is noted here that the relative changes in reactance and capacitance values can also be plotted and used to monitor and reflect the attachment of cells to the electrode surface (see FIG. 25 (D)). Further, the method of expressing impedance with parallel resistance and reactance may also be used to describe the change in impedance due to cell attachment to the electrode surface.

[00186] The peak value of the resistance ratio (ratio of the resistance of cells attached to the electrode to the resistance of no cells attached to the electrode) and the frequency at the peak value depend on factors such as how many cells are attached to the electrode surface, how close this attachment is, the size of the cells, the plasma membrane of the cells and the dielectric properties of intracellular components, among other things. In many cell types we have tested, we have found that for the same type of cell under similar physiological conditions, more cells adhering to the electrode surface will result in a higher peak in the ratio and a higher frequency at this peak.

[00187] FIGS. 26(A), 26(B), and 26(C) show the spectra of the resistance, reactance, and resistance ratios of similar "round on line" electrodes with more cells in the wells containing "round on line" electrode structures in the bottom wells, compared to the results in FIGS. 25(A), 25(B), and 25(C) with fewer cells attached to the electrodes.

[00188] FIG. 28A shows the spectrum of the resistance ratio under different numbers of cells added to wells containing the same "round on line" electrode. For example, seeding about 500 cells causes a maximum of 17% change in series resistance at a frequency of 2kHz, and seeding about 3,200 and 7,000 cells causes 182% and 517% change in series resistance at frequencies of 5 and 30kHz, respectively. Likewise, changes in series reactance may also be used to account for the relationship between cell number and the magnitude of the change in reactance. (see FIG. 28B, for example: a reactance value at 250kHz can be used to demonstrate the relationship between cell number and magnitude of reactance change). Further, if the measured impedance is expressed in terms of parallel resistance and reactance, then the dependence of the number of cells on the magnitude of the change in parallel resistance and/or parallel reactance may also be demonstrated.

Derivation of an index relating to cell number

[00189] Based on the measured impedance, the interdependence between the cell number (more precisely, the number of living cells, or the number of attached cells) and the state of cell attachment, a parameter called "cell number index" (or cell index) can be derived from the measured impedance spectrum. Various methods can be used to calculate this "cell number index". In the following, we illustrate several methods for calculating the cell number index based on the change in resistance or reactance when a cell is attached to an electrode structure relative to when a cell is not attached to an electrode structure. The impedance (resistance and reactance) without cell attachment but with the same cell culture medium on the electrode structure is sometimes referred to as the baseline impedance. The baseline impedance may be obtained by one or more of the following methods: (1) (ii) the impedance measured when a cell-free medium is placed in a well with an electrode structure, wherein the medium is the same medium used to measure impedance when monitoring cell attachment conditions; (2) impedance measured shortly (e.g., 10 minutes) after addition of the cell-containing medium to the well containing the electrode structure at the bottom of the well (cells have not had sufficient time to attach to the electrode surface shortly after addition of the cell-containing medium; the "short" time period depends on the cell type and/or surface treatment or modification of the electrode surface); (3) the impedance of the electrode structure measured after killing all cells in the well by a treatment (e.g., high temperature treatment) or reagent (e.g., detergent) (e.g., in this manner, the treatment and/or reagent should not affect the dielectric properties of the medium on the electrode).

[00190] In one example, the cell number index can be calculated as follows:

(1) at each measurement frequency, the measured resistance value (when the cell is attached to the electrode) is divided by the baseline resistance to give a resistance ratio,

(2) the maximum value of the resistance ratio in the frequency spectrum is sought or determined,

(3) the maximum resistance ratio is reduced by one.

[00191] In this context, a "cell number index" of zero or close to zero means that no cells or only very few cells are present or attached to the electrode surface. A high "cell number index" means that, for the same type of cell, and for cells under the same physiological conditions, there are more cells attached to the electrode surface.

[00192] In another example, the cell number index can be calculated as follows:

(1) at each measurement frequency, the resistance ratio was obtained by dividing the measured resistance value (when the cell was attached to the electrode) by the baseline resistance,

(2) the maximum value of the resistance ratio in the frequency spectrum is sought or determined,

(3) the log of the maximum resistance ratio is calculated (e.g., based on 10 or e-2.718).

[00193] In this context, a "cell number index" of zero or close to zero means that no cells or only very few cells are present or attached to the electrode surface. A high "cell number index" means that, for the same type of cell, and for cells under the same physiological conditions, there are more cells attached to the electrode surface.

[00194] In another example, the cell number index may be calculated as follows:

(1) at each measurement frequency, the reactance ratio was obtained by dividing the measured reactance value (when the cell was attached to the electrode) by the baseline reactance,

(2) the maximum value of the reactance ratio in the frequency spectrum is sought or determined,

(3) the maximum reactance ratio is decreased by one.

