WO2013012498A1

WO2013012498A1 - Electric cell- substrate impedance sensing (ecis) of biological samples in shear stress flow

Info

Publication number: WO2013012498A1
Application number: PCT/US2012/042505
Authority: WO
Inventors: Brett Blackman; Nicole HASTINGS; Michael SIMMERS
Original assignee: Hemoshear, Llc
Priority date: 2011-06-14
Filing date: 2012-06-14
Publication date: 2013-01-24
Also published as: WO2013012498A9

Abstract

A system for characterizing the response of cells (201) to a condition, such as exposure to a pharmaceutical or other compound, includes a container (141) and an electrode array (101). The system is particularly suitable for performing electric cell-substrate impedance sensing (ECIS).The electrode array has a substrate (109) supporting a plurality of contact pads (105), a plurality of electrodes (103), and a plurality of electrical conductors (107) connecting the electrodes to corresponding ones of the contact pads. One or more electrically insulating materials (109, 115) covers the electrical conductors and has one or more openings (121) on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrodes are positioned inside the container (141) and the contact pads are positioned outside the container. Cells (201) in the container can be exposed to hemodynamic shear forces by rotating a body (181) extending into a culture medium (191) in the container. Electrical impedance measurements between the electrodes can be used to assess the response of cells after exposure to the condition. A porous membrane (193) can be added to the container for allowing co-culture of cells.

Description

ELECTRIC CELL- SUBSTRATE IMPEDANCE SENSING (ECIS) OF

BIOLOGICAL SAMPLES IN SHEAR STRESS FLOW

FIELD OF INVENTION

[0001] The present invention relates generally to systems and methods for characterizing the response of biological samples (e.g., cells) to various conditions, and more particularly to systems and methods for characterizing the response of biological samples to a condition while the sample is in an environment including hemodynamic shear forces.

BACKGROUND

[0002] It can be difficult to study how an organism will respond to pharmaceutical compounds and/or materials that may be used inside the body (e.g., materials used in stents or other medical devices) . Research aimed at identifying new pharmaceutical compounds and/or new or improved materials for medical devices can include experiments in which biological samples (e.g., in the form of cultured cells) are studied after they are exposed to a particular set of conditions. The condition (s) being tested can vary. For example, cultured cells can be exposed to a new drug candidate and their response to the drug candidate can be studied to help evaluate the drug candidate. Other non-limiting examples of conditions that can be tested include the presence of a pharmaceutical, the concentration of a pharmaceutical, combinations of two or more pharmaceuticals, the absence of a pharmaceutical or other substance, the presence and/or concentration or absence of proteins or other factors that may affect cell response, the presence and/or amount or absence of a particular cell type (e.g., immune cells), or the hemodynamic shear forces the sample is subjected to, and combinations thereof.

[0003] One of the difficulties is that once cells are taken out of the organism for testing in the laboratory, they often do not behave or respond in the same manner as when in vivo. This has been found to be true, for example, of all human cells that have been removed from their organ source. Thus, results from studies performed on isolated cultured cells can be misleading. In particular, it is difficult to understand from cell culture studies how drug candidates or other conditions being evaluated will affect living organisms because of the potential for differences in the response or behavior of the cultured cells compared to the cells in the living organism.

[0004] There have been some attempts to mimic in vivo conditions for cultured cells to cause the cultured cells to behave more like the in vivo cells. Many microfluidic systems flow or perfuse medium across a cell culture on a plate or chip. Some even "pulse" the flow in an effort to mimic arterial blood flow. This is an improvement over static cell cultures, but still has shortcomings when trying to recreate a response that accurately reflects the response in a living animal or human. In a human or animal, blood in most of the circulation does not flow evenly in one direction with constant force, nor does it pulse in a consistent manner. Blood moves and pulses with varying force, and may even "pool" or circle in some parts of the vascular system. The in vivo shear forces resulting from this type of blood flow are of fundamental importance for recreating and/or maintaining accurate region-specific cell phenotypes in cultured cell studies.

[0005] United States Patent No. 7,811,782, issued October 12, 2010 (Blackman, et al . ) , which is commonly owned with the present application and hereby incorporated by reference, discloses an in vitro biomechanical model used to apply hemodynamic (i.e., blood flow) patterns modeled after human or other animal circulation to human or animal cells in culture. The model can replicate hemodynamic flow patterns that are measured directly from the circulation using non-invasive magnetic resonance imaging, ultrasound, or other methods and translated to a motor that controls rotation of a cone that is submerged in fluid (i.e., cell culture media) and brought into close proximity to the surface of the cultured cells. The controlled time-varying rotation of the cone transduces momentum on the fluid and creates time-varying shear stresses on the cells. This model closely mimics the physiological hemodynamic forces imparted on endothelial cells (cells lining the blood vessels) in vivo and overcomes previous flow devices limited in applying more simplified nonphysiological flow patterns.

[0006] The present inventors have made certain improvements to the teachings of the ^λ782 patent, which will be described in detail below.

SUMMARY

[0007] One aspect of the invention is a system for characterizing the response of cells to a condition. The system includes a container for holding cultured cells and an electric cell-substrate impedance sensing electrode array. The electric cell-substrate impedance sensing electrode includes a substrate that supports a plurality of contact pads, electrodes and electrical conductors. The electrical conductors connect the electrodes to corresponding ones of the contact pads. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials have one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electric cell-substrate impedance sensing electrode array extends through a sidewall of the container so the electrodes are positioned inside the container and the contact pads are positioned outside the container.

[0008] Another aspect of the invention is a system for characterizing the response of cells to a condition. The system includes a container for holding cultured cells and an electric cell-substrate impedance sensing electrode array. The electric cell-substrate impedance sensing electrode array includes a substrate and a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electric cell-substrate impedance sensing electrode array is positioned so the electrodes are positioned inside the container and the contact pads are positioned outside the container. The system for characterizing the response of cells to a condition includes a system for exposing cells in the container to fluid shear forces. The system for exposing the cells to fluid shear forces includes a body adapted for being positioned in the container and a motor adapted to rotate the body .

[0009] One aspect of the invention is a method of characterizing the response of cells to a condition. The method includes placing an electric cell-substrate impedance sensing electrode array in a container. The electric cell-substrate impedance sensing electrode array includes a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electric cell-substrate impedance sensing electrode array is positioned so it extends through a sidewall of the container so the electrodes are positioned inside the container and the contact pads are positioned outside the container. Cells are cultured in the container. The cells are subjected to fluid shear forces while they are in the container. An electric cell-substrate impedance sensing instrument is connected to the contact pads of the electrode array to measure electrical impedance between pairs of electrodes of the electrode array after the cells have been subjected to the condition .

[0010] Another aspect of the invention is a method of characterizing the response of cells to a condition. The method includes placing an electric cell-substrate impedance sensing electrode array in a container. The electrode array includes a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electric cell-substrate impedance sensing electrode array is positioned so the electrodes are positioned inside the container and the contact pads are positioned outside the container. Cells are cultured in the container. A body is rotated in the container and the cells are subjected to fluid shear forces produced by the rotating body while the cells are in the container. An electric cell-substrate impedance sensing instrument connected to the contact pads of the ECIS electrode array is used to measure electrical impedance between pairs of electrodes of the electrode array after the cells have been subjected to the condition.

[0011] Yet another aspect of the invention is a system for characterizing the response of cells to a condition. The system includes a plurality of electric cell-substrate impedance sensing sample testing stations. Each sample testing station includes a container for holding cultured cells and an electric cell-substrate impedance sensing electrode array including a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes, and electrical conductors are supported by the substrate. One or more electrically insulating materials coves the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrode array is positioned to extend through a sidewall of the container so the electrodes are positioned inside the container and the contact pads are positioned outside the container. An electric cell- substrate impedance sensing analyzer is electrically connected to the contact pads of the electrode array at each of the sample testing stations. The electric cell-substrate impedance sensing analyzer is adapted to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station.

[0012] Still another aspect of the invention is a system for characterizing the response of cells to a condition. The system includes a plurality of electric cell-substrate impedance sensing sample testing stations. Each sample testing station includes a container for holding cultured cells and an electric cell-substrate impedance sensing electrode array. The electric cell-substrate impedance sensing electrode arrays include a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrode arrays are positioned so the electrodes are positioned inside the respective container and the contact pads are positioned outside the respective container. The system includes a body and a motor adapted to rotate the body while the body extends into the container to subject cells in the container to fluid shear forces. The system includes an electric cell-substrate impedance sensing analyzer electrically connected to the contact pads of the electrode array at each of the sample testing stations. The electric cell- substrate impedance sensing analyzer is adapted to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station after the cells have been exposed to the condition.

[0013] Another aspect of the invention is a method of characterizing the response of cells to a condition. The method includes culturing cells at a plurality of electric cell- substrate impedance sensing sample testing stations. Each ECIS sample testing station includes a container for culturing cells and an electric cell-substrate impedance sensing electrode array including a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials have one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrode array extends through a sidewall of the respective container so the electrodes are positioned inside the container and the contact pads are positioned outside the container. The method includes using an electric cell-substrate impedance sensing analyzer electrically connected to the contact pads of the electrode array at each of the sample testing stations to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station after the cells have been exposed to the condition.

[0014] Yet another aspect of the invention is a method of characterizing the response of cells to a condition. The method includes culturing cells at a plurality of electric cell- substrate impedance sensing sample testing stations. Each sample testing station includes a container for culturing cells and an electric cell-substrate impedance sensing electrode array including a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrode array is positioned so the electrodes are positioned inside the respective container and the contact pads are positioned outside the respective container. The method includes rotating a body in the container to subject the cells in the container to fluid shear forces. An electric cell-substrate impedance sensing analyzer electrically connected to the contact pads of the ECIS electrode array at each of the sample testing stations is used to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station after the cells have been exposed to the condition.

[0015] Another aspect of the invention is a system for characterizing the response of cells to a condition. The system includes a container for holding culture media and optionally cultured cells. A porous membrane is positioned in the container to separate the container into an upper volume and a lower volume. The porous membrane is suitable for plating cells thereon. The system includes an electric cell-substrate impedance sensing electrode array having a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes, and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrodes are positioned inside the container in the lower volume and the contact pads are positioned outside the container.

[0016] Another aspect of the invention is a method for characterizing the response of cells to a condition. The method includes adding a culture media to a container having a porous membrane is suspended in the container such that a first side of the membrane is proximal and in spaced relation to a surface of the container, the porous membrane being adapted to permit fluid communication of the culture media. A first cell type is plated on the first side of a porous membrane or a second cell type is plated on a second side of the porous membrane, thereby defining within the container a lower volume comprising the first cell type or an upper volume comprising the second cell type. A third cell type is optionally plated on the surface of the container. Culture media is perfused into and out of the upper volume. Culture media is perfused into and out of the lower volume. A fourth cell type is optionally suspended in the culture media in the upper volume. A fifth cell type is optionally suspended in the culture media in the lower volume. All of the cell types are within the culture media. One or more of the cell types that are present is exposed to the condition. An electric cell-substrate impedance sensing electrode array is used to measure electrical impedance associated with the cells. The electric cell-substrate impedance sensing electrode array includes a substrate, a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads. The contact pads, electrodes, and electrical conductors are supported by the substrate. One or more electrically insulating materials covers the electrical conductors. The one or more electrically insulating materials has one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings. The electrodes are positioned inside the container. The electrical impedance is measured between electrode pairs of the electrodes of the electric cell-substrate impedance sensing electrode array to determine the response of one or more of the cell types to the condition.

[0017] Other objects and features of the invention will be in part apparent and in part pointed out hereinafter.

BRIEF DESCRPTION OF THE DRAWINGS

[0018] FIG. 1 is a perspective of one embodiment of a an Electric Cell-substrate Impedance Sensing (ECIS®) electrode array according to the present invention;

[0019] FIG. 2 is a top plan view of the electrode array illustrated in Fig. 1 ;

[0020] FIG. 3 is an exploded perspective of the electrode array illustrated in Fig. 1 in combination with a container having an opening for receiving the electrode array;

[0021] FIG. 4 is perspective showing the electrode array illustrated in Fig. 3 being inserted into the container through the opening;

[0022] FIG. 5 is a perspective of the combination of electrode array and container after the electrode array has been extended into the container through the opening; [0023] FIGS. 6-12 illustrate a sequence in which the electrode array and container illustrated in Fig. 5 are loaded into one embodiment of a system for replicating in vivo hemodynamic conditions and connected to an electrical impedance analyzer ;

[0024] FIG. 13 is a schematic diagram of one embodiment of an ECIS® analyzer and multiple sample test stations;

[0025] FIG. 14 is a schematic diagram of one embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array;

[0026] FIG. 15 is a schematic diagram of a second embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array and on the bottom of a porous membrane positioned above the ECIS® array;

[0027] FIG. 16 is a schematic diagram of a third embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array and on the top of a porous membrane positioned above the ECIS® array;

[0028] FIG. 17 is a schematic diagram of a fourth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array and on both the top and bottom of a porous membrane positioned above the ECIS® array;

[0029] FIG. 18 is a schematic diagram of a fifth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on both the top and bottom of a porous membrane positioned above the ECIS® array and in a volume of cell culture media above the porous membrane;

[0030] FIG. 19 is a schematic diagram of a sixth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on both the top and bottom of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media below the porous membrane; [0031] FIG. 20 is a schematic diagram of a seventh embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array, on both the top and bottom of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media above the porous membrane;

[0032] FIG. 21 is a schematic diagram of an eighth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array, on both the top and bottom of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media below the porous membrane;

[0033] FIG. 22 is a schematic diagram of a ninth embodiment of a culture dish, ECIS® electrode array, and cell sample including cells on the ECIS® electrode array and cells in cell culture media above the ECIS® electrode array;

[0034] FIG. 23 is a schematic diagram of a tenth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array, on the bottom of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media above the porous membrane;

[0035] FIG. 24 is a schematic diagram of an eleventh embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array, on the bottom of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media between the porous membrane and the ECIS® electrode array;

[0036] FIG. 25 is a schematic diagram of a twelfth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array, on the top of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media above the porous membrane; and

[0037] FIG. 26 is a schematic diagram of a thirteenth embodiment of a culture dish, ECIS® electrode array, and cell sample, wherein cells are cultured on the ECIS® array, on the top of a porous membrane positioned above the ECIS® array, and in a volume of cell culture media between the porous membrane and ECIS® electrode array.

