WO2025006933A2 - High throughput transwell device incorporating shear stress for drug screening of blood brain barrier function - Google Patents

Abstract

An apparatus may include a multi-well plate defining a plurality of wells including a first well and a second well. The multi-well plate comprises a top plate, a bottom plate, and at least one support plate. A cell culture mesh is supported in between two support plates. The apparatus may include one or more shafts, cones, or fins that fit within the first well and/or the second well of the multi-well plate, the shafts being positioned in close proximity to the cell culture mesh. A rotational motor may be connected to drive the one or more shafts to rotate and create fluid flow or shear stress (e.g., oscillatory or constant) within the first and/or second well. The bottom plate defines a conduit or a lower chamber such that the first well is in fluid communication with the second well.

Description

HIGH THROUGHPUT TRANSWEUU DEVICE INCORPORATING SHEAR STRESS FOR DRUG SCREENING OF BLOOD BRAIN BARRIER FUNCTION

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0001] This invention was made with government support under Grant no. R01 HL141761 awarded by the National Institutes of Health. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application 63/510,762 filed June 28, 2023, which is incorporated herein by reference in its entirety.

BACKGROUND

[0003] The blood brain barrier (BBB) regulates the active and passive transport of molecules between the circulation and neural space. The core element of this intricate anatomical structure is the neurovascular unit (NVU) composed of microvascular endothelial cells interacting with other cell types including astrocytes, pericytes, neurons, and microglia. The BBB regulates transport into the neural space through specialized transport proteins and metabolic enzymes, facilitating homeostasis in the brain. The BBB is also a key structure in determining the access of pharmaceutical compounds to the brain. For therapeutic applications for the brain, the BBB is a major bottleneck in developing compounds that can treat neural disorders. For other applications, drug permeability through the BBB may be a major mode of toxicity and a mechanism of drug side effects. In addition, the BBB is involved mechanistically in many neural disorders including traumatic brain injury (TBI), Alzheimer’s disease (AD), and many other disorders of the brain.

[0004] Shear stress from blood flow plays an important role in regulating BBB barrier function and selective permeability, helping to mimic the function of the BBB in ex vivo models. Shear stress upregulates the expression of tight junction proteins of the BBB and increases transendothelial electrical resistance (TEER), both key factors in the selective permeability of the BBB9. Shear stress also increases the expression of multidrug resistance transporters (MRT), ion channels, and p450 enzymes, which are crucial for regulating molecule transport and maintaining homeostasis in the BBB. A limitation of many in vitro BBB models is that they are either low throughput or do not incorporate a physiological level of shear stress.

[0005] Thus, there is a need to integrate shear stress into cell culture models of the blood brain barrier to effectively recreate proper barrier function and selective molecule transport across the blood brain barrier.

SUMMARY

[0006] In some implementations, an apparatus is disclosed, the apparatus including: a multi-well plate defining a plurality of wells including a first well and a second well, the multi-well plate including a top plate, a bottom plate, and at least one support plate disposed between the top plate and the bottom plate. The at least one support plate includes a first support plate and a second support plate. A cell culture mesh is supported in between the first support plate and the second support plate. One or more shafts, cones, or fins fit within at least one of the first well and the second well of the multi-well plate and can be positioned in close proximity to the cell culture mesh. A rotational motor connected to drive the one or more shafts, cones, or fins to rotate, such that rotation of the shafts, cones, or fins creates fluid flow or shear stress (e.g., oscillatory or constant) within at least one of the first well and the second well. The bottom plate defines a conduit or a lower chamber such that the first well is in fluid communication with the second well.

[0007] In other implementations, the cell culture mesh is disposed in the first well while the second well is left open, wherein the cell culture mesh separates the first well into an upper chamber and the lower chamber, the lower chamber being in fluid communication with the second well.

[0008] In some implementations, the cell culture mesh is seeded with cells. In some implementations, the cells are endothelial cells or epithelial cells including lung, intestine, or mucosa cells. In some implementations, the cell culture mesh models a blood brain barrier (BBB), and the cells are endothelial cells found within the BBB. In some implementations, an apparatus, wherein cells are grown (i) on both sides the cell culture mesh, (ii) in the bottom of the first well or the second well, (iii) on the sides of the first well or the second well, and/or (iv) in gels placed within the first well or the second well. In some implementations, the cells migrate through the cell culture mesh (e.g., for transmigration assays, models for the immune system, viral/bacterial infection, or cancer metastasis).