[00195] In this context, a "cell number index" of zero or close to zero means that no cells or only very few cells are present or attached to the electrode surface. A high "cell number index" means that, for the same type of cell, and for cells under the same physiological conditions, there are more cells attached to the electrode surface.

[00196] In another example, the cell number index can be calculated by:

(1) at each measurement frequency, the measured resistance value (when the cell is attached to the electrode) is divided by the baseline resistance to give a resistance ratio,

(2) in each measuring frequency, the resistivity ratio value is reduced by one to obtain the relative change value of the resistance,

(3) all relative change values are integrated.

[00197] In this case, the "cell number index" is obtained based on multiple frequency points, rather than at a single peak frequency as in the example above. Similarly, a "cell number index" of zero or close to zero indicates that no cells are present on the electrode surface. A high "cell number index" means that, for cells of the same type and under the same physiological conditions, there are more cells attached to the electrode.

[00198] It is noted that the use of impedance information to monitor the state of cells at the electrodes is not necessary to calculate the "cell number index" described above. In practice, one may choose to use the impedance value directly (e.g., at a single fixed frequency, or at the highest relative change frequency, or at multiple frequencies) as an indication of the cell state.

[00199] However, it is preferred that the "cell number index" be calculated and used to monitor the cell state. The use of a "cell number index" to monitor the state of attachment and/or viability has several advantages:

[00200] First, the performance of different electrode shapes can be compared using a cell number index.

[00201] Second, for a given electrode shape, a "calibration curve" can be established by measuring the impedance values obtained when different numbers of cells are placed on the electrode to show the correlation between cell number and cell number index (in such experiments it is important to ensure that the seeded cells adhere well to the electrode surface). With such a calibration curve, when a new impedance measurement is made, the cell number can be estimated from the newly measured cell number index.

[00202] Third, the cell number index can also be used to compare different surface conditions. Surface treatment with a larger cell number index for the same electrode shape and the same cell number indicates that the electrode surface is more suitable for cell attachment and/or indicates a better surface for cell attachment.

C. Examples of the embodiments

[00203] The following examples are intended to illustrate but not limit the invention.

Example one

Resistive and capacitive electrodes for 8 different types of electrodes with or without cell attachment Resist against

[00204] FIG. 3 is a graphical representation of the impedance and capacitive reactance of 8 different types of electrodes with or without NIH3T3 cell attachment. The diameter of the electrodes 2AA, 2AB, 2AC, 2AD, and 3A is 1 mm; the diameter of the electrodes 2BE, 3B, and 3C was 3 mm. The characteristics of each electrode type were different and are shown in table 2. The electrode surface is coated with chemical and biological molecules. Fibronectin was used in this experiment. After coating, NIH3T3 cells were seeded on the surface of the electrodes. The resistance and reactance (capacitive reactance) were measured 0 hours after inoculation (immediately after inoculation of the cells) and 2 hours after inoculation. (A, B) the resistance and capacitive reactance of 8 different types of electrodes, which are a function of frequency. In all 5 electrode types with NIH3T3 cells attached (2 hours after seeding the cells), an increase in electrical resistance and a decrease in capacitive reactance were seen compared to the corresponding microelectrode without cell attachment (0 hours after seeding the cells). (B) The bar graph summarizes the resistive and capacitive reactance changes at a given frequency as shown. Here, the capacitive reactance value is an absolute value. Resistance and capacitive reactance changes were only visible on electrodes with NIH3T3 cells attached.

Table 2 summary of some electrodes examined.

Name of electrode structure	Substrate material	Type of electrode structure	Size (micron)	Diameter of active area
					2CF	Glass	Interleaving	48/28	6mm
2BE	Glass	Interleaving	48/18	3mm
					2AA	Glass	Interleaving	80/50	1mm
2AB	Glass	Interleaving	80/15	1mm
					2AC	Glass	Interleaving	50/30	1mm
2AD	Glass	Interleaving	50/10	1mm
3C	Glass	On the line "	60/160/180	3mm
					3B	Glass	On the line "	30/80/90	3mm
3A	Glass	On the line "	30/80/90	1mm
						Plastic (Kapton)	Interleaving	50/50

[00205] Electrodes 2AA, 2AB, 2AC, 2AD, 2BE and 2CF are interdigitated electrodes with values for electrode width and gap width of 80/50, 80/15, 80/30, 50/10, 48/18 and 48/28, respectively.

[00206] The electrodes 3A, 3B and 3C are round on rod (or "round on line") electrodes having a rod (or line) width and a rod (or line) gap, and the electrode circle diameters have values of 30/80/90, 30/80/90 and 60/160/180.