[0038] Corresponding reference numbers indicate corresponding parts throughout the drawings.

DETAILED DESCRIPTION

[0039] One embodiment of an Electric Cell-substrate Impedance Sensing (ECIS®) electrode array, generally designated 101, is shown in Figs. 1 and 2.

[0040] The ECIS® electrode array 101 includes a plurality of gold-film electrodes 103, gold-film contact pads 105, and gold-film conductive traces 107 connecting the electrodes to the contact pads. The gold-film for the electrodes 103, contact pads 105, and traces 107 is suitably deposited on an electrically insulating flat substrate 109 using techniques known to those skilled in the art. It is understood that other electrically conductive materials can be used instead of gold within the scope of the invention. Another insulating layer 115 is deposited on top of almost the entirety of the gold-film electrodes 103 and traces 107, leaving only the contact pads 105, a central electrode 117, and small diameter openings 121 (e.g., 250 micrometer) on the other electrodes 133 uncovered by the insulating over-layer 115. Thus, the conductive traces 107 are embedded in insulating materials 109, 115 while the contact pads 105, central electrode 117, and small portions of the other electrodes 133 have exposed un-insulated upper surfaces. Several small un-insulated openings 121 can be associated with each electrode 133. For example, there are suitably about 10 openings 121 in the insulating over-layer 115 for each electrode 133 in the illustrated embodiment. The electric cell-substrate impedance sensing electrode array 101 has a substantially flat upper surface because the electrodes 103, contact pads, 105, traces 107, and insulating overlayer are thin materials deposited on the flat substrate 109.

[0041] The contact pads 105 are suitably positioned in a line along the edge of one end of the substrate 115. The electrodes 103 are spread out over a sensing portion 125 of the ECIS® array 101 that is spaced from the contact pads 105. The electrodes 103 include the central electrode 117, which in the illustrated embodiment has an annular shape with a circular perimeter and hollow center. The ECIS® array 101 has a perimeter that includes an arcuate segment 131 partially circumscribing the sensing portion 125 of the ECIS® array. The central electrode 117 and the arcuate perimeter segment 131 are positioned concentrically with one another.

[0042] The electrodes 103 also include a plurality of electrodes 133 positioned radially outward of the central electrode 117. Some of these electrodes 133 are spaced farther from the central electrode 117 than others. For example, a first set of the electrodes 133a is suitably spaced one distance from the central electrode 117 and a second set of electrodes 133b is suitably spaced a shorter distance from the central electrode. The electrodes 133a in the first set are positioned along a first concentric circle having a relatively larger radius and the electrodes in the second set 133b are positioned along a second concentric circle having a relatively shorter radius. The distance between the electrodes of the second set 133b and the central electrode 117 is suitably no more than about 70 percent of the distance between the electrodes of the first set 133a and the central electrode, more suitably no more than about 60 percent of the distance between the electrodes of the first set and the central electrode, and still more suitably about 50 percent of the distance between the electrodes of the first set and the central electrode.

[0043] The electrodes of the first and second set 133a, 133b form a ring of electrodes 133 circumscribing the central electrode 117. The electrodes of the first set 133a are positioned equi-angularly around the central electrode 117. The electrodes of the second set 133b are suitably also positioned equi-angularly around the central electrode 117. The electrodes of the first set 133a are suitably offset angularly from the electrodes of the second set 133b so no two electrodes lie on the same radial line extending from the center of the central electrode 117. One electrode of the second set 133b is suitably positioned on a radial line extending between each adjacent pair of electrodes of the first set 133a. Similarly, one electrode of the first set 133a is suitably positioned on a radial line extending between each adjacent pair of electrodes of the second set 133b. Accordingly, the electrodes 133 in the ring formed by the first and second sets of electrodes 133a, 133b alternate between electrodes of the first set and electrodes of the second set as they extend around the central electrode 117.

[0044] One advantage provided by the arrangement of electrodes 103 in the illustrated embodiment is that the electrodes are spread out over a large portion of the sensing portion 125 of the ECIS® electrode array 101. This helps reduce the risk that localized anomalies in the sample may unduly influence the assessment of the response of the sample to the condition being tested. However, other configurations of electrodes are possible within the broad scope of the invention. In the illustrated embodiment, there are four electrodes in each of the first 133a and second 133b sets of electrodes. However, the number of electrodes can vary within the scope of the invention. Further, the number of electrodes in the first set can differ from the number of electrodes in the second set within the scope of the invention. Moreover, the electrodes can all be spaced the same distance from the central electrode without departing from the broad scope of the invention. If desired the electrodes can be arranged in other configurations within the broad scope of the invention, including configurations in which none of the electrodes is a central electrode .

[0045] The ECIS® array 101 is configured so the sensing portion 125 can be positioned inside a culture dish 141 while the end of the ECIS® array having the contact pads 105 thereon extends through a slot shaped opening 143 made in the sidewall of the dish to the exterior of the dish as illustrated in Figs. 3-5. As illustrated in these Figs., the contact pads 105 are positioned outside the container 141. The electric cell- substrate impedance sensing array 101 is configured so the electrodes 103 are not divided into distinct wells by the electric cell-substrate impedance sensing array. Instead, the entire upper surface of the sensing portion 125 of the electrode array 101 is in a single well formed by the container 141. The electric cell-substrate impedance sensing array 101 is configured so the electrodes 133 are not divided into distinct wells by the electric cell-substrate impedance sensing array 101. The electrode array 101 can be positioned so the electrodes 103 are in the cell culture dish 141 or other container in other ways within the scope of the invention, such as for example by being completely contained within the container, inserted through or formed as part of the bottom of the container, etc. within the broad scope of the invention.

[0046] It is challenging to gain an understanding of how cells of a complex organism will respond in vivo to various substances and conditions by studying the cells in the laboratory. For example, once cells are taken out of humans or other animals for testing in the laboratory, they do not behave or respond in the same manner as when in the human body. Thus, results from drug and chemical studies on isolated cultured cells in static conditions can be misleading. United States Patent No. 7,811,782, the contents of which are incorporated by reference, describes a system for replicating in the laboratory the hemodynamic conditions from within the human circulatory system. Briefly, cells cultured in a co-culture dish having an artificial porous membrane (often referred to as a TRANSWELL®) are subjected to shear forces generated by placing a rotating body (e.g., a plate or other structure having a cone-shaped surface) in the dish. The shear forces produced by the body (e.g., by its conical surface) can efficiently and reliably transform cells from their static cell-culture state and recalibrate them to their in vivo (inside the living organism) behavior by applying region-specific hemodynamic (blood flow) forces. The technology can create a wide range of healthy and disease-prone (e.g., pro-inflammatory) states by applying hemodynamics derived from different vascular regions to the cell-culture system. This facilitates more accurate investigation and understanding of organismal biology and allows for simulated in vivo drug testing for efficacy and toxicity in organ systems in healthy and disease-prone states.

[0047] Figures 6-12 illustrate the ECIS® array 101 and culture dish 141 being loaded in a base plate of one example of a system 161 for replicating in vivo hemodynamic conditions. The base plate 163 has a recess 165 including a circular portion 167 sized and shaped to receive the culture dish. The recess 165 also includes a rectangular slot 169 portion extending from the circular portion 167 to a side of the plate. The rectangular slot portion 169 is adapted to receive a clamp 171 used to interface the electrode array 101 to a commercially available ECIS® instrument 151 or other suitable impedance analyzer. Once a culture dish 141 containing a sample is loaded on the plate 163 and the ECIS® array 101 is interfaced to the analyzer 151 through the clamp 171, the rotating body 181 of the hemodynamic replicating system 161 is lowered into the culture dish 141 as illustrated in Figs. 11 and 12.

[0048] Once the body 181 is lowered into position, the motor 183 rotates the body in order to apply hemodynamic shear forces to cells in the container 141. In particular, the motor 183 adjusts the rate of rotation of the body 181 in a way that results in time-varying shear forces that replicate or mimic shear forces that would be encountered in vivo. The operation of the rotating body 181 can be adjusted to select shear forces that replicate or approximate those that would be encountered in a healthy organism or those that would be encountered in a disease state.

[0049] The contact pads 105 are suitably configured to interface with an impedance measuring instrument 151 (e.g., an ECIS® Z Theta or ECIS® Z instrument from Applied Biophysics of Troy, New York) using an suitable interface, as will be described in more detail below. The instrument 151 selectively applies electrical signals (e.g., AC electrical signals) to the electrodes causing current to flow between two or more electrodes 103 through the openings 121 in the insulating over- layer 115 and through the cells and culture medium. Commercially available ECIS® and other impedance analyzers provide users the option to select any of various types of impedance measurements. For example, the impedance can be measured at one or more fixed frequencies. The impedance can also be monitored as the frequency of the signal applied to the electrodes is swept over a range of frequencies. Some impedance analyzers allow measurement of a phase shift between the impedance and the signal applied to the electrodes. Any of these measurements can be taken if they are needed or desired for any particular experiment .

[0050] The cells growing over the electrodes 103 during an experiment (e.g., as illustrated in Fig. 14) can affect the electrical impedance between the electrodes. For example, the impedance can be correlated with the density of the cells growing over the electrodes 103, with higher cell densities yielding higher impedance values. The small openings 121 in the insulating over-layer 115 require the electrical current to flow through a relatively small cross sectional area at the electrode 133. As the openings 121 become covered by cells, the cells at the openings increase the impedance. The effects associated with cells at the openings 121 have a much greater impact on the change in the impedance signal than cells elsewhere on the ECIS® array 101 (e.g., any cells on the central electrode 117) because there is much larger cross sectional area for current to flow between the upper surface of any cell layer adhered to the ECIS® array at the openings and the central electrode 117. Thus, any change in the impedance signals over the course of an experiment are substantially attributable to the influences of cells at the small openings 121 in the insulating layer 115 covering the outer electrodes 133. Accordingly, changes in the electrical signal over time during an experiment are also substantially attributable to changes in the cells at the small openings 121 on the electrodes 133.

[0051] In addition to cell density, an electrode array 101 can be used to study other phenomena, including without limitation binding of molecules to cell surface receptors, signal transduction pathways, cell death, cell viability, cell proliferation, cell attachment, cell migration, cell micromotion and contraction, cell spreading, wound healing, cell invasion and extravasation, the barrier function of the cell layer, spacing between the ventral side of the cell and the substratum, and cell membrane capacitance.

[0052] It is possible to run different experiments sequentially using a single sample testing station having a single electrode array 101 connected to an impedance analyzer 151. However, it can be desirable to perform multiple experiments in parallel using a plurality of sample stations, each having its own cell culture dish and electrode array connected to a single impedance analyzer adapted to perform impedance measurements at each of the plurality of sample testing stations. [0053] The schematic diagram in Fig. 13 illustrates one embodiment of a system for running a plurality of experiments in parallel at a plurality of sample testing stations 153. The ECIS® analyzer 151 is connected to a distribution box 155. The distribution box 155 is connected by ribbon cables 157 to each of a plurality of ECIS® electrode arrays 101 (each of which can be substantially identical to the ECIS® electrode array described above) at a plurality of different sample test stations 153. The distribution box 155 includes electrical circuitry (not shown) connecting each of the cables 157 extending between the distribution box and the sample test stations 153 to a subset of the pins of the cable 159 connecting the distribution box to the ECIS analyzer 151, which can be, for example, a standard cable that comes with a commercially available ECIS® analyzer instrument 151.

[0054] In the schematic diagram in Fig. 13, there are eight sample test stations 153 connected to a single ECIS® analyzer 151. However, the number of sample test stations can vary within the scope of the invention. In one embodiment there are two or more sample test stations, such as 2-3 sample test stations. In another embodiment, there are suitably 4-12 sample test stations connected to a single impedance analyzer and still more suitably 8-12 sample test stations connected to a single impedance analyzer .