[0009] In some implementations, an apparatus, wherein both of the upper chamber of the first well and the lower chamber via the second well are accessible from the top plate such that a standard robotic pipetting apparatus having standard well geometry may access and interact with either side of the cell culture mesh.

[0010] In some implementations, an apparatus, further including one or more electrodes configured to measure transendothelial electrical resistance (TEER) across the cell culture mesh, the one or more electrodes including a first electrode placed in the first well and a second electrode placed in the second well.

[0011] In some implementations, an apparatus, further including a detector capable of measuring cells within at least one of the first well and the second well during at least one of before, during, or after rotation of the one or more shafts, cones, or fins to cause fluid flow within the first well or the second well. In some implementations, the detector is a laser speckle imager capable of quantifying the velocity of fluid at or about the bottom of the first well or the second well, or the detector is at least one of plate reader, a cell sorter, a cell counter, a microscope, or a camera.

[0012] In some implementations, an apparatus, wherein the one or more shafts, cones, or fins are capable of fluid flow within the well of between 0.1 dyn/cm2 to 20 dyn/cm2, 0.5 dyn/cm2 to 15 dyn/cm2, 1.0 dyn/cm2 to 12 dyn/cm2, 0.2 dyn/cm2 to 12 dyn/cm2, and 5.0 dyn/cm2 to 12 dyn/cm2. In some implementations, an apparatus, wherein the flow is an oscillatory flow.

[0013] In some implementations, an apparatus further including a computer connected to at least one of the rotational motor, the detector, or both, and that includes one or more code segments that: calculate the fluid flow within each of the one or more wells; that measure cell death on exposure to fluid flow; or that measure cell adhesion.

[0014] In some implementations, an apparatus, wherein the multi-well plate includes a 2, 4, 6, 8, 10, 12, 24, 48, 96, 394, or 1536 well plate.

[0015] In some implementations, the one or more shafts, cones, or fins include at least one of biocompatible material, sterile, smooth, rough, trapezoidal, conical, flat, cut, or polygonal. In some implementations, the one or more shafts, cones, or fins include at least one of steel, steel alloy, stainless steel, titanium, plastic, polymer, glass, quartz, or wood. [0016] In some implementations, an apparatus, wherein the multi-well plate includes a polyacrylate, a polymethylacrylate, a polycarbonate, a polysulphone, a polyhydroxy acid, a polyanhydride, a polyorthoester, a polypropylene, a polyphosphazene, a polyphosphate, a polyester, a nylon or a mixture thereof.

[0017] Additional advantages will be set forth in part in the description which follows or may be learned by practice. The advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 shows a high-throughput cone-and-plate (HT-CAP) device, according to one implementation.

[0019] FIG. 2 shows a detailed diagram of the tip of a drive shaft of FIG. 1 including example dimensions.

[0020] FIG. 3 shows an image of a prototype device wherein the gearbox and drive shafts are visible with the tips disposed within a plate, according to one implementation.

[0021] FIG. 4 shows a high-throughput apparatus according to another implementation.

[0022] FIG. 5 shows a cross-section of the multi -well plate of FIG. 4 showing the distribution and layering of the plates.

[0023] FIG. 6 shows a perspective view of a prototype multi-well plate, according to one implementation.

[0024] FIG. 7 provides a side view of the prototype of FIG. 6.

[0025] FIG. 8 provides a top view of the prototype of FIG. 6.

[0026] FIG. 9 shows the same prototype of FIG. 6 within a prototype apparatus having a gearbox, a motor, and a plurality of cone-tipped drive shafts inserted into the wells of the multi-well plate.

[0027] FIG. 10A shows an example of blood brain barrier metastasis where cells or soluble molecules (e.g., cancer cells) are introduced into the first well.

[0028] FIG. 10B shows an example of chemotherapy screening. [0029] FIG. 10C shows an example of a model of a blood tumor barrier.

[0030] FIG. 11 shows a graph displaying a permeability assay to assess the barrier contributions of the device to the model, according to one implementation.

[0031] FIG. 12 shows a graph of daily measurement of TEER for the cells grown in the system with or without shear stress (*p<0.05; n=10), according to one implementation.

[0032] FIG. 13 shows images from immunostaining for actin and paxillin for both the shear stress-treated and static groups, according to one implementation.

[0033] FIG. 14 shows a graph of Actin relative intensity measurements from 6.2 dyn/cm² for 8 hours per day for seven days, according to one implementation.

[0034] FIG. 15 shows a graph of quantification of actin/paxillin colocalization shown as the average Pearson correlation coefficient (*p<0.05; n = 50), according to one implementation.