Example two

Quantitative cell detection with 3B electrode

[00207] FIG. 4 shows the quantitative detection of cells using 3B electrodes. Serial dilutions of NIH3T3 cells (cell numbers 10,000, 5,000, 2,500, 1,250, and 625) were added to the fibronectin-coated 3B electrode surface. The resistive and capacitive reactances were measured at 0 hours (immediately) and 16 hours after inoculation. The curves show the resistive and capacitive reactance data at the given frequencies shown. Curve T0 is the baseline resistance and capacitive reactance of the electrode without cell attachment. Curves T0-T16 show the change in resistance and capacitive reactance after the cell is attached to the electrode. The 3B electrode can measure less than 600 cells. The dynamic quantification range of the 3B electrode on NIH3T3 cells was between 10,000 and 500.

EXAMPLE III

Real-time monitoring of NIH3T3 and PAE cell proliferation with 3C and 3B

[00208] Figure 5 shows real-time monitoring of NIH3T3 and Porcine Aortic Endothelial (PAE) cell proliferation with 3C and 3B electrodes. 2,500 NIH3T3 cells and 2,500 PAE cells were seeded on the coated electrode. NIH3T3 cells were seeded on fibronectin-coated electrodes and PAE cells on gelatin-coated electrodes. Resistance and capacitive reactance were measured daily to monitor cell proliferation. Day 0 refers to measurements immediately after cell inoculation. The values of the capacitive reactance are shown in this figure as absolute values. In both cell types, the resistance and capacitive reactance values increased with increasing culture time (days), indicating cell proliferation. NIH3T3 cells reached a plateau on day 4 and PAE cells reached a plateau on day 5, suggesting that NIH3T3 cells proliferated faster than PAE cells.

Example four

Comparison of the impedance of 4 different types of cells with 3C electrodes

[00209] FIG. 6 shows a comparison of the impedance of 4 different types of cells using 3C electrodes. The electrical resistance of the 4 cell types was measured with a 3C electrode. These 4 cell types were NIH3T3 cells (mouse fibroblasts), HEP-G2 cells (human hepatocytes), PAE cells (porcine endothelial cells), and HUVEC cells (human endothelial cells). For NIH3T3 and HEP-G2, the electrodes were coated with fibronectin; PAE and HUVEC electrodes were coated with gelatin. Two electrodes were used for each cell type shown. For NIH3T3 and HEP-G2, 10,000 cells were seeded on each electrode; for HUVEC and PAE, 20,000 cells were seeded on each electrode. The resistance and capacitive reactance were measured 0 hours, and 3 or 4 hours after inoculation (only resistance data is shown here). For HEP-G2, the resistance was measured 119 hours after inoculation. Significant increases in the electrical resistance of NIH3T3 cells, HUVEC cells and PAE cells were seen at 3 or 4 hours. In contrast, the resistance of HEP-G2 increased only slightly 4 hours after inoculation, indicating that hepatocytes attached slowly to the electrodes. HEP-G2 stabilized increasing in resistance after overnight incubation (data not shown) and reached a plateau 119 hours after inoculation.

EXAMPLE five

Repeatability of resistance measurements

[00210] Fig. 7 shows the repeatability of the resistance measurement. Reproducibility was tested on 7 sets of electrodes (3B) seeded with HUVEC cells. Electrodes were gelatin coated and 15,000 HUVEC cells were seeded on each group of electrodes. The resistance of each set of electrodes was measured immediately after inoculation (t0), at 20 hours and 30 minutes. The resistance value was clearly increased after 20 hours of culture, indicating that the cells were attached to the electrodes. the average resistance value at t0 was 47.4, and the standard deviation was 3.9; at 20 hours and 30 minutes, the average resistance value was 284.8 and the standard deviation was 17.2. the coefficient of variation at t0 was 8.3%, and the coefficient of variation at 20 hours and 30 minutes was 6.1%.

EXAMPLE six

Cell migration device with integrated electrode structure on bottom of lower chamber

Invasion and migration of defined non-invasive NIH3T3 cells and invasive HT1080 cells Comparison of sex