[0055] The number of sample test stations that can be connected to a single impedance analyzer depends in part on the number of electrodes in each electrode array and also on the number of electrodes that the analyzer is designed to energize. Some commercially available ECIS® analyzers are designed to take impendence measurements in a 96 well test plate. Operation of the ECIS® electrode array 101 described above involves energizing the eight outer electrodes 133. Accordingly, it is possible to connect up to twelve of the ECIS® electrode arrays 101 to a single 96 well ECIS® analyzer 151 using a distribution box 155, thereby allowing up to 12 sample test stations 153 to conduct up to 12 experiments in parallel using a single ECIS® analyzer having a 96 well capability.

[0056] The motor 183 and rotating body 181 of the system 161 for applying hemodynamic shear forces to the sample can create electronic noise. It has been observed that noise in the measurements from the ECIS® analyzer 151 increases when the motor 183 for the rotating body 181 is turned on. The electronic noise increases further whenever the rotating body 181 is lowered into the cell culture container 141. When the number of sample stations 153 is relatively low (e.g., three or less), the noise is not very problematic. As the number of sample stations 153 increases, the noise becomes more problematic.

[0057] To alleviate noise problems caused by the multiple motors 183 and multiple rotating bodies 181, the electrical cables 157 connecting the sample stations 153 to the distribution box 155 are suitably shielded with a fine wire mesh wrapped around the cables. Electrical tape at the ends of the cables 157 can be used to hold the wire mesh in place. The mesh reduces crosstalk and noise across the multiple sample stations 153.

[0058] Another way to alleviate noise problems is to modify the software of the ECIS® instrument 151. For example, the software is suitably adapted to include updated data collection processes, such that the wells being utilized for measurement are able to properly read and store the data and communication errors are minimized by being modified to treat the electrodes of each electrode array as a separate dataset. The measurements are multiplexed in separate threads. There is only one measurement occurring at any time. The settings on the lock-in amplifier can be adjusted to turn up the current and increase the time-constant to reduce the noise by overcoming interference from the motors used to rotate the rotating bodies producing the shear forces. Also, noise reduction, and running averaging functions can be added to data collection to computationally yield improved and smoothed readings over long time periods.

[0059] There are many different types of experiments that can be performed using the system described above. Several of the configurations of the various cell types are provided below. When more than one sample testing station 153 is connected to a single impedance analyzer, the sample testing stations can each run the same type of experiment or different types of experiments. It is also understood that the configurations provided below do not illustrate all the experiments that may be run using the sample test stations 153.

[0060] Figure 14 is a schematic diagram of one embodiment of a culture dish, ECIS® array and cell sample.

A layer of cultured cells 201a is adhered directly to the ECIS® array. The cell layer 201a can include any cell type, for example vascular cells, smooth muscle cells, astrocytes, glial cells, or any of the cell types listed herein. In the example in the diagram the cells 201a are endothelial cells. The endothelial cells 201a could encompass numerous types of endothelial cells including microvascular endothelial cells or sinusoidal endothelial cells. A perfusion system including one or more clips 185 (e.g., two clips as in the diagram) supporting conduits 187 for cell culture media 191 perfuses fresh cell culture media into the dish 141 and removes old cell culture media from the dish, as indicated by the arrows. The rotating body 181 rotates in the media 191 above the cell layer 201a and thereby produces shear forces on the cell layer, e.g., replicating blood flow forces in a blood vessel that may be encountered in a human body or other complex organism.

[0061] The system can also be used to study cell samples in a dish including a porous membrane 193, for example a porous membrane of a TRANSWELL® insert, as illustrated in Fig. 15. This configuration is similar to that of Fig. 14, but in this configuration a layer of cultured cells 201b is adhered to the bottom of the porous membrane 193 in addition to the layer of cells 201a adhered to the top of the ECIS® array 101 under the membrane. The ECIS® array 101 detects changes in the cell layer 201a that is adhered to it. However, the presence of the cells 201b on the porous membrane 193 may affect the cells 201a on the ECIS® array 101. The cells 201a, 201b can be substantially similar in cell type or the cells 201a on the array 101 can include primarily cells of a first cell type and the cells 201b on the porous membrane can include primarily cells of a second cell type different from the first cell type. Additional details about culturing cells in a cell culture container having a porous membrane suspended therein and under hemodynamic shear producing systems are provided in U.S. Patent No. 7,811,782, already incorporated by reference.

[0062] The configuration in Fig. 16 is similar to that of Fig. 15 except cells 201b are cultured on top of the porous membrane instead of on the bottom. Again, the cells 201a, 201b can be substantially similar in cell type or the cells 201a on the array 101 can include primarily cells of a first cell type and the cells 201b on the porous membrane can include primarily cells of a second cell type different from the first cell type.

[0063] The configuration illustrated in Fig. 17 is similar to Figs. 15 and 16, but in this configuration cultured cells 201b, 201c are adhered to the top and bottom of the porous membrane 193. The cells 201c adhered to the top of the porous membrane 193 can be primarily a different cell type than the cells 201b adhered to the bottom of the porous membrane. The cells 201a on the electrode array 101 and the cells 201b on the bottom of the porous membrane 193 can include primarily the same type of cells or the cells 201a on the array 101 can include primarily cells of a first cell type and the cells 201b on the bottom of the porous membrane can include primarily cells of a second cell type different from the first cell type. [0064] Figure 18 illustrates a configuration involving three different types of cells. A first type of cells 201a is adhered to the bottom of the porous membrane and a second type of cells 201b different from the first type of cells is adhered to the top of the porous membrane. A third type of cells 201c is added to the cell culture media 191 above the porous membrane 193 and is not adhered to any structure at the beginning of the experiment .

[0065] Figure 19 represents a configuration that is substantially similar to the experiment described in Fig. 18. One difference between this configuration and that of Fig. 18 is that the third cell type 201c is added to the cell culture media 191 below the porous membrane instead of above the porous membrane. As the rotating body 181 produces shear forces that replicate conditions that may be encountered in vivo, the ECIS® array 101 can detect and monitor adherence of the third cell type 201c to the surface of the ECIS. The impedance between the electrodes 103 of the electrode array can detect cells adhering to and spreading on the electrode array 101.

[0066] In the configuration of Fig. 20, cells 201b, 201c are adhered to the top and bottom of the porous membrane 193. Cells 201a are also adhered to the top of the ECIS® electrode array 101. In this sense this configuration is similar to that of Fig. 17. One difference between this configuration and that of Fig. 17 is that non-adhered cells 201d are added to the culture media 191 in the upper chamber above the porous membrane, similar to what is described for the configuration of Fig. 18. The impedance between the electrodes 103 of the electrode array 101 can detect cells adhering to and/or diapedesing through the cells 201a already on the array 101.

[0067] The configuration of Fig. 21 is substantially similar to that of Fig. 20 except that the non-adhered cells 201b are added to the cell culture media 191 in the lower chamber below the porous membrane. Cells 201a, 201c, and 201d are adhered to the ECIS electrode array 101, the bottom of the porous membrane 193, and the top of the porous membrane, respectively .

[0068] Another configuration is illustrated in Fig. 22. This configuration is similar to Fig. 14, but also includes the addition of cells 201b to the culture media 191. The same types of cells that can be added to the culture media in Figs. 18-21 can be added to the culture media in this configuration.

[0069] Another configuration is illustrated in Fig. 23. This configuration is similar to that of Fig. 15, but in this configuration cells 201c are added to the cell culture media 191 in the chamber above the porous membrane 193. The cells 201c can be the same type of cells as are added to the culture media in Figs. 18-22.

[0070] The configuration of Fig. 24 is similar to that of Fig. 15, but cells 201b are added to the culture media 191 below the porous membrane 193. Cells 201a are adhered to the ECIS® electrode array 101 and cells 201c are adhered to the bottom of the porous membrane 193. The cells 201b added to the culture media can be the same type of cells as are added to the culture media in Figs. 18-23.

[0071] Another configuration is illustrated in Fig. 25. This configuration is similar to Fig. 16, but cells 201c are added to the culture media 191 above the membrane 193. Cells 201a are adhered to the ECIS® electrode array 101 and cells 201b are adhered to the top of the membrane 193. The cells 201c that are added to the culture media can be the same type of cells added to the culture media in Figs. 18-24.

[0072] The configuration of Fig. 26 is similar to that of Fig. 16, but cells 201b are added to the culture media 191 below the membrane 193. Cells 201a are adhered to the ECIS® electrode array 101 and cells 201c are adhered to the top of the membrane 193. The cells 201b added to the culture media can be the same types of cells as those that are added to the culture media in Figs. 18-25.

[0073] It is also noted that in any of the configurations in which there is a porous membrane and cells are added to the cell culture media in either the upper or lower chamber that cells could also be added to the cell culture media in the opposite chamber so the cells are added to the cell culture media of both the upper and lower chambers.

[0074] It is also noted that any of the various groups of cells 201a, 201b, 201c, 201d, etc. referenced in the configurations can include cells that are primarily of the same type as the type of cells in any of the other groups of cells or that any one or all of the groups of cells can include cells that are primarily a different type of cell than the types of cells in any one or all of the other groups of cells.

[0075] Accordingly, one general method for characterizing the response of cells to a condition includes adding culture media 191 to the container 141. A first cell type is plated on one first side of the porous membrane 193 and/or a second cell type is plated on the second side of the porous membrane. The cells of the first cell type can be any of various different cell types include renal cells, cells of the airways, or cells of the blood-brain barrier to provide a few non-limiting examples. Additional examples of cells that can constitute cells of the first cell type include smooth muscle cells, glial cells, astrocytes, neurons, or epithelial podocytes. The cells of the second cell type can be any of various cells types. For example, the cells of the second cell type can suitably be vascular cells (e.g., endothelial cells).

[0076] The porous membrane 193 is suspended in the container 141 such that the first side is proximal and in spaced relation to a surface of the container, thereby defining within the container a lower volume comprising the first cell type or an upper volume comprising the second cell type. The porous membrane 193 is adapted to permit fluid communication of the culture media 191. If cells of both the first and second cell types are present the porous membrane can be adapted to permit physical interaction and communication between cells of the first cell type and cells of the second cell type.

[0077] A third cell type is optionally plated on the surface of the container 141. For example, the cells of the third cell type are suitably adhered to an upper surface of the electric cell-substrate impedance sensing electrode array 101 so they extend over multiple electrodes 103 of the electric cell- substrate impedance sensing electrode array. The electric cell- substrate impedance sensing electrode array can have a substantially flat upper surface. The electric cell-substrate impedance sensing array can be configured so the electrodes are not divided into distinct wells by the electric cell-substrate impedance sensing array. The contact pads of the electric cell- substrate impedance sensing array suitably are positioned outside the container.

[0078] The cells of the third cell type can be renal cells, cells of the airways, or cells of the blood-brain barrier, for example. Additional examples of cells that are suitable for the third cell type include smooth muscle cells, glial cells, astrocytes, neurons, macrophages, or leukocytes.

[0079] Culture media 191 is perfused into and out of the upper volume and perfused into and out of the lower volume. A fourth cell type is optionally suspended in the culture media 191 in the upper volume. A fifth cell type is optionally suspended in the culture media 191 in the lower volume. Cells of that are suitable for being suspended in the cell culture media 191 include, for example, blood cells or immune cells, such as monocytes, macrophages, dendritic cells, red blood cells, neutrophils, lymphocytes, basophils, eosinophils, leukocytes, platelets or combinations thereof. [0080] The cells of the first cell type, cells of the second cell type, cells of the third cell type, cells of the fourth cell type, and cells of the fifth cell type can each independently be primary cells or immortalized cells. The primary or immortalized cells can comprise cells isolated from at least one normal subject or at least one subject having a pathological condition, cells isolated from at least one subject having a risk factor for a pathological condition, cells isolated from at least one subject with a single nucleotide polymorphism linked to a pathological condition, cells isolated from at least one subject with an identified genotype linked to drug toxicity, or cells isolated from at least one subject with a single nucleotide polymorphism linked to drug toxicity.

[0081] When the cells are isolated from at least one subject having a risk factor for the pathological condition, the risk factor can include, but is not limited to, age, gender, race, epigenetic imprinting, an identified genotype linked to the pathological condition, an identified single nucleotide polymorphism linked to the pathological condition, diabetes, hypertension, atherosclerosis, atherosclerosis plaque rupture, atherosclerosis plaque erosion, thoracic aortic aneurysm, abdominal aortic aneurysm, cerebral aneurysm, heart failure, stroke, Marfan Syndrome, carotid intima-medial thickening, atrial fibrillation, kidney disease, pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary artery disease, pulmonary hypertension, hyperlipidemia, familial hypercholesterolemia, peripheral artery disease, deep vein thrombosis, vascular restenosis, vascular calcification, myocardial infarction, obesity, hypertriglyceridemia, hypoalphalipoproteinemia, fatty liver disease, hepatitis C, hepatitis B, liver fibrosis, bacterial infection, viral infection, cirrhosis, liver fibrosis, or alcohol-induced liver disease . [0082] The primary cells can include a cell lineage derived from stem cells (e.g., adult stem cells, embryonic stem cells, inducible pluripotent stem cells, or bone marrow-derived stem cells) or stem-like cells. The cell lineage derived from stem cells or stem-like cells can comprise endothelial cells, smooth muscle cells, cardiac myocytes, hepatocytes, neuronal cells, or endocrine cells.