[0035] FIG. 16 shows a graph of relative intensity values for paxillin immunostaining (*p<0.05; n = 10), according to one implementation.

[0036] FIG. 17 shows higher-resolution images of actin and paxillin staining for static and shear stress acclimated BMECs, according to one implementation.

[0037] FIG. 18 shows images of the MRP-1 and BCRP immunofluorescent staining after seven days of shear stress or static culture (Scale bar = 40 pm), according to one implementation.

[0038] FIG. 19 shows a graph of relative intensity values for MRP-1 expression (*p<0.05; n = 40), according to one implementation.

[0039] FIG. 20 shows a graph of the quantified BCRP relative fluorescent intensity values (*p<0.05; n = 40), according to one implementation.

[0040] FIG. 21 shows immunofluorescent pictures of claudin-5 and ZO-1 for flow-treated and static HBMECs (Scale bar = 40 pm), according to one implementation.

[0041] FIG. 22 shows a graph of luantification of claudin-5 positive cell border shown as a percentage of total cell border (*p<0.05; n = 20), according to one implementation.

[0042] FIG. 23 shows a graph of quantification of ZO-1 positive cell border shown as a percentage of total cell border (*p<0.05; n = 20), according to one implementation. [0043] FIG. 24 shows images of JAM-A and occludin immunofluorescent staining after 7- day shear stress treatment alongside the static group (scale bar = 40 pm), according to one implementation.

[0044] FIG. 25 shows a graph of the percentage of JAM-A positive cell border staining (*p<0.05; n = 10), according to one implementation.

[0045] FIG. 26 shows a graph of quantification of positive cell border percentage from occludin staining (*p<0.05; n = 10), according to one implementation.

[0046] FIG. 27 shows images of cocultured BMECs and pericytes immunostained for PEC AM- 1 (red), PDGFRP (green), and JAG-1 (blue), according to one implementation.

[0047] FIG. 28 shows images of immunostaining for tight junction proteins claudin-5 and ZO-1 in cocultures of BMECs and pericytes (Scale bar = 40 pm), according to one implementation.

[0048] FIG. 29 shows a graph of quantification of claudin-5 positive cell border shown for co-culture as a percentage of total cell border (*p<0.05; n = 10), according to one implementation.

[0049] FIG. 30 shows a graph of quantification of ZO-1 positive cell border shown as a percentage of total cell border (*p<0.05; n = 10), according to one implementation.

[0050] FIG. 31 shows images of immunostaining for tight junction proteins JAM-A and occuludin in the coculture model (Scale bar = 40 pm), according to one implementation.

[0051] FIG. 32 shows a graph of the percentage of JAM-A positive cell border staining (n = 10), according to one implementation.

[0052] FIG. 33 shows a graph of the quantification of positive cell border percentage from occludin staining (n = 10), according to one implementation.

[0053] FIG. 34 displays a graph of measurements of TEER on the cultures over the seven days of culture (*p<0.05 vs static endothelial control; fp<0.05 vs respective static control; n=31-32), according to one implementation.

[0054] FIGS. 35 and 36 show graphs after seven days wherein the permeability of all monoculture and co-culture conditions were assessed with lOkDa dextran, according to one implementation. [0055] FIGS. 37 and 38 show graphs after seven days wherein the permeability of all monoculture and co-culture conditions were assessed with 20kDa dextran, according to one implementation.

[0056] FIGS. 39 and 40 show graphs after seven days wherein the permeability of all monoculture and co-culture conditions were assessed with 66 kDa BSA (p<0.05; n=15-16), according to one implementation.

[0057] FIG. 41 shows a graph of shear stress of 6.2 dyn/cm², according to one implementation.

[0058] FIG. 42 shows a graph of shear stress of 10 dyn/cm², according to one implementation.

[0059] FIG. 43 shows a graph of shear stress of 20 dyn/cm², according to one implementation.

[0060] FIG. 44 shows a graph of permeability coefficients for 10 kDa dextran, according to one implementation.

[0061] FIG. 45 shows a graph of permeability coefficients for 20 kDa dextran, according to one implementation.

[0062] FIG. 46 shows a bar graph representation of 66 kDa BSA permeability coefficients, according to one implementation.

[0063] FIG. 47 shows a plot of the normalized permeability coefficients of 20 kDa dextran vs 66 kDa BSA, according to one implementation.

[0064] FIG. 48 shows a plot of the normalized permeability coefficients of 20 kDa dextran vs 10 kDa dextran, according to one implementation.