[00211]FIG. 22 is a comparison of the invasion and migration activities of non-invasive NIH3T3 cells and invasive HT1080 cells in our electronic cell migration device, as shown in FIG. 1, in which the electrode structure is integrated into the bottom surface of the lower chamber. In the experiment, cellsThe transfer device takes the form of a conventional transwell transfer device, but has an electrode sensing chip (i.e., a glass substrate fitted with an electrode structure) mounted on the bottom surface of each lower chamber. After the cells migrate through the microporous membrane, they fall onto and attach to the electrode structure, and the cell-substrate impedance is measured and quantified by an impedance analyzer. Two cell lines, NIH3T3 and HT1080, were used to test cell migration devices. The NIH3T3 cell line is a non-invasive cell line, whereas the HT1080 cell line is a proven invasive cell line and has strong invasion and migration activity in both in vitro and in vivo migration experiments. Both NIH3T3 cells (purchased from ATCC) and HT1080 cells (purchased from ATCC) were cultured in DMEM medium containing 10% FBS. In the migration experiment, both types of cells were digested with pancreatin and counted. Then a cell suspension is prepared. Because the electrode sensor chip (i.e., the glass substrate on which the electrode structures are mounted) is integrated or included on a 24-well transwell migration device, standard transwell migration detection methods can also be implemented on our electron cell migration device. Briefly, the electronic sensors located on the lower chamber and microporous membrane of the insert were first coated with extracellular matrix proteins. In the experiment, the electrode sensor and the microporous membrane were coated with 50. mu.g/ml of fibronectin for one hour at room temperature. After washing with phosphate buffered saline, cell culture medium (10% FBS DMEM) was added to the lower chamber, where the pre-coated electrode sensors were installed. An insert is then placed into the lower chamber. 2X 10 of each insert⁵Individual cell NIH3T3 or HT1080 cells. The electrode structure is connected with an impedance analyzer through a connecting pad on the electronic sensing chip. Invasion and migration activity can be monitored in real time without disturbing the experiment. In the figure, the Y-axis shows the cell migration index, which is calculated based on the measured electrode structure resistance at different frequencies at different times during the course of the experiment and at the beginning of the experiment (baseline electrical set of experiment, no cell attachment). The method of calculating the cell migration index is similar to the method of calculating the "cell number index", which is based on the maximum ratio of the measured resistance at a given time to the baseline resistance. Cell migration index and number of cells reaching the basal compartment and cell invasion and migration activity dense phaseAnd off. The X-axis is the time interval of each measurement. As shown, the invasion and migration activities of HT1080 cells steadily increased over time. In contrast, non-invasive NIH3T3 cells showed undetectable invasive activity throughout the experiment. This result is consistent with previously reported cell lines using conventional cross-well methods. Moreover, the cell migration apparatus of the present invention for use herein can provide real-time, continuous, label-free measurement of cancer cell invasion and migration activity.

EXAMPLE seven

Cell migration device with electrode structure on microporous membrane of insert

Invasion and migration of defined non-invasive NIH3T3 cells and invasive HT1080 cells Comparison of sex

[00212]Fig. 23(a) shows a comparison of the invasion and migration activities of non-invasive NIH3T3 cells and invasive HT1080 cells on a cell migration device (fig. 40(B)) with an electrode structure placed on a microporous membrane of an insert (similar to the structure shown in fig. 17). In this cell migration device, one electrode structural unit is attached to the bottom surface of the insert (fig. 23 (B)). Thus, when cells invade through the extracellular matrix layer and migrate through the microporous membrane, the invaded cells attach directly to the electrode structure, and where the cell-electrode interaction can be detected, the impedance between the electrode structures can be measured and quantified. Two cell lines, NIH3T3 and HT1080, were used to test cell migration devices. The NIH3T3 cell line is a non-invasive cell line, and the HT1080 cell line is an invasive cell line, and the cell line has strong invasion and migration activities in an in vitro migration experiment and an in vivo migration experiment. Both NIH3T3 cells (purchased from ATCC) and HT1080 cells (purchased from ATCC) were cultured in DMEM containing 10% FBS. In the migration experiment, both cells were trypsinized and counted. Then a cell suspension is prepared. Since microporous membranes with assembled electrode structures can be bonded to insertion wells suitable for commercial 24-well migration devices, standard transwell migration detection methods can be implemented on our cell migration device.Briefly, the microporous membrane of the insert and the built-in electrode sensor array are first coated with extracellular matrix proteins. In this experiment, both the membrane electrode sensor and the microporous membrane were coated with 50 μ g/ml fibronectin for one hour at room temperature. After washing with phosphate buffered saline, cell culture medium (10% FBS DMEM) was added to the lower chamber, where the pre-coated electrode sensors were installed. An insert is then placed into the lower chamber. 2X 10 of each insert⁵Individual cell NIH3T3 or HT1080 cells. As shown in fig. 23(B), the electric wire was bonded to the connection pad on the film by a conductive adhesive, and then connected to the impedance analyzer. The bonding areas of the wires and the connection pads are covered with a biocompatible, silicone-based adhesive. Invasion and migration activity can be monitored in real time without disturbing the experiment. To verify the measurement results of the sensor, cells attached to the electrode sensor on the microporous membrane after membrane migration were fixed with 100% methanol for 5 minutes and then stained with giemsa stain (Sigma Diagnostics) for 30 minutes after the end of the experiment. (A) Invasion and migration activities of NIH3T3 and HT1080 cells were measured. The Y-axis shows the cell migration index, which is calculated based on the measured resistance of the electrode structure at different frequencies at different times during the experiment and at the beginning of the experiment (baseline resistance of the experiment, no cell attachment). The method of calculating the cell migration index is similar to the method of calculating the "cell number index", which is based on the maximum ratio of the measured resistance at a given time to the baseline resistance. The cell migration index is closely related to the number of cells reaching the basal compartment and the cell invasion and migration activity. The X-axis is the time interval of each measurement. As the figure shows, the migratory activity of HT1080 cells increases with time. The migratory activity of non-invasive NIH3T3 cells was weaker than that of HT 1080. (B) Giemsa staining stains migrating cells on the electronic sensor array on the other side of the microporous membrane. As shown, the color density of HT1080 was higher than that of NIH3T3 cells, which is consistent with the results obtained from measurements of the impedance between electrode structures on a microporous membrane. Unlike the device described in FIG. 22, in which the electrode structure measures cells that migrate and fall to the bottom surface of the lower chamber, the device of this example can detect and measure cells once they have passed through the micro chamberCell migration upon pore membrane migration. Thus, detection of cell migration using such a cell migration device with electrode structures mounted on a membrane can be more straightforward and rapid than the device described in FIG. 22.