[0083] Cell types for use in the systems and methods of the present invention include renal cells, cells of the airways, blood-brain barrier cells, vascular cells, hepatic cells, pancreatic cells, cardiac cells, muscle cells, spleen cells, gastrointestinal tract cells, skin cells, liver cells, immune cells, or hematopoietic cells.

[0084] Specific cell types for use in the systems and methods include astrocytes, endothelial cells, glomerular fenestrated endothelial cells, renal epithelial podocytes, alpha cells, β-cells, delta cells, pancreatic polypeptide (PP) cells, epsilon cells, glial cells, hepatocytes, neurons, nonparenchymal hepatic cells, podocytes, smooth muscle cells, mesangial cells, pericytes cells, cardiac muscle cells, skeletal muscle cells, leukocytes, monocytes, myocytes, macrophages, neutrophils, dendritic cells, T-cells, B-cells, endothelial progenitor cells, stem cells, circulating stem cells, circulating hematopoietic cells, endocardial cells, fibroblasts, chondrocytes, or osteoblasts. The nonparenchymal hepatic cells comprise hepatic stellate cells, sinusoidal endothelial cells, and/or Kupffer cells. Preferably, the specific cell types can include endothelial cells, smooth muscle cells, hepatocytes, sinusoidal endothelial cells, or a combination thereof.

[0085] The cell types for use in the systems and methods of the invention can be animal cell types, such as cells from a genetically modified animal. The animal cell types are preferably human cell types. The human cell types can be selected on the basis of age, gender, race, epigenetics, disease, nationality, the presence or absence of one or more single nucleotide polymorphisms, a risk factor as described herein, or some other relevant characteristic, for example, a characteristic that to a pathological condition. For example, the cell types can be cells from a normal subject, a subject having diabetes, a hypertensive subject, a smoker, a subject having abdominal aortic aneurysm, a subject having fatty liver disease, an aged subject, or an animal genetically-modified to model diabetes, hypertension, fatty liver disease or aging, or modified to model abdominal aortic aneurysm.

[0086] At least one of the cell types that are present can be vascular or organ cells from one or more patients with an identified genotype linked to pharmaceutical toxicity or a pathophysiological endpoint. For example, the cells can be from one or more patients that have a single nucleotide polymorphism linked to pharmaceutical toxicity or a pathophysiological endpoint .

[0087] All of the cell types that are present are suitably within the culture media 191. One or more of the cell types that are present are exposed to the condition (s) . The electric cell-substrate impedance sensing electrode array 101 is used to measure electrical impedance to determine the response of one or more of the cell types to the conditions.

[0088] In some embodiments of present method, cells of the first cell type are present. In other embodiments, cells of the second cell type are present.

[0089] In another embodiment of the method, cells of the first cell type and cells of the third cell type are present.

[0090] In another embodiment of the method, cells of the second cell type and cells of the third cell type are present.

[0091] In another embodiment of the method, cells of the first cell type, cells of the second cell type, and cells of the third cell type are present. [0092] In another embodiment of the method, cells of the first cell type, cells of the second cell type, cells of the third cell type and cells of the fourth cell type are present.

[0093] In another embodiment of the method, cells of the first cell type, cells of the second cell type, cells of the third cell type and cells of the fifth cell type are present.

[0094] In another embodiment of the method, cells of the first cell type, cells of the third cell type and cells of the fourth cell type are present.

[0095] In another embodiment of the method, cells of the first cell type, cells of the third cell type and cells of the fifth cell type are present.

[0096] In another embodiment of the method, cells of the second cell type, cells of the third cell type, and cells of the fifth cell type are present.

[0097] In another embodiment of the method, cells of the second cell type, cells of the third cell type, and cells of the fourth cell type are present.

[0098] In another embodiment of the method, cells of the second cell type, cells of the third cell type, cells of the fourth cell type and cells of the fifth cell type are present.

[0099] In another embodiment of the method, cells of the first cell type, cells of the third cell type, cells of the fourth cell type and cells of the fifth cell type are present.

[00100] In another embodiment of the method, cells of the first cell type, cells of the second cell type and cells of the fourth cell type are present.

[00101] In another embodiment of the method, cells of the first cell type, cells of the second cell type and cells of the fifth cell type are present.

[00102] In another embodiment of the method, cells of the first cell type and cells of the fifth cell type are present.

[00103] In another embodiment of the method, cells of the second cell type and cells of the fifth cell type are present. [00104] In another embodiment of the method, cells of the first cell type and cells of the second cell type are present.

[00105] In another embodiment of the method, cells of the first cell type and cells of the fourth cell type are present.

[00106] In another embodiment of the method, cells of the second cell type and cells of the fourth cell type are present.

[00107] In another embodiment of the method, cells of the first cell type, cells of the second cell type, cells of the fourth cell type and cells of the fifth cell type are present.

[00108] In another embodiment of the method, cells of the first cell type, cells of the fourth cell type, and cells of the fifth cell type are present.

[00109] In another embodiment of the method, cells of the second cell type, cells of the fourth cell type and cells of the fifth cell type are present.

[00110] In another embodiment of the method, cells of the first cell type, cells of the second cell type, cells of the third cell type, cells of the fourth cell type, and cells of the fifth cell type are present.

[00111] The conditions the cells are exposed to suitably include a shear force applied to the cells in the upper volume of the container 141 or to a surface of the porous membrane in the upper volume of the container 141. The shear force is suitably induced by a system comprising a body 181 adapted for being positioned in the culture media in the upper volume of the container 141 and a motor 183 adapted to rotate the body. The body 181 suitably has a conical surface.

[00112] The shear force suitably mimics hemodynamic flow. The hemodynamic flow is suitably a time-variant hemodynamic flow. The hemodynamic flow may be derived from a previously measured hemodynamic pattern, for example a hemodynamic pattern derived from an animal (e.g., a genetically modified animal or a human) . The hemodynamic pattern can be derived from a subject or subjects having a pathological condition or a disease-promoting condition. For example, the disease-promoting condition may comprise an anatomical condition such as atrophy, calculi, choristoma, pathologic constriction, pathologic dilation, diverticulum, hypertrophy, polyps, prolapse, rupture, an arteriovenous fistula, or an appendage.

[00113] Alternatively, the hemodynamic pattern can be derived from a healthy subject.

[00114] The hemodynamic pattern can be derived from at least a portion of an artery, an arteriole, a vein or an organ using ultrasound, magnetic resonance imaging (MRI), or any other suitable measurement technique. When the hemodynamic pattern is derived from an artery or an arteriole, the hemodynamic pattern can be an atheroprone or atheroprotective hemodynamic pattern.

[00115] When a hemodynamic pattern is derived from at least a portion of an artery, the artery can comprise a carotid artery, thoracic artery, abdominal artery, pulmonary artery, a femoral artery, renal efferent artery, renal afferent artery, a coronary artery, a brachial artery, a internal mammary artery, a cerebral artery, the aorta, a precapillary arteriole, a coronary artery, hepatic artery, anterior cerebral artery, middle cerebral artery, posterior cerebral artery, basilar artery, external carotid artery, internal carotid artery, vertebral artery, subclavian artery, aortic arch, axillary artery, internal thoracic artery, branchial artery, deep branchial artery, radial recurrent artery, superior epigastric artery, descending aorta, inferior epigastric artery, interosseous artery, radial artery, ulnar artery, palmar carpal arch, dorsal carpal arch, superficial/deep palmar arches, digital artery, descending branch of the femoral circumflex artery, descending genicular artery, superior genicular arteries, inferior genicular arteries, anterior tibial artery, posterior tibial artery, peroneal artery, deep plantar arch, arcuate artery, common carotid arteries, intercostal arteries, left/right gastric artery, celiac trunk, splenic artery, common hepatic artery, superior mesenteric artery, renal artery, inferior mesenteric artery, testicularis artery, common iliac artery, internal iliac artery, external iliac artery, femoral circumflex artery, perforating branches, deep femoral artery, popliteal artery, dorsal metatarsal artery, or dorsal digital arteries.

[00116] When a hemodynamic pattern is derived from at least a portion of an vein, the vein can comprise a post-capillary venule, saphenous vein, hepatic portal vein, superior vena cava, inferior vena cava, coronary vein, Thesbian vein, superficial vein, perforator vein, systemic vein, pulmonary vein, jugular vein, sigmoid sinus, external jugular vein, internal jugular vein, inferior thyroid vein, subclavian vein, internal thoracic vein, axillary vein, cephalic vein, branchial vein, intercostal vein, basilic vein, median cubital vein, thoracoepigastric vein, ulnar vein, median antebranchial vein, inferior epigastric vein, deep palmar arch, superficial palmar arch, palmar digital veins, inferior vena cava, hepatic vein, renal vein, abdominal vena cava, testicularis vein, common iliac vein, perforating branches, external iliac vein, internal iliac vein, external pudendal vein, deep femoral vein, great saphenous vein, femoral vein, accessory saphenous vein, superior genicular vein, popliteal vein, inferior genicular vein, great saphenous vein, small saphenous vein, anterior/posterior tibial vein, deep plantar vein, dorsal venous arch, or dorsal digital vein.

[00117] When a hemodynamic pattern is derived from at least a portion of an organ, the organ can comprise a liver, a kidney, a lung, a brain, a pancreas, a spleen, a large intestine, a small intestine, a heart, a skeletal muscle, an eye, a tongue, a reproductive organ, or an umbilical cord. When the hemodynamic pattern is derived from the heart, the hemodynamic pattern can be derived, for example, from a chamber of the heart, a cardiac sinus, a left atrial appendage during sinus rhythm, an atrial fibrillation, or a ventricular fibrillation. The chamber of the heart can be a left atrium, a right atrium, a left ventricle or a right ventricle.

[00118] The hemodynamic pattern can result from a physical change resulting from a pathological condition.

[00119] The hemodynamic pattern can be altered as a direct or indirect effect of administration of a pharmaceutical to a subject as compared to the flow or the hemodynamic pattern for the subject absent administration of the pharmaceutical.

[00120] The condition (s) can suitably involve the presence of a pharmaceutical or a compound. For example, the condition (s) can include a particular concentration of a pharmaceutical or a compound. The pharmaceutical or compound being tested can be perfused into at least one of the upper volume and the lower volume. For example, the pharmaceutical or the compound is suitably added to the culture media 191 while the hemodynamic shear forces are being applied to the cells. Alternatively, the pharmaceutical or compound can be added to the culture media 191 before the hemodynamic shear force is applied to the cells.

[00121] Various pharmaceuticals and compounds that are suitable for testing as a condition that may affect the cells include, without limitation, a cyclooxygenase inhibitor (e.g., celecoxib) ; a taxane (e.g., paclitaxel) ; a tyrosine kinase inhibitor (e.g., imatinib) ; a low molecular weight heparin (e.g., enoxaparin) ; an anti-thrombogenic agent (e.g., bivalirudin, dipyridamole, urokinase, r-urokinase, r- prourokinase, reteplase, alteplase, streptokinase, rt-PA, TNK- rt-PA, monteplase, staphylokinase, pamiteplase, unfractionated heparin, or APSAC) ; a calcium channel blocker (e.g., amlodipine or nifedipine); an anti-platelet agent (e.g., clopidogrel, abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban or ticlopinin) ; an anticlotting agent (e.g., fondaparinux) ; a chelating agent (e.g., penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA, deferoxamine mesylate or batimastat) ; an anti-inflammatory agent (e.g., rofecoxib) ; a rho kinase inhibitor (e.g., Y27632); a PDGF inhibitor (e.g., AG1295), a cholesterol lowering agent (e.g., a statin); an anti-restenosis agent; an antibiotic (e.g., actinomycin-D) ; an anti-neoplastic agent (e.g., c-myc antisense or dexamethasone) ; an anti-hypertensive agent (e.g., an ACE inhibitor) ; a synthetic polysaccharide; an agent that raises HDL; or a combination thereof.

[00122] Additional non-limiting examples of pharmaceuticals the presence of concentration of which may be tested as one of the conditions include atorvastatin, sirolimus, tacrolimus, everolimus, wortmannin, or a combination thereof.

[00123] Further non-limiting examples of substances that are suitable pharmaceuticals or compounds that can be tested include radiocontrast agents, radio-isotopes, propharmaceuticals, antibody fragments, antibodies, live cells, therapeutic pharmaceutical delivery microspheres or microbeads, nanoparticles , gels or cell-impregnated gels, or a combination thereof .

[00124] The drug or compound can be an anti-inflammatory agent, an antineoplastic agent, an anti-diabetic agent, a protein kinase inhibitor, an anti-thrombotic agent, a thrombolytic agent, an anti-platelet agent, an anti-coagulant, a calcium channel blocker, a chelating agent, a rho kinase inhibitor, an anti-hyperlipidemic agent, an agent that raises HDL, an anti-restenosis agent, an antibiotic, an immunosuppressant, an anti-hypertensive agent, a diuretic, an anorectic, an appetite suppressant, an anti-depressant, an anti^¬ psychotic, a contraceptive, a calcimimetic, a biologic medical product, or a combination thereof.