[0065] FIG. 49 shows a plot of the normalized permeability coefficients of 10 kDa Dextran vs 66 kDa BSA, according to one implementation.

[0066] FIG. 50 shows a scatter plot of the normalized permeability coefficients to the static DMSO control of 20 kDa dextran vs 66 kDa BSA, according to one implementation.

[0067] FIG. 51 shows a scatter plot of the normalized permeability coefficients to the static DMSO control of 20 kDa dextran vs 10 kDa dextran, according to one implementation.

[0068] FIG. 52 shows a scatter plot of the normalized permeability coefficients to the static DMSO control of 10 kDa Dextran vs 66 kDa BSA, according to one implementation. [0069] FIG. 53 shows a plot of permeability coefficients for 10 kDa dextran, 20 kDa dextran, and BSA (*p<0.05 versus static DMSO; n = 9), according to one implementation.

[0070] FIG. 54 shows a plot of measurements of TEER for the model under shear stress and drug treatment (*p<0.05 versus static DMSO; n = 40), according to one implementation.

[0071] FIG. 55 shows a graph of TEER measurements for a device utilizing various pore size polycarbonate mesh after 48 hours of shear stress exposure, according to one implementation.

[0072] FIG. 56 shows a graph of permeability of trastuzumab of an example device with BMEC seeded on the mesh, according to one implementation.

[0073] FIG. 57 shows a graph of cancer endothelial adhesion assay measuring the effects of different compounds on cell adhesion, according to one implementation.

[0074] Various objects, aspects, features, and advantages of the disclosure will become more apparent and better understood by referring to the detailed description taken in conjunction with the accompanying drawings, in which like reference characters identify corresponding elements throughout. In the drawings, like reference numbers generally indicate identical, functionally similar, and/or structurally similar elements.

DETAILED DESCRIPTION

Definitions

[0075] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting and/or capital letters has no influence on the scope and meaning of a term; the scope and meaning of a term are the same, in the same context, whether or not it is highlighted and/or in capital letters. It will be appreciated that the same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms discussed herein, nor is any special significance to be placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any terms discussed herein, is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

[0076] It will be understood that when an element is referred to as being “on” another element, it can be directly on the other element or intervening elements may be present therebetween. In contrast, when an element is referred to as being “directly on” another element, there are no intervening elements present. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.

[0077] It will be understood that, although the terms first, second, third, etc. may be used herein to describe various elements, components, regions, layers and/or sections, these elements, components, regions, layers and/or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer or section from another element, component, region, layer or section. Thus, a first element, component, region, layer or section discussed below can be termed a second element, component, region, layer or section without departing from the teachings of the present invention.

[0078] It will be understood that when an element is referred to as being “on,” “attached” to, “connected” to, “coupled” with, “contacting,” etc., another element, it can be directly on, attached to, connected to, coupled with or contacting the other element or intervening elements may also be present. In contrast, when an element is referred to as being, for example, “directly on,” “directly attached” to, “directly connected” to, “directly coupled” with or “directly contacting” another element, there are no intervening elements present. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed “adjacent” to another feature may have portions that overlap or underlie the adjacent feature.

[0079] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” or “includes” and/or “including” or “has” and/or “having” when used in this specification specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

[0080] Furthermore, relative terms, such as “lower” or “bottom” and “upper” or “top,” may be used herein to describe one element's relationship to another element as illustrated in the figures. It will be understood that relative terms are intended to encompass different orientations of the device in addition to the orientation shown in the figures. For example, if the device in one of the figures is turned over, elements described as being on the “lower” side of other elements would then be oriented on the “upper” sides of the other elements. The exemplary term “lower” can, therefore, encompass both an orientation of lower and upper, depending on the particular orientation of the figure. Similarly, if the device in one of the figures is turned over, elements described as “below” or “beneath” other elements would then be oriented “above” the other elements. The exemplary terms “below” or “beneath” can, therefore, encompass both an orientation of above and below.

[0081] As used herein, “around,” “about,” “substantially” or “approximately” shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range. Numerical quantities given herein are approximate, meaning that the terms “around,” “about,” “substantially” or “approximately” can be inferred if not expressly stated.

[0082] As used herein, the terms “comprise” or “comprising,” “include” or “including,” “carry” or “carrying,” “has/have” or “having,” “contain” or “containing,” “involve” or “involving” and the like are to be understood to be open-ended, i.e., to mean including but not limited to.

[0083] As used herein, the phrase