Example eight

Cell migration device on microporous membrane of insert with electrode structure

Real-time monitoring of inhibition effect of doxycycline on cancer cell invasion and migration

[00213]Fig. 24 is a graph showing real-time monitoring of inhibition effect of doxycycline on cancer cell invasion and migration using a cell migration device with an electrode structure on a microporous membrane of an insert (similar to the structure shown in fig. 17). (A) An inhibition of HT1080 cell invasion and migration by doxycycline, which is time and dose dependent. (B) The dynamic inhibition of HT1080 cell invasion and migration by doxycycline was monitored in real time using fully automated equipment with software-controlled data acquisition of measured impedance. As shown in fig. 24(B), the migration process was continuously monitored every 15 minutes. Cell migration devices with electrodes mounted on microporous membranes with insertion holes (as depicted in figure 23) were used for dynamic monitoring of drug inhibition of cancer cell invasion and migration. The dose-and time-dependent inhibition of HT1080 cell invasion and migration by doxycycline (Sigma) was measured on a cell migration device. Briefly, microporous membranes (pore diameter 10 μm) and electrode structures were coated with fibronectin (50 μ g/ml) at room temperature for 1 hour and washed with phosphate buffered saline. HT1080 cells were trypsinized and made into 10⁶Cells/ml suspension. 1ml of medium containing doxycycline at various concentrations was added to the lower chamber. Solvent DMSO of doxycycline was used as a vehicle control. To each insert, a 200. mu. lHT1080 cell suspension (2X 10) containing doxycycline or vector was added⁵Individual cells). An impedance analyzer is used to automatically and electronically switch to the electrode configuration of the different insertion holes. After the electrode structure is connected with the cell, the invasion and migration activities of doxycycline and doxycycline-free electrode structure can be achieved without beatingThe conditions of the experiments were monitored at different time intervals. The migration index (or cell migration index) was calculated in the same manner as shown in FIGS. 22 and 23.

Example nine

Device for monitoring migration in real time

1. Electronic cell chip design

[00214] FIG. 8 shows a design of an electronic cell chip. This figure shows 5 representative electronic cell chip designs. Gold electrodes of different shapes and sizes are located in the middle of the glass substrate. The size of the glass substrate is 1cm × 1 cm. The gold electrode is connected to the electrical detection interface via a connection electrode pad at the edge of the glass substrate.

2. Mechanism for detecting cells on electronic cell chip

[00215]The electrodes can be represented by a series resistance-capacitance Circuit (RC Circuit) (Warburg, E., Ann. Phys.1901, 6, 125-135.) the resistance and capacitance of the conductor electrolyte can be varied from f- κ, where 0 < κ < 1 and f is the frequency. For bare electrodes in the cell-free case (FIG. 9), R_solSimply equal to the asymptotic value of the measured resistance at high frequencies (Z-Z0). The electrical set or impedance is nearly identical for a cell-free electrode coated with chemicals or proteins (Luong, J.H.T., M.Habi-Rezaei, J.Meghrous, C.Xiao, and A.Ka men.2001.monitoring mobility, scanning and reporting of adhesive cells using an impedance sensor. anal.chem.73, 1844-1848.). In contrast, the impedance of the electrode increases significantly when the cells attach to the electrode and diffuse (Z ═ Zcell). The impedance increase is related to the cell-electrode adhesion and covered area on the electrode. Thus, for a given cell type (adherent cells), a change in the number of cells can be represented by a corresponding change in the impedance value.

3. Detection of cells attached to electrodes by measuring impedance

[00216] As a basis for comparative studies, a test apparatus was fabricated. The device comprises an electronic cell chip (FIG. 8), an electronic interface fixed on a printed circuit board, and a culture well mounted on the chip. Therefore, the basic test device only includes one detection cell.