[00125] When the drug is an anti-inflammatory agent, the anti-inflammatory agent can comprise a steroid (e.g., prednisone, hydrocortisone, prednisolone, betamethasone, or dexamethasone) , a non-steroidal anti-inflammatory drug (NSAID) (e.g., a salicylate, ibuprofen, acetaminophen, naproxen, or ketoprofen) , a selective cyclooxygenase inhibitor (e.g., celecoxib or rofecoxib) , a non-selective cyclooxygenase inhibitor, an immune selective anti-inflammatory agent (e.g., phenylalanine-glutamine-glycine tripeptide) , or a combination thereof .

[00126] When the drug comprises an anti-neoplastic agent, the anti-neoplastic agent can comprise an alkylating agent (e.g., cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucide or ifosfamide) , an anti^¬ metabolite (e.g., azathioprine or mercaptopurine) , a plant alkaloid (e.g., a taxane such as paclitaxel or docetaxel) , a vinca alkaloid such as vincristine, vinblastine or vindesine, or a podophyllotoxin such as etoposide or teniposide) , a topoisomerase inhibitor (e.g., irinotecan, topotecan or amsacrine) , a cytotoxic antibiotic (e.g., actinomycin, bleomycin, plicamysin, mitomycin, doxorubicin, daunorubicin, valrubicin, idarubicin or epirubicin) , or a combination thereof.

[00127] When the drug is an anti-diabetic agent, the anti-diabetic agent can comprise a biguanide (e.g., metformin), a thiazolidinedione (e.g., rosiglitazone, troglitazone or pioglitazone) , a sulfonylurea (e.g., tolbutamine, acetohexamide, tolazamide, chlorpropamide, glipazide, glyburide, glimepiride, gliclazide, glycopyramide or gliquidone) , an incretin mimetic (e . g . , exenatide, liraglutide or taspoglutide) , a dipeptidyl peptidase IV inhibitor (e.g., vildagliptin, sitagliptin, saxaglitpin, linagliptin, allogliptin or septagliptin) , a sodium-glucose co-transporter 2 inhibitor (e.g., dapagliflozin, canagliflozin, empagliflozin, ipragliflozin, remogliflozin or sergliflozin) , or a glucokinase activator (e.g., piragliatin) .

[00128] When the drug comprises a protein kinase inhibitor, the protein kinase inhibitor can comprise a serine/threonine-specific kinase inhibitor, a tyrosine-specific kinase inhibitor (e.g., imatinib, bevacizumab, cetuximab, axitinib, lapatinib, ruxolitinib or sorafenib) , an epidermal growth factor (EGF) receptor inhibitor, a fibroblast growth factor (FGF) receptor inhibitor, a platelet-derived growth factor (PDGF) receptor inhibitor, or a vascular endothelial growth factor (VEGF) receptor inhibitor.

[00129] When the drug comprises the anti-thrombotic agent, the anti-thrombotic agent can comprise dipyridamole, urokinase, r-urokinase, r-prourokinase, reteplase, alteplase, streptokinase, rt-PA, TNK-rt-PA, monteplase, staphylokinase, pamiteplase, unfractionated heparin or APSAC.

[00130] When the drug comprises the thrombolytic agent, the thrombolytic agent can comprise a streptokinase, a urokinase or a tissue plasminogen activator.

[00131] When the drug comprises the anti-platelet agent, the anti-platelet agent can comprise a glycoprotein Ilb/IIIa inhibitor, a thromboxane inhibitor, an adenosine diphosphate receptor inhibitor, a prostaglandin analogue, or a phosphodiesterase inhibitor. For example, the anti-platelet agent can comprise clopidogrel, abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban or ticlopinin.

[00132] When the drug comprises the anti-coagulant, the anti-coagulant can comprise a vitamin K antagonist (e.g., warfarin), a factor Xa inhibitor (e.g., apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban, fondaparinux or idraparinux) , or a direct thrombin inhibitor (e.g., hirudin, bivalirudin, lepirudin, desirudin, dabigatran, ximelagatran, melagatran or argatroban) .

[00133] When the drug comprises the calcium channel blocker, the calcium channel blocker can comprise verapamil, diltiazem, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, isradipine, efonidipine, felodipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine.

[00134] When the drug comprises the chelating agent, the chelating agent can comprise penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA, deferoxamine mesylate or batimastat .

[00135] When the drug comprises the rho kinase inhibitor, the rho kinase inhibitor can comprise Y27632.

[00136] When the drug comprises the anti-hyperlipidemic agent, the antihyperlipidemic agent can comprise a statin (e.g., atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin or simvastatin) , a fibrate (e.g., bezafibrate, ciprofibrate, clofibrate, gemfibrozil or fenofibrate) , a selective inhibitor of dietary cholesterol absorption (e.g., ezetimibe) , or a cholesterylester transfer protein inhibitor (e.g., anacetrapib, dalcetrapib, torcetrapib or evacetrapib) .

[00137] When the drug comprises the agent that raises HDL, the agent that raises HDL can comprise an inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9) , such as AMG145.

[00138] When the drug comprises the anti-restenosis agent, the anti-restenosis agent can comprise dexamethasone ticlopidine, clopidogrel, sirolimus, paclitaxel, zotarolimus, everolimus, or umirolimus.

[00139] When the drug comprises the antibiotic, the antibiotic can comprise actinomycin-D .

[00140] When the drug comprises the immunosuppressant, the immunosuppressant can comprise a glucocorticoid, methotrexate, azathioprine, mercaptopurine, dactinomycin, mitomycin C, bleomycin, mithramycin, ciclosporin, tacrolimus, sirolimus, an interferon, infliximab, etanercept or adalimumab.

[00141] When the drug comprises the anti-hypertensive agent, the antihypertensive agent can comprise a beta adrenergic receptor antagonist (e.g., alprenolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutalol, pindolol, propranolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, metoprolol or nebivolol) , an angiotensin II receptor antagonist (e.g., losartan, olmesartan, valsartan, telmisartan, irbesartan, or azilsartan) , or an angiotensin converting enzyme inhibitor (e.g., captopril, enalapril, lisinopril, quinapril, zofenopril, imidapril, benazepril, trandolapril or ramipril) .

[00142] When the drug comprises the diuretic, the diuretic can comprise furoseamide, amiloride, spironolactone, or hydrochlorothiazide .

[00143] When the drug comprises the anorectic, the anorectic can comprise phentermine, fenfluramine, dexfenfluramine, sibutramine, lorcaserin, topiramate, or a combination thereof.

[00144] When the drug comprises the anti-depressant, the anti-depressant can comprise imipramine, desipramine, amitryptiline, paroxetine, citalopram, fluoxetine, or escitalopram.

[00145] When the drug comprises the anti-psychotic, the anti-psychotic can comprise aripiprazole, risperidone, olanzapine, quetiapine, cariprazine, lurasidone or asenopine.

[00146] When the drug comprises the contraceptive, the contraceptive can comprise a combination of drospirenone and ethinyl estradiol.

[00147] When the drug comprises the calcimimetic, the calcimimetic can comprise cinacalcet.

[00148] When the drug comprises the biologic medical product, the biologic medical product can comprise a synthetic polysaccharide, a synthetic, partially synthetic or humanized immunoglobulin, or a recombinant therapeutic protein. [00149] The compound can suitably be capable of inhibiting, activating or altering the function of proteins or genes in any of the cell types in the container 141.

[00150] Additional non-limiting examples of materials that can be tested include vascular stent materials, such as a nanoporous metal, a polymer, or a carbon material. For example, the compound can be evaluated for elution from a vascular stent material, and one or more of the cell types can be tested for compatibility with, cellular adhesion to, or phenotypic modulation by the vascular stent material. The pharmaceutical or compound can eluted from a vascular stent material adjacent to the second cell type.

[00151] The compound can be a protein and/or a compound that is known to affect cell response.

The condition (s) may affect the cells in various different ways. Thus, the response of the cells to the condition (s) that is assessed in the method may include adherence of cells to the electric cell-substrate impedance sensing electrode array 101, adherence of cells to cells on the electric cell-substrate impedance sensing electrode array, diapedesis of cells through the cells on the electric cell-substrate impedance sensing electrode array, binding of molecules to cell surface receptors, permeability, a change in cell permeability, a change in cell density, a change in cell layer thickness, a change in a signal transduction pathway, cell death, a change in cell viability, a change in cell proliferation, a change in cell attachment, a change in cell migration, a change in cell micromotion and/or contraction, a change in cell spreading, a change in wound healing, a change in cell invasation and extravasation, a change in the barrier function of the cell layer, a change in spacing between the ventral side of the cell and the substratum, or a change in cell membrane capacitance.

[00152] For example, the measured electrical impedance after applying the shear force for a period of time in the presence of the pharmaceutical or the compound can be compared to the electrical impedance after applying the shear force for a period of time in the absence of the pharmaceutical or the compound to determine the effect of the pharmaceutical or compound on one or more of the cell types.

[00153] Assessing cell response to the condition (s) can suitably include analyzing the cells (e.g., the cells of the first and/or second type) after the shear forces have been applied for a period of time.

[00154] Assessing cell response to the condition (s) can also include analyzing the culture media. For example, the cell culture media can be analyzed to assess cytokine or humoral factor secretion.

[00155] Assessing cell response to the condition (s) can also include assessing adherence of cells that were suspended in the cell culture media to the electric cell-substrate impedance sensing electrode array 101, adherence of cells that were suspended in the cell culture media to cells that were plated on the electric cell-substrate impedance sensing electrode array, or diapedesis of cells that were suspended in the cell culture media through cells on the electric cell-substrate impedance sensing electrode array.

[00156] Assessing cell response to the condition (s) can suitably include culturing any or all of the cell types that are present .

[00157] When introducing elements of the present invention of the preferred embodiments thereof, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements. The terms "comprising", "including", and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements . [00158] In view of the foregoing, it will be seen that the several objects of the invention are achieved and other advantageous results attained.

[00159] As various changes could be made in the above constructions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

Claims

We claim:

1. A system for characterizing the response of cells to a condition, the system comprising:

a container for holding cultured cells; and

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads, the

contact pads, electrodes and electrical conductors being supported by the substrate;

one or more electrically insulating materials covering the electrical conductors, the one or more electrically insulating materials having one or more openings on the

electrodes so the electrodes are not insulated by the insulating materials at the openings,

the electric cell-substrate impedance sensing electrode array extending through a sidewall of the container so the electrodes are positioned inside the container and the contact pads are positioned outside the container.

2. A system as recited in claim 1 further comprising a system for exposing cells cultured in the container to fluid shear forces.

3. A system as recited in claim 2 wherein the system for exposing cells to fluid shear forces comprises a body and a motor adapted to rotate the body while the body extends into the container .

4. A system as recited in claim 3 wherein the body has a conical surface.

5. A system for characterizing the response of cells to a condition, the system comprising:

a container for holding cultured cells;

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

the electric cell-substrate impedance sensing electrode array being positioned so the electrodes are positioned inside the

container and the contact pads are positioned outside the container; and

a system for exposing cells in the container to fluid shear forces comprising a body adapted for being positioned in the container and a motor adapted to rotate the body.

6. A system as set forth in claim 5 wherein the body has a conical surface.

7. A system as set forth in claim 6 wherein the system for exposing cells to fluid shear forces is adapted for positioning the conical surface of the body in the container and so the conical surface is at the bottom of the body.

8. A system as recited in any one of claims 1-7 wherein the electric cell-substrate impedance sensing electrode array has a substantially flat upper surface.

9. A system as recited in any one of claims 1-8 wherein the electric cell-substrate impedance sensing array is configured so the electrodes are not divided into distinct wells by the electric cell-substrate impedance sensing array.

10. A method of characterizing the response of cells to a condition, the method comprising:

(a) placing an electric cell-substrate impedance sensing electrode array in a container, the electric cell-substrate impedance sensing electrode array comprising

a substrate;

and

the electric cell-substrate impedance sensing electrode array being positioned so it extends through a sidewall of the container so the electrodes are positioned inside the container and the contact pads are positioned outside the container,

(b) culturing cells in the container;

(c) subjecting the cells to fluid shear forces while they are in the container; and (d) using an electric cell-substrate impedance sensing instrument connected to the contact pads of the electrode array to measure electrical impedance between pairs of electrodes of the electrode array after the cells have been subjected to the condition .

11. A method as recited in claim 10 wherein subjecting the cells to fluid shear forces comprises rotating a body extending into a fluid culture medium contained in the container .

12. A method of characterizing the response of cells to a condition, the method comprising:

(a) placing an electric cell-substrate impedance sensing electrode array in a container, the electrode array comprising:

a substrate;

and

the electric cell-substrate impedance sensing electrode array being positioned so the electrodes are positioned inside the container and the contact pads are positioned outside the container,

(b) culturing cells in the container; (c) rotating a body in the container and subjecting the cells to fluid shear forces produced by the rotating body while the cells are in the container; and

(d) using an electric cell-substrate impedance sensing instrument connected to the contact pads of the ECIS electrode array to measure electrical impedance between pairs of electrodes of the electrode array after the cells have been subjected to the condition.

13. A method as recited in any one of claims 11 and 12 wherein the body has a conical lower surface.

14. A system for characterizing the response of cells to a condition, the system comprising:

a plurality of electric cell-substrate impedance sensing sample testing stations, each sample testing station comprising:

a container for holding cultured cells; and

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

and

the electrode array being positioned to extend through a sidewall of the container so the electrodes are positioned inside the

container and the contact pads are positioned outside the container; and

an electric cell-substrate impedance sensing analyzer electrically connected to the contact pads of the electrode array at each of the sample testing stations, the electric cell- substrate impedance sensing analyzer being adapted to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station .

15. A system as recited in claim 14 where at least one electric cell-substrate impedance sensing sample station further comprises a system for exposing cells cultured in the container to fluid shear forces.

16. A system as recited in 15 wherein the system for exposing cells to fluid shear forces comprises a body and a motor adapted to rotate the body while the body extends into the container .

17. A system for characterizing the response of cells to a condition, the system comprising:

a container for holding cultured cells; and

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

a plurality of contact pads, a plurality of

electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads, the

one or more electrically insulating materials covering the electrical conductors,

the one or more electrically insulating materials having one or more openings on the

electrodes so the electrodes are not insulated by the insulating materials at the openings, the electrode array being positioned so the electrodes are positioned inside the container and the contact pads are positioned outside the container, and

a system comprising a body and a motor

adapted to rotate the body while the body extends into the container to subject cells

in the container to fluid shear forces; and

an electric cell-substrate impedance sensing analyzer electrically connected to the contact pads of the electrode array at each of the sample testing stations, the electric cell- substrate impedance sensing analyzer being adapted to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station.

18. A system as recited in any one of claims 16 and 17 wherein the body has a conical surface.

19. A system as recited in any one of claims 14-18 wherein there are 2-3 sample testing stations.

20. A system as recited in any one of claims 14-18 wherein there are 4-12 sample testing stations.

21. A system as recited in any one of claims 14-18 wherein there are 8-12 sample testing stations.

22. A system as recited in any one of claims 14-21 wherein the electric cell-substrate impedance sensing array for at least one sample station is configured so its electrodes are not divided into distinct wells.

23. A system as recited in any one of claims 14-22 wherein the electrode array for at least one sample station has a substantially flat upper surface.

24. A method of characterizing the response of cells to a condition, the method comprising:

culturing cells at a plurality of electric cell-substrate impedance sensing sample testing stations, each ECIS sample testing station comprising:

a container for culturing cells; and

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

the electrode array extending through a sidewall of the container so the electrodes are positioned inside the container and the contact pads are positioned outside the container,

the method further comprising using an electric cell- substrate impedance sensing analyzer electrically connected to the contact pads of the electrode array at each of the sample testing stations to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station after the cells have been exposed to the condition .

25. A method as recited in 24 further comprising exposing cells cultured in the containers to fluid shear forces.

26. A system as recited in 25 wherein exposing the cells to fluid shear forces comprises rotating a body positioned to extend into a fluid culture medium contained in the container.

27. A method of characterizing the response of cells to a condition, the method comprising:

(a) culturing cells at a plurality of electric cell- substrate impedance sensing sample testing stations, each sample testing station comprising:

a container for culturing cells; and

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

and

the electrode array being positioned so the electrodes are positioned inside the container and the contact pads are positioned outside the container; and (b) rotating a body in the container to subject the cells in the container to fluid shear forces; and

(c) using an electric cell-substrate impedance sensing analyzer electrically connected to the contact pads of the ECIS electrode array at each of the sample testing stations to selectively energize the electrodes to measure electrical impedance between electrode pairs at each sample testing station after the cells have been exposed to the condition.

28. A method as recited in any one of claims 24-27 wherein there are 2-3 sample testing stations.

29. A method as recited in any one of claims 24-27 wherein there are 4-12 sample testing stations.

30. A method as recited in any one of claims 24-27 wherein there are 8-12 sample testing stations.

31. A method as recited in any one of claims 10-13 and 24-30 wherein culturing the cells comprises culturing a layer of cells adhered to the upper surface of the electrode array, the layer of cells extending over multiple electrodes of the electrode array.

32. A method as recited in any one of claims 10-13 and 24-31 wherein culturing the cells comprises culturing the cells in fluid culture medium that extends continuously over multiple electrodes of the electrode array.

33. A system as recited in any one of claims 24-32 wherein the body has a conical lower surface.

34. A system for characterizing the response of cells to a condition, the system comprising:

a container for holding culture media and optionally cultured cells; a porous membrane positioned in the container to separate the container into an upper volume and a lower volume, the porous membrane being suitable for plating cells thereon;

an electric cell-substrate impedance sensing electrode array comprising:

a substrate;

a plurality of contact pads, a plurality of electrodes, and a plurality of electrical conductors connecting the electrodes to corresponding ones of the contact pads, the contact pads, electrodes and electrical conductors being supported by the substrate ;

one or more electrically insulating materials covering the electrical conductors, the one or more electrically insulating materials having one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings, the electrodes being positioned inside the container in the lower volume and the contact pads being positioned outside the container.

35. A method for characterizing the response of cells to a condition, the method comprising:

adding a culture media to a container;

plating a first cell type on a first side of a porous membrane or plating a second cell type on a second side of the porous membrane, wherein said porous membrane is suspended in the container such that the first side is proximal and in spaced relation to a surface of the container, thereby defining within the container a lower volume comprising the first cell type or an upper volume comprising the second cell type, the porous membrane being adapted to permit fluid communication of the culture media; optionally plating a third cell type on the surface of the container;

perfusing culture media into and out of the upper volume; perfusing culture media into and out of the lower volume; optionally suspending a fourth cell type in the culture media in the upper volume;

optionally suspending a fifth cell type in the culture media in the lower volume;

wherein all of the cell types are within the culture media;

exposing one or more of the cell types that are present to the conditions; and

using an electric cell-substrate impedance sensing electrode array to measure electrical impedance, the electric cell-substrate impedance sensing electrode array comprising:

a substrate;

one or more electrically insulating materials covering the electrical conductors, the one or more electrically insulating materials having one or more openings on the electrodes so the electrodes are not insulated by the insulating materials at the openings, the electrodes being positioned inside the container, the electrical impedance being measured between electrode pairs of the electrodes of the electric cell-substrate impedance sensing electrode array to determine the response of one or more of the cell types to the conditions.

36. A method of claim 35 wherein cells of the first cell type are present.

37. A method of claim 35 wherein cells of the second type are present.

38. A method of claim 35 wherein cells of the first cell type and cells of the third cell type are present.

39. A method of claim 35 wherein cells of the second cell type and cells of the third cell type are present.

40. A method of claim 35 wherein cells of the first cell type, cells of the second cell type and cells of the third cell type are present.

41. A method of claim 35 wherein cells of the first cell type, cells of the second cell type, cells of the third cell type and cells of the fourth cell type are present.

42. A method of claim 35 wherein cells of the first cell type, cells of the second cell type, cells of the third cell type and cells of the fifth cell type are present.

43. A method of claim 35 wherein cells of the first cell type, cells of the third cell type and cells of the fourth cell type are present.

44. A method of claim 35 wherein cells of the first cell type, cells of the third cell type and cells of the fifth cell type are present.

45. A method of claim 35 wherein cells of the second cell type, cells of the third cell type, and cells of the fifth cell type are present.

46. A method of claim 35 wherein cells of the second cell type, cells of the third cell type, and cells of the fourth cell type are present.

47. A method of claim 35 wherein cells of the second cell type, cells of the third cell type, cells of the fourth cell type and cells of the fifth cell type are present.

48. A method of claim 35 wherein cells of the first cell type, cells of the third cell type, cells of the fourth cell type and cells of the fifth cell type are present.

49. A method of claim 35 wherein cells of the first cell type, cells of the second cell type and cells of the fourth cell type are present.

50. A method of claim 35 wherein cells of the first cell type, cells of the second cell type and cells of the fifth cell type are present.

51. A method of claim 35 wherein cells of the first cell type and cells of the fifth cell type are present.

52. A method of claim 35 wherein cells of the second cell type and cells of the fifth cell type are present.

53. A method of claim 35 wherein cells of the first cell type and cells of the second cell type are present.

54. A method of claim 35 wherein cells of the first cell type and cells of the fourth cell type are present.

55. A method of claim 35 wherein cells of the second cell type and cells of the fourth cell type are present.

56. A method of claim 35 wherein cells of the first cell type, cells of the second cell type, cells of the fourth cell type and cells of the fifth cell type are present.

57. A method of claim 35 wherein cells of the first cell type, cells of the fourth cell type, and cells of the fifth cell type are present.

58. A method of claim 35 wherein cells of the second cell type, cells of the fourth cell type and cells of the fifth cell type are present.

59. A method of claim 35 wherein cells of the first cell type, cells of the second cell type, cells of the third cell type, cells of the fourth cell type, and cells of the fifth cell type are present.

60. A method of any one of claims 35 to 59 wherein the cells of the third cell type are adhered to an upper surface of the electric cell-substrate impedance sensing electrode array.

61. A method of claim 60 wherein the cells of the third cell type adhered to the upper surface of the electric cell- substrate impedance sensing electrode array extend over multiple electrodes of the electric cell-substrate impedance sensing electrode array.

62. A method of any one of claims 35 to 61 wherein the conditions comprise a shear force applied to the cells in the upper volume or to a surface of the porous membrane in the upper volume of the container.

63. A method of claim 62 wherein the shear force mimics hemodynamic flow.

64. A method of claim 63 wherein said hemodynamic flow is time-variant .

65. A method of claim 63 wherein the hemodynamic flow is derived from a previously measured hemodynamic pattern.

66. A method of claim 65 wherein the previously measured hemodynamic pattern is derived from an animal.

67. A method of claim 66 wherein the animal is a genetically modified animal.

68. A method of claim 66 wherein the animal is a human.

69. A method of any one of claims 65-68 wherein said pattern is derived from a subject or subjects having a pathological condition or a disease-promoting condition.

70. A method of claim 69, wherein the disease-promoting condition comprises atrophy, calculi, choristoma, pathologic constriction, pathologic dilation, diverticulum, hypertrophy, polyps, prolapse, rupture, an arteriovenous fistula, or an appendage .

71. A method of any one of claims 65 to 70 wherein the hemodynamic pattern is derived from at least a portion of an artery, an arteriole, a vein or an organ.

72. A method of any one of claims 65 to 71, wherein the hemodynamic pattern is derived from an artery or an arteriole and is optionally atheroprone or atheroprotective .

73. A method of claim 71 or 72, wherein the hemodynamic pattern is derived from at least a portion of an artery, the artery comprising a carotid artery, thoracic artery, abdominal artery, pulmonary artery, a femoral artery, renal efferent artery, renal afferent artery, a coronary artery, a brachial artery, a internal mammary artery, a cerebral artery, the aorta, a precapillary arteriole, a coronary artery, hepatic artery, anterior cerebral artery, middle cerebral artery, posterior cerebral artery, basilar artery, external carotid artery, internal carotid artery, vertebral artery, subclavian artery, aortic arch, axillary artery, internal thoracic artery, branchial artery, deep branchial artery, radial recurrent artery, superior epigastric artery, descending aorta, inferior epigastric artery, interosseous artery, radial artery, ulnar artery, palmar carpal arch, dorsal carpal arch, superficial/deep palmar arches, digital artery, descending branch of the femoral circumflex artery, descending genicular artery, superior genicular arteries, inferior genicular arteries, anterior tibial artery, posterior tibial artery, peroneal artery, deep plantar arch, arcuate artery, common carotid arteries, intercostal arteries, left/right gastric artery, celiac trunk, splenic artery, common hepatic artery, superior mesenteric artery, renal artery, inferior mesenteric artery, testicularis artery, common iliac artery, internal iliac artery, external iliac artery, femoral circumflex artery, perforating branches, deep femoral artery, popliteal artery, dorsal metatarsal artery, or dorsal digital arteries.

74. A method of claim 71, wherein the hemodynamic pattern is derived from at least a portion of vein, the vein comprising a post-capillary venule, saphenous vein, hepatic portal vein, superior vena cava, inferior vena cava, coronary vein, Thesbian vein, superficial vein, perforator vein, systemic vein, pulmonary vein, jugular vein, sigmoid sinus, external jugular vein, internal jugular vein, inferior thyroid vein, subclavian vein, internal thoracic vein, axillary vein, cephalic vein, branchial vein, intercostal vein, basilic vein, median cubital vein, thoracoepigastric vein, ulnar vein, median antebranchial vein, inferior epigastric vein, deep palmar arch, superficial palmar arch, palmar digital veins, inferior vena cava, hepatic vein, renal vein, abdominal vena cava, testicularis vein, common iliac vein, perforating branches, external iliac vein, internal iliac vein, external pudendal vein, deep femoral vein, great saphenous vein, femoral vein, accessory saphenous vein, superior genicular vein, popliteal vein, inferior genicular vein, great saphenous vein, small saphenous vein, anterior/posterior tibial vein, deep plantar vein, dorsal venous arch, or dorsal digital vein.

75. A method of claim 71, wherein the hemodynamic pattern is derived from at least a portion of an organ, the organ comprising a liver, a kidney, a lung, a brain, a pancreas, a spleen, a large intestine, a small intestine, a heart, a skeletal muscle, an eye, a tongue, a

reproductive organ, or an umbilical cord.