[00217] Two sets of differently shaped chip designs were first tested. Design 1 contained one electrode with a diameter of 1mm and design 2 contained one electrode with a diameter of 3 mm. The detection areas of the electrodes are different in these two designs. NIH3T3 cells (purchased from ATCC) were used for detection. For each test, two devices were used, one to place the medium and the other to place the cells. The electrodes of the test device were coated with fibronectin (50. mu.g/ml) for 1 hour at room temperature. After coating, 100. mu.l of NIH3T3 cell suspension (10,000 cells) or 100. mu.l of culture medium was added to the test device. The resistance and capacitive reactance at different frequencies were measured by a 1,260 impedance analyzer (Solartron inc. uk) immediately after inoculation (t0) and 2 hours after inoculation (t 2). At t0, the resistance and capacitive reactance of the device with cells and the device without cells are similar. At t2, the resistance value of the device with cells at different frequencies was seen to rise significantly, while the resistance of the device without cells was unchanged, indicating the attachment and spreading of cells on the electrodes. Both designs showed similar results (data not shown).

4. Measuring different kinds of cells with a test device

[00218] The device was further tested with 4 different cell types, including two primary human cell types, HUVEC (purchased from ATCC) and primary human hepatocytes (purchased from ScienCell inc. san Diego, California) and two cell lines, NIH3T3 cell line and PAE cell line (purchased from ATCC). All cell types showed a significant increase in resistance values after 3 or 4 hours of seeding as shown in FIG. 6.

5. Cell proliferation

[00219] Cell proliferation was detected on the test device using PAE cells. In this study, PAE cells were seeded at different densities (8,000 and 1,000 cells) to continuously monitor the rate of cell proliferation at different cell seeding densities. The resistance was then measured at different time intervals without disturbing the culture. As shown in FIG. 10, in both devices, the resistance value steadily increased with the increase in the culture time, indicating the increase in the number of cells during the culture. Notably, the resistance values of the devices with high cell seeding density increased faster than those of the devices with low cell seeding density, as confirmed by observation under an optical microscope (data not shown). These results indicate that the device can continuously monitor cell proliferation in real time without the need to label such measurements.

6. Quantification and sensitivity in measuring cells on a test device

[00220]The quantification capacity and sensitivity of the device in performing cell assays was compared experimentally to the MTT assay. MTT assay is a method commonly used for cell quantification⁴⁸. Serial dilutions of NIH3T3 cells were added to the test device or 96-well microtiter plate (fig. 11). At 37 ℃ 5% CO₂Measurements were made by measuring electrical impedance after overnight incubation under conditions, or by MTT staining. As shown in fig. 28, the data measured by the two methods are very close. The resistance measured with the device can be used for linear quantification. In fact, the sensitivity level of the present device is comparable to the MTT assay (less than 600 cells).

7. Repeatability of testing device detection

[00221] The reproducibility of the resistance measurements of the test device was experimentally determined by three different cell types. Here, representative results showing the performance of the device were obtained with primary human hepatocytes. In the experiment, 6 devices were used, and the resistance was measured immediately after cell inoculation (t0) and 4 hours (t 4). And calculate CVs at t0 and t4 (fig. 7). CV _ t0 represents the degradation of the device, while CV _ t4 represents the degradation of the device plus cell attachment and diffusion. As shown in FIG. 7, the CV at t0 and t4 for the resistance measurements of the test device was small and the repeatability was good. With the modified arrangement described below, then the CV would be further reduced.

D. Method of producing a composite material

[00222] In the migration assay, plastic wells on a porous substrate are pre-filled with a buffer solution containing a chemoattractant. Transmembrane wells with plastic plates (purchased from BDBiosciences) were then inserted into plastic wells in the assay device. After addition of cells to the transmembrane pore, the impedance of the electrode was measured in the frequency range of 1kHz to 1 MHz. In order to minimize or eliminate the influence of the electric field on the cells, 5 or 10mV is applied to the electrodes. The detailed protocol is described below.

Protocol for migration analysis on an electronic device

1. Coating the electrodes:

[00223] The electrodes are coated with cell adhesion molecules. Fibronectin coating conditions 50. mu.l of 50. mu.g/ml fibronectin at room temperature was added to each unit for one hour, followed by washing 3 times with PBS. Gelatin coating conditions were 50. mu.l of 1% gelatin added to each unit at 37 ℃ for 30 minutes, 50. mu.l of 0.5% glutaraldehyde added at room temperature for 25 minutes to immobilize the coated gelatin, which was then washed with PBS, and 50. mu.l of 0.1M glycine added at room temperature for 30 minutes to block free aldehyde groups. Washed again with PBS 3 times. Optionally, a pre-coated device is provided.

ECM coating

[00224] Mu.l of ECM solution was added to the inserts and incubated overnight at room temperature. ECM materials useful in the assay include BD magtlarge matrix (BDBiosciences), fibronectin (Sigma), collagen (Sigma or BD Biosciences), and laminin (BD Biosciences), depending on the assay requirements. The inserts were washed with PBS. A pre-coated insert may also be provided.