76. A method of claim 75, wherein the hemodynamic pattern is derived from a chamber of the heart, a cardiac sinus, a left atrial appendage during sinus rhythm, an atrial fibrillation, or a ventricular fibrillation.

77. A method of claim 76, wherein the hemodynamic pattern is derived from a chamber of the heart, the chamber of the heart comprising a left atrium, a right atrium, a left ventricle or a right ventricle.

78. A method of any one of claims 65 to 77, wherein the hemodynamic pattern results from a physical change resulting from a pathological condition.

79. A method of any one of claims 65 to 77, wherein the hemodynamic pattern has been altered as a direct or indirect effect of administration of a pharmaceutical to a subject as compared to the flow or the hemodynamic pattern for the subject absent administration of the pharmaceutical.

80. A method of any one of claims 65 to 79 wherein said hemodynamic pattern is derived from analysis of ultrasound data.

81. A method of any one of claims 65 to 80 wherein said hemodynamic pattern is derived from analysis of magnetic resonance imaging (MRI) data.

82. A method of any one of claims 62 to 81 wherein the shear force is induced by a system comprising a body adapted for being positioned in the culture media in the upper volume of the container and a motor adapted to rotate the body.

83. A method of claim 82 wherein the body has a conical surface .

84. A method of claim 83 wherein the system is adapted for positioning the conical surface of the body in the container and so the conical surface is at the bottom of the body.

85. A method of any one of claims 35 to 84 wherein the conditions comprise presence of a pharmaceutical or a compound.

86. A method of any one of claims 35 to 84 wherein the conditions comprise a concentration of a pharmaceutical or a compound .

87. A method of claim 85 or 86 wherein the pharmaceutical or the compound is perfused into at least one of the upper volume and the lower volume.

88. A method of claim 87 wherein the pharmaceutical or the compound is added to the culture media while applying the shear force .

89. A method of claim 87 wherein the pharmaceutical or the compound is added to the culture media before applying the shear force .

90. A method of any one of claims 85 to 89 wherein the pharmaceutical is a cyclooxygenase inhibitor; a taxane; a tyrosine kinase inhibitor; a low molecular weight heparin; an anti-thrombogenic agent; a calcium channel blocker; an anti- platelet agent; an anticlotting agent; a chelating agent; an anti-inflammatory agent; a rho kinase inhibitor; a PDGF inhibitor, a cholesterol lowering agent; an anti-restenosis agent; an antibiotic; an anti-neoplastic agent; an anti^¬ hypertensive agent; a synthetic polysaccharide; an agent that raises HDL; or a combination thereof.

91. A method of claim 90, wherein the cyclooxygenase inhibitor is celecoxib; the taxane is paclitaxel; the tyrosine kinase inhibitor is imatinib; the low molecular weight heparin is enoxaparin; the anti-thrombogenic agent is bivalirudin, dipyridamole, urokinase, r-urokinase, r-prourokinase, reteplase, alteplase, streptokinase, rt-PA, TNK-rt-PA, monteplase, staphylokinase, pamiteplase, unfractionated heparin, or APSAC; the calcium channel blocker is amlodipine or nifedipine; the anti-platelet agent is clopidogrel, abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban or ticlopinin; the anticlotting agent is fondaparinux; the chelating agent is penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA, deferoxamine mesylate or batimastat; the anti-inflammatory agent is rofecoxib; the rho kinase inhibitor is Y27632; the PDGF inhibitor is AG1295; the cholesterol lowering agent is a statin; the antibiotic is actinomycin-D; the anti-neoplastic agent is c- myc antisense or dexamethasone ; or the anti-hypertensive agent is an ACE inhibitor.

92. A method of any one of claims 85 to 91 wherein the pharmaceutical is atorvastatin, sirolimus, tacrolimus, everolimus, wortmannin, or a combination thereof.

93. A method of any one of claims 85 to 91 wherein the pharmaceutical or the compound is a radiocontrast agent, a radio-isotope, a propharmaceutical, an antibody fragment, an antibody, a live cell, a therapeutic pharmaceutical delivery microsphere or microbead, nanoparticle, gel or cell-impregnated gel, or a combination thereof.

94. A method of any one of claims 85 to 89 wherein the drug is an anti-inflammatory agent, an anti-neoplastic agent, an anti-diabetic agent, a protein kinase inhibitor, an anti^¬ thrombotic agent, a thrombolytic agent, an anti-platelet agent, an anticoagulant, a calcium channel blocker, a chelating agent, a rho kinase inhibitor, an antihyperlipidemic agent, an agent that raises HDL, an anti-restenosis agent, an antibiotic, an immunosuppressant, an anti-hypertensive agent, a diuretic, an anorectic, an appetite suppressant, an anti-depressant, an anti- psychotic, a contraceptive, a calcimimetic, a biologic medical product, or a combination thereof.

95. A method of claim 94, wherein the drug is an anti^¬ inflammatory agent, the antiinflammatory agent comprising a steroid, a non-steroidal anti-inflammatory drug (NSAID) , a selective cyclooxygenase inhibitor, a non-selective cyclooxygenase inhibitor, an immune selective anti-inflammatory agent, or a combination thereof.

96. A method of claim 95, wherein the anti-inflammatory agent comprises the steroid, the steroid comprising prednisone, hydrocortisone, prednisolone, betamethasone, or dexamethasone .

97. A method of claim 95, wherein the anti-inflammatory agent comprises the non-steroidal anti-inflammatory drug, the non-steroidal anti-inflammatory drug comprising a salicylate, ibuprofen, acetaminophen, naproxen, or ketoprofen.

98. A method of claim 95, wherein the anti-inflammatory agent comprises the selective cyclooxygenase inhibitor, the selective cyclooxygenase inhibitor comprising celecoxib or rofecoxib .

99. A method of claim 95, wherein the anti-inflammatory agent comprises the immune selective anti-inflammatory agent, the immune selective anti-inflammatory agent comprising phenylalanine-glutamine-glycine tripeptide .

100. A method of claim 94, wherein the drug comprises an anti-neoplastic agent, the anti-neoplastic agent comprising an alkylating agent, an anti-metabolite, a plant alkaloid, a topoisomerase inhibitor, a cytotoxic antibiotic, or a combination thereof.

101. A method of claim 100, wherein the anti-neoplastic agent comprises the alkylating agent, the alkylating agent comprising cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucide or ifosfamide.

102. A method of claim 100, wherein the anti-neoplastic agent comprises the anti-metabolite, the anti-metabolite comprising azathioprine or mercaptopurine.

103. A method of claim 100, wherein the anti-neoplastic agent comprises the plant alkaloid, the plant alkaloid comprising a taxane, a vinca alkaloid, or a podophyllotoxin .

104. A method of claim 103, wherein the plant alkaloid comprises the taxane, the taxane comprising paclitaxel or docetaxel .

105. A method of claim 103, wherein the plant alkaloid comprises the vinca alkaloid, the vinca alkaloid comprising vincristine, vinblastine or vindesine.

106. A method of claim 103, wherein the plant alkaloid comprises the podophyllotoxin, the podophyllotoxin comprising etoposide or teniposide.

107. A method of claim 100, wherein the anti-neoplastic agent comprises the topoisomerase inhibitor, the topoisomerase inhibitor comprising irinotecan, topotecan or amsacrine.

108. A method of claim 100, wherein the anti-neoplastic agent comprises the cytotoxic antibiotic, the cytotoxic antibiotic comprising actinomycin, bleomycin, plicamysin, mitomycin, doxorubicin, daunorubicin, valrubicin, idarubicin or epirubicin .

109. A method of claim 94, wherein the drug is an anti^¬ diabetic agent, the anti-diabetic agent comprising a biguanide, a thiazolidinedione, a sulfonylurea, an incretin mimetic, a dipeptidyl peptidase IV inhibitor, a sodium-glucose co- transporter 2 inhibitor, or a glucokinase activator.

110. A method of claim 109, wherein the anti-diabetic agent comprises the biguanide, the biguanide comprising metformin.

111. A method of claim 109, wherein the anti-diabetic agent comprises the thiazolidinedione, the thiazolidinedione comprising rosiglitazone, troglitazone or pioglitazone .

112. A method of claim 109, wherein the anti-diabetic agent comprises the sulfonylurea, the sulfonylurea comprising tolbutamine, acetohexamide, tolazamide, chlorpropamide, glipazide, glyburide, glimepiride, gliclazide, glycopyramide or gliquidone.

113. A method of claim 109, wherein the anti-diabetic agent comprises the incretin mimetic, the incretin mimetic comprising exenatide, liraglutide or taspoglutide .

114. A method of claim 109, wherein the anti-diabetic agent comprises the dipeptidyl peptidase IV inhibitor, the dipeptidyl peptidase IV inhibitor comprising vildagliptin, sitagliptin, saxaglitpin, linagliptin, allogliptin or septagliptin .

115. A method of claim 109, wherein the anti-diabetic agent comprises the sodium-glucose co-transporter 2 inhibitor, the sodium-glucose co-transporter 2 inhibitor comprising dapagliflozin, canagliflozin, empagliflozin, ipragliflozin, remogliflozin or sergliflozin .

116. A method of claim 109, wherein the anti-diabetic agent comprises the glucokinase activator, the glucokinase activator comprising piragliatin.

117. A method of claim 94, wherein the drug comprises a protein kinase inhibitor, the protein kinase inhibitor comprising a serine/threonine-specific kinase inhibitor, a tyrosine-specific kinase inhibitor, an epidermal growth factor

(EGF) receptor inhibitor, a fibroblast growth factor (FGF) receptor inhibitor, a platelet-derived growth factor (PDGF) receptor inhibitor, or a vascular endothelial growth factor

(VEGF) receptor inhibitor.

118. A method of claim 117, wherein the protein kinase inhibitor comprises the tyrosine-specific kinase inhibitor, the tyrosine-specific kinase inhibitor comprising imatinib, bevacizumab, cetuximab, axitinib, lapatinib, ruxolitinib or sorafenib .

119. A method of claim 94, wherein the drug comprises the anti-thrombotic agent, the anti-thrombotic agent comprising dipyridamole, urokinase, r-urokinase, r-prourokinase, reteplase, alteplase, streptokinase, rt-PA, TNK-rt-PA, monteplase, staphylokinase, pamiteplase, unfractionated heparin or APSAC.

120. A method of claim 94, wherein the drug comprises the thrombolytic agent, the thrombolytic agent comprising a streptokinase, a urokinase or a tissue plasminogen activator.

121. A method of claim 94, wherein the drug comprises the anti-platelet agent, the antiplatelet agent comprising a glycoprotein Ilb/IIIa inhibitor, a thromboxane inhibitor, an adenosine diphosphate receptor inhibitor, a prostaglandin analogue, or a phosphodiesterase inhibitor.

122. A method of claim 94, wherein the drug comprises the anti-platelet agent, the antiplatelet agent comprising clopidogrel, abciximab, tirofiban, orbofiban, xemilofiban, sibrafiban, roxifiban or ticlopinin.

123. A method of claim 94, wherein the drug comprises the anti-coagulant, the anticoagulant comprising a vitamin K antagonist, a factor Xa inhibitor or a direct thrombin inhibitor .

124. A method of claim 123, wherein the anti-coagulant comprises the vitamin K antagonist, the vitamin K antagonist comprising warfarin.

125. A method of claim 123, wherein the anti-coagulant comprises the factor Xa inhibitor, the factor Xa inhibitor comprising apixaban, betrixaban, edoxaban, otamixaban, rivaroxaban, fondaparinux or idraparinux.

126. A method of claim 123, wherein the anti-coagulant comprises the direct thrombin inhibitor, the direct thrombin inhibitor comprising hirudin, bivalirudin, lepirudin, desirudin, dabigatran, ximelagatran, melagatran or argatroban.

127. A method of claim 94, wherein the drug comprises the calcium channel blocker, the calcium channel blocker comprising verapamil, diltiazem, amlodipine, aranidipine, azelnidipine, barnidipine, benidipine, cilnidipine, clevidipine, isradipine, efonidipine, felodipine, lacidipine, lercanidipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nisoldipine, nitrendipine or pranidipine.

128. A method of claim 94, wherein the drug comprises the chelating agent, the chelating agent comprising penicillamine, triethylene tetramine dihydrochloride, EDTA, DMSA, deferoxamine mesylate or batimastat.

129. A method of claim 94, wherein the drug comprises the rho kinase inhibitor, the rho kinase inhibitor comprising Y27632.

130. A method of claim 94, wherein the drug comprises the anti-hyperlipidemic agent, the anti-hyperlipidemic agent comprising a statin, a fibrate, a selective inhibitor of dietary cholesterol absorption, or a cholesterylester transfer protein inhibitor.

131. A method of claim 130, wherein the anti-hyperlipidemic agent comprises the statin, the statin comprising atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin or simvastatin.

132. A method of claim 130, wherein the anti-hyperlipidemic agent comprises the fibrate, the fibrate comprising bezafibrate, ciprofibrate, clofibrate, gemfibrozil or fenofibrate.