3. Seeding cells

[00225]100 μ l cell suspension (10)⁵Individual cells) were added to the pre-coated insert while 100 μ l of conditioned medium containing the chemoattractant was added to the electrode cell chip chamber. After placing the insert in the chamber, the impedance is measured at t 0. The device is at 37 ℃ and 5% CO₂Incubation under 100% humidity chamber conditions. By measuring the impedance at different time intervals, migration can be monitored in real time.

4. Data acquisition

[00226] Data storage and analysis was performed using a software program (125505S Z plot) bundled onto a purchased 1260 impedance analyzer (Solartron Analytical).

E. Reference to the literature

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[00280] The above examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Many variations from the above-described embodiments are possible. Since modifications and variations of the above embodiments will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the following claims.

Claims (44)

1. An apparatus for measuring the electrical impedance, resistance, or capacitance of a cell-substrate interface comprising two or more electrodes mounted on one side of a biocompatible membrane having at least one aperture, wherein the apparatus has a surface adapted for cell attachment or growth.

2. The device recited in claim 1 wherein said biocompatible membrane comprises glass, sapphire, silicon dioxide on silicon, one or more plastics, or one or more polymers, and has a thickness of between 2 microns and 500 microns.

3. The device recited in claim 2, wherein said biocompatible membrane has a coating material for attaching one or more cells thereto, wherein said coating material optionally comprises an extracellular matrix component.

4. The device recited in claim 1, wherein at least two of said two or more electrodes have substantially the same surface area.

5. The device recited in claim 4, wherein said at least two electrodes having substantially the same surface area have an alternating or concentric configuration.

6. The device as recited in claim 5, wherein the at least two electrodes are shaped to: circular on the line, diamond on the line, tooth-shaped, or sinusoidal.

7. The device as recited in claim 5, wherein the electrodes thereon have a width of 20 microns to 250 microns; wherein the electrode member gap width is 3 to 80 microns; and wherein the ratio of the electrode section width to the gap width ranges between 1: 3 and 20: 1, wherein the electrode section gap is 0.2 to 3 times the cell width for detecting electrical impedance, resistance, or capacitance at the cell-substrate interface.

8. The device of claim 4, disposed in a fluid container.

9. The device recited in claim 8, further comprising an impedance analyzer connected to at least two of the electrodes.

10. The device recited in claim 9, wherein the surface area of the exposed face of the biocompatible membrane fitted with the electrodes comprises a substantially uniform distribution of electrode components, wherein the electrodes are distributed over at least 50% of the surface area of the exposed face of the biocompatible membrane fitted with two or more electrodes.

11. The device recited in claim 1, wherein said two or more electrodes comprise at least four electrodes, wherein said at least four electrodes are arranged in an electrode structure array of two or more interleaved electrode structure elements (IDES) or concentric electrode structure elements (ccs), each element comprising at least two electrodes.

12. A device comprising the device of claim 11, wherein said biocompatible membrane is reversibly or irreversibly attached to a structure providing a plurality of spaced apart fluid reservoirs, such that at least one fluid reservoir carries a single IDES or CCES structural element, wherein, for each spaced apart fluid reservoir carrying a single IDES or CCES, the surface area of the exposed side of the biocompatible membrane to which said electrodes are attached comprises a substantially uniform distribution of electrodes or electrode components.

13. A system for analyzing the electrical impedance, resistance, or capacitance of a cell-substrate interface comprising the apparatus of claim 12, further comprising an impedance analyzer connected to at least four electrodes.

14. The device as recited in claim 8, wherein the device separates an upper chamber from a lower chamber of the fluid container.

15. The device recited in claim 14, wherein at least two electrodes are mounted on the upper side of the membrane.

16. The device recited in claim 15 wherein said membrane has a layer of cells on the upper side, wherein said cells are Caco-2 cells.

17. The device recited in claim 16 wherein said membrane is provided with a layer of cells on the upper side, said cells being either epithelial or endothelial cells.

18. The device of claim 17, wherein said device is for detecting migration or invasion of one or more cells through said epithelial or endothelial cell layer, and wherein said lower compartment, or said upper compartment, or both said lower and upper compartments comprise at least one compound known or suspected to modulate said migration or invasion of said one or more cells.

19. The device recited in claim 14, wherein at least two electrodes are mounted on the underside of said membrane, wherein said at least one aperture has a diameter of between 1 micron and 25 microns.

20. A device as claimed in claim 19 wherein the membrane comprises at least one substance to facilitate cell attachment on the underside of the membrane.

21. The device recited in claim 19 wherein said membrane comprises on its upper side at least one biomolecule coating, at least one extracellular matrix component, an epithelial cell layer or endothelial cell layer, or a combination thereof.

22. The device as claimed in claim 21, wherein the device is for detecting migration or invasion of one or more cells and the lower compartment or the upper compartment or both the lower and upper compartments thereof comprises at least one compound known or suspected to modulate cell migration or invasion.

23. An apparatus for measuring the electrical impedance, resistance, or capacitance of a cell-substrate interface comprising a plate having two or more wells, wherein at least two of the wells comprise the apparatus of claim 1, wherein each apparatus separates each well into an upper chamber and a lower chamber.

24. A device as claimed in claim 23, wherein two or more electrodes are located on the underside of the membrane.

25. The device recited in claim 23 wherein two or more electrodes are located on the upper side of the membrane.

26. A device for measuring electrical impedance, resistance, or capacitance of a cell-substrate interface comprising the device of claim 12, wherein the biocompatible membrane is reversibly or irreversibly attached to a first plate having two or more wells providing a cell transfer unit lower chamber; and reversibly or irreversibly attached to a second plate that provides a tubular structure that provides the upper chamber of the cell migration unit, such that each cell migration unit contains a single IDES or CCES.

27. The device recited in claim 26 wherein said array of electrode structures is located on the underside of a biocompatible membrane.

28. The device recited in claim 26 wherein said array of electrode structures is located on the upper side of the biocompatible membrane.

29. An apparatus for analyzing electrical impedance, resistance, or capacitance of a cell-substrate interface, comprising:

a) a plate with two or more holes; and

b) an insert tray adapted to fit said plate, wherein the insert tray has one or more insert chambers, each insert chamber having:

i) a fluid impermeable wall; and

ii) the device of claim 1, forming a bottom surface of each of the one or more insert chambers;

and wherein each insert chamber may fit into one well of said plate, such that the wells of the plate constitute the lower chamber and the inserts constitute the upper chamber of the cell migration/invasion unit.

30. The device recited in claim 29, wherein the at least two electrodes are mounted on an upper side of the biocompatible membrane.

31. The device recited in claim 29, wherein said at least two electrodes are mounted on the underside of said biocompatible membrane, wherein said at least one aperture has a diameter of between 1 micron and 25 microns.

32. A method of monitoring cell migration or invasion, comprising:

a) providing a device as claimed in claim 13;

b) placing a cell in an upper chamber of the device; and

c) the change in impedance between the electrodes is monitored to monitor cell migration or invasion.

33. The method of claim 32, further comprising adding a known or suspected modulator of cell migration or cell invasion to the lower chamber of the device or adding a known or suspected modulator of cell migration or cell invasion to the upper chamber of the device.

34. A method of monitoring cell migration or invasion, comprising:

a) providing a device as claimed in claim 26;

b) placing a cell in an upper chamber of the device; and

c) the change in impedance between the electrodes is monitored to monitor cell migration or invasion.

35. The method of claim 34, further comprising adding a known or suspected modulator of cell migration or cell invasion to the lower chamber of the device or adding a known or suspected modulator of cell migration or cell invasion to the upper chamber of the device.

36. A method of monitoring cell migration or invasion, comprising:

a) providing an apparatus as claimed in claim 29;

b) placing a cell in an upper chamber of the device; and

c) the change in impedance between the electrodes is monitored to monitor cell migration or invasion.

37. The method of claim 36, further comprising adding a known or suspected modulator of cell migration or cell invasion to the lower chamber of the device or adding a known or suspected modulator of cell migration or cell invasion to the upper chamber of the device.

38. The device as recited in claim 8, wherein the device separates a lower chamber from an upper chamber of the fluid container, and wherein at least two electrodes are mounted on an upper side of the membrane, and wherein the pores of the biocompatible membrane have a diameter of less than 3 microns.

39. The apparatus recited in claim 38, further comprising an impedance analyzer.

40. A method of analyzing the integrity of a cell monolayer, comprising: culturing cells in the upper chamber of the device of claim 39; monitoring the integrity of the cell monolayer in the at least one chamber by monitoring the impedance between the electrodes mounted on the membrane using an impedance analyzer.

41. A system for measuring cell-substrate interface electrical impedance, resistance, or capacitance, comprising:

a) the device of claim 26;

b) an impedance analyzer;

c) an electronic interface comprising electronic switches to control and switch said impedance analyzer, connected to the different electrode structure units on the device.

42. The system set forth in claim 41, further comprising a software program capable of measuring and monitoring in real time values of inter-electrode or inter-electrode structure impedance on said device.

43. The system as recited in claim 42, wherein said software program includes at least one of the following functions:

a) controlling an electronic switch to connect the impedance analyzer to one of a plurality of electrode structure units on the device;

b) controlling an impedance analyzer to measure impedance between the electrode structures at one or more frequencies;

c) processing the obtained impedance data to obtain appropriate biologically relevant parameters (e.g., cell number index);

d) displaying the results on a monitor, or storing the results; and

e) the functions (a) to (d) above are automatically performed at regular or irregular intervals.

44. The device recited in claim 10, wherein the electrodes are distributed over at least 90% of the surface area of the exposed face of the biocompatible membrane that is equipped with two or more electrodes.