133. A method of claim 130, wherein the anti-hyperlipidemic agent comprises the selective inhibitor of dietary cholesterol absorption, the selective inhibitor of dietary cholesterol absorption comprising ezetimibe.

134. A method of claim 130, wherein the anti-hyperlipidemic agent comprises the cholesterylester transfer protein inhibitor, the cholesterylester transfer protein inhibitor comprising anacetrapib, dalcetrapib, torcetrapib or evacetrapib.

135. A method of claim 94, wherein the drug comprises the agent that raises HDL, the agent that raises HDL comprising an inhibitor of proprotein convertase subtilisin/kexin type 9 (PCSK9) .

136. A method of claim 135, wherein the PCSK9 inhibitor comprises AMG145.

137. A method of claim 94, wherein the drug is the anti- restenosis agent, the anti-restenosis agent comprising dexamethasone ticlopidine, clopidogrel, sirolimus, paclitaxel, zotarolimus, everolimus, or umirolimus.

138. A method of claim 94, wherein the drug comprises the antibiotic, the antibiotic comprising actinomycin-D .

139. A method of claim 94, wherein the drug comprises the immunosuppressant, the immunosuppressant comprising a glucocorticoid, methotrexate, azathioprine, mercaptopurine, dactinomycin, mitomycin C, bleomycin, mithramycin, ciclosporin, tacrolimus, sirolimus, an interferon, infliximab, etanercept or adalimumab .

140. A method of claim 94, wherein the drug comprises the anti-hypertensive agent, the anti-hypertensive agent comprising a beta adrenergic receptor antagonist, an angiotensin II receptor antagonist, or an angiotensin converting enzyme inhibitor .

141. A method of claim 140, wherein the anti-hypertensive agent comprises the beta adrenergic receptor antagonist, the beta adrenergic receptor antagonist comprising alprenolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, oxprenolol, penbutalol, pindolol, propranolol, sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol, metoprolol or nebivolol .

142. A method of claim 140, wherein the anti-hypertensive agent comprises the angiotensin II receptor antagonist, the angiotensin II receptor antagonist comprising losartan, olmesartan, valsartan, telmisartan, irbesartan, or azilsartan.

143. A method of claim 140, wherein the anti-hypertensive agent comprises the angiotensin converting enzyme inhibitor, the angiotensin converting enzyme inhibitor comprising captopril, enalapril, lisinopril, quinapril, zofenopril, imidapril, benazepril, trandolapril or ramipril.

144. A method of claim 94, wherein the drug comprises the diuretic, the diuretic comprising furoseamide, amiloride, spironolactone, or hydrochlorothiazide.

145. A method of claim 94, wherein the drug comprises the anorectic, the anorectic comprising phentermine, fenfluramine, dexfenfluramine, sibutramine, lorcaserin, topiramate, or a combination thereof.

146. A method of claim 94, wherein the drug comprises the anti-depressant, the antidepressant comprising imipramine, desipramine, amitryptiline, paroxetine, citalopram, fluoxetine, or escitalopram.

147. A method of claim 94, wherein the drug comprises the anti-psychotic, the antipsychotic comprising aripiprazole, risperidone, olanzapine, quetiapine, cariprazine, lurasidone or asenopine .

148. A method of claim 94, wherein the drug comprises the contraceptive, the contraceptive comprising a combination of drospirenone and ethinyl estradiol.

149. A method of claim 94, wherein the drug comprises the calcimimetic, the calcimimetic comprising cinacalcet.

150. A method of claim 94, wherein the drug comprises the biologic medical product, the biologic medical product comprising a synthetic polysaccharide, a synthetic, partially synthetic or humanized immunoglobulin, or a recombinant therapeutic protein.

151. A method of any one of claims 85 to 150 wherein the compound is capable of inhibiting, activating or altering the function of proteins or genes in said cell types.

152. A method of any one of claims 85 to 151 wherein the compound is to be evaluated for elution from a vascular stent material, and the method further comprises testing at least one of the cell types for compatibility with, cellular adhesion to, or phenotypic modulation by the vascular stent material.

153. A method of claim 152, wherein the vascular stent material comprises a nanoporous metal, a polymer, or a carbon material .

154. A method of claim 85 to 153, wherein the pharmaceutical or compound is eluted from a vascular stent material adjacent to the second cell type.

155. A method of any one of claims 85 to 154 wherein the compound is a protein.

156. A method of any one of claims 85 to 155 wherein the compound is known to affect cell response.

157. A method of any one of claims 35 to 156 wherein the response of one or more of the cell types to the conditions comprises adherence of cells to the electric cell-substrate impedance sensing electrode array, adherence of cells to cells on the electric cell-substrate impedance sensing electrode array, diapedesis of cells through the cells on the electric cell-substrate impedance sensing electrode array, binding of molecules to cell surface receptors, a change in cell permeability, a change in cell density, a change in cell layer thickness, a change in a signal transduction pathway, cell death, a change in cell viability, a change in cell proliferation, a change in cell attachment, a change in cell migration, a change in cell micromotion and/or contraction, a change in cell spreading, a change in wound healing, a change in cell invasation and extravasation, a change in the barrier function of the cell layer, a change in spacing between the ventral side of the cell and the substratum, or a change in cell membrane capacitance.

158. A method of claim 157 wherein the response comprises adherence of cells to the electric cell-substrate impedance sensing electrode array.

159. A method of any one of claims 62 to 158 wherein the conditions comprise the addition of the pharmaceutical or the compound and the application of the shear force, and the method further comprises comparing the measured electrical impedance after applying the shear force for a period of time in the presence of the pharmaceutical or the compound to the electrical impedance after applying the shear force for a period of time in the absence of the pharmaceutical or the compound, to determine the effect of the pharmaceutical or compound on one or more of the cell types.

160. A method of any one of claims 62 to 159, further comprising the step of analyzing at least one of the first cell type and the second cell type after applying the shear force for a period of time.

161. A method of any one of claims 35 to 160, further comprising analyzing said culture media for cytokine or humoral factor secretion.

162. A method of any one of claims 35 to 161, wherein the cells of the first cell type are present, and the cells of the first cell type are renal cells, cells of the airways, or cells of the blood-brain barrier.

163. A method of any one of claims 35 to 161, wherein the cells of the first cell type are present, and the cells of the first cell type are smooth muscle cells, glial cells, astrocytes, neurons, or epithelial podocytes.

164. A method of any one of claims 35 to 161, wherein the cells of the second cell type are present, and the cells of the second cell type are vascular cells.

165. A method of claim 164, wherein the vascular cells are endothelial cells.

166. A method of any one of claims 35 to 161, wherein the cells of the third cell type are present, and the cells of the third cell type are renal cells, cells of the airways, or cells of the blood-brain barrier.

167. A method of any one of claims 35 to 161, wherein the cells of the third cell type are present, and the cells of the third cell type are smooth muscle cells, glial cells, astrocytes, neurons, macrophages, or leukocytes.

168. A method of any one of claims 35 to 161, wherein the cells of the fourth cell type are present, and the cells of the fourth cell type are blood cells or immune

cells .

169. A method of any one of claims 35 to 161, wherein the cells of the fifth cell type are present, and the cells of the fifth cell type are blood cells or immune cells.

170. A method of claim 168 or 169, wherein the blood cells or immune cells are monocytes, macrophages, dendritic cells, red blood cells, neutrophils, lymphocytes, basophils, eosinophils, leukocytes, platelets or combinations thereof.

171. A method of any one of claims 35 to 170, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type comprises primary cells.

172. A method of claim 171, wherein the primary cells comprise a cell lineage derived from stem cells or stem-like cells .

173. A method of claim 172, wherein the primary cells comprise the cell lineage derived from stem cells, the cell lineage derived from stem cells comprising adult stem cells, embryonic stem cells, inducible pluripotent stem cells, or bone marrow-derived stem cells.

174. A method of claim 173, wherein the cell lineage derived from stem cells comprises endothelial cells, smooth muscle cells, cardiac myocytes, hepatocytes, neuronal cells, or endocrine cells.

175. A method of any one of claims 35 to 170, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type comprises immortalized cells.

176. A method of any one of claims 171 to 175, wherein the primary cells or the immortalized cells comprise cells isolated from at least one normal subject or at least one subject having a pathological condition, cells isolated from at least one subject having a risk factor for a pathological condition, cells isolated from at least one subject with a single nucleotide polymorphism linked to a pathological condition, cells isolated from at least one subject with an identified genotype linked to drug toxicity, or cells isolated from at least one subject with a single nucleotide polymorphism linked to drug toxicity.

177. A method of claim 176, wherein the primary cells or the immortalized cells comprise cells isolated from at least one subject having a risk factor for the pathological condition, the risk factor comprising smoking, age, gender, race, epigenetic imprinting, an identified genotype linked to the pathological condition, an identified single nucleotide polymorphism linked to the pathological condition, diabetes, hypertension, atherosclerosis, atherosclerosis plaque rupture, atherosclerosis plaque erosion, thoracic aortic aneurysm, abdominal aortic aneurysm, cerebral aneurysm, heart failure, stroke, Marfan Syndrome, carotid intima-medial thickening, atrial fibrillation, kidney disease, pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary artery disease, pulmonary hypertension, hyperlipidemia, familial hypercholesterolemia, peripheral artery disease, deep vein thrombosis, vascular restenosis, vascular calcification, myocardial infarction, obesity, hypertriglyceridemia, hypoalphalipoproteinemia, fatty liver disease, hepatitis C, hepatitis B, liver fibrosis, bacterial infection, viral infection, cirrhosis, liver fibrosis, or alcohol-induced liver disease.

178. A method of claim any one of claims 35 to 177, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type comprises renal cells, cells of the airways, blood-brain barrier cells, vascular cells, hepatic cells, pancreatic cells, cardiac cells, muscle cells, spleen cells, gastrointestinal tract cells, skin cells, liver cells, immune cells, or hematopoietic cells.

179. A method of any one of claims 35 to 178, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type comprises astrocytes, endothelial cells, glomerular fenestrated endothelial cells, renal epithelial podocytes, alpha cells, β- cells, delta cells, pancreatic polypeptide (PP) cells, epsilon cells, glial cells, hepatocytes, neurons, nonparenchymal hepatic cells, podocytes, smooth muscle cells, mesangial cells, pericytes cells, cardiac muscle cells, skeletal muscle cells, leukocytes, monocytes, myocytes, macrophages, neutrophils, dendritic cells, T-cells, B-cells, endothelial progenitor cells, stem cells, circulating stem cells, circulating hematopoietic cells, endocardial cells, fibroblasts, chondrocytes, or osteoblasts .

180. A method of claim 179, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type comprises the nonparenchymal hepatic cells, the nonparenchymal hepatic cells comprising hepatic stellate cells, sinusoidal endothelial cells, or Kupffer cells.

181. A method of any one of claims 35 to 180, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type comprises endothelial cells, smooth muscle cells, hepatocytes, or sinusoidal endothelial cells.

182. A method of claim any one of claims 35 to 181, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type, the primary cells, or the immortalized cells are cells from an animal .

183. A method of claim 182, wherein the cells from an animal are from a genetically modified animal.

184. A method of claim 182, wherein the cells from an animal are cells from a human.

185. The method of claim 184, wherein the cells from a human are selected on the basis of age, gender, race, epigenetics, disease, nationality, or the presence or absence of one or more single nucleotide polymorphisms.

186. The method of any one of claims 35 to 185, wherein at least one of the first cell type, the second cell type, the third cell type, the fourth cell type, and the fifth cell type is from a normal subject, a subject having diabetes, a hypertensive subject, a smoker, a subject having abdominal aortic aneurysm, a subject having fatty liver disease, an aged subject, or an animal genetically-modified to model diabetes, hypertension, fatty liver disease or aging, or modified to model abdominal aortic aneurysm.

187. A method of any one of claims 35 to 186, wherein the response of one or more of the cell types to the conditions comprises adherence of cells of the fourth cell type or cells of the fifth cell type to the electric cell-substrate impedance sensing electrode array, adherence of cells of the fourth cell type or cells of the fifth cell type to cells on the electric cell-substrate impedance sensing electrode array, or diapedesis of cells of the fourth cell type or cells of the fifth cell type through the cells on the electric cell-substrate impedance sensing electrode array.

188. A method of any one of claims 35 to 187, wherein at least one of the cell types that are present are vascular or organ cells from one or more patients with an identified genotype linked to pharmaceutical toxicity or a pathophysiological endpoint.

189. A method of claim 188, wherein said one or more patients have a single nucleotide polymorphism linked to pharmaceutical toxicity or a pathophysiological endpoint.

190. A method of any one of claims 35 to 189, wherein the cells of the first type and the cells of the second type are present, and the porous membrane is adapted to permit physical interaction and communication between cells of the first cell type and cells of the second cell type.

191. A method of any one of claims 35 to 190, further comprising the step of culturing all of the cell types present.

192. A method of any one of claims 35 to 191 wherein the electric cell-substrate impedance sensing electrode array has a substantially flat upper surface.

193. A method of any one of claims 35 to 192 wherein the electric cell-substrate impedance sensing array is configured so the electrodes are not divided into distinct wells by the electric cell-substrate impedance sensing array.

194. A method of any one of claims 35 to 192 wherein the contact pads are positioned outside the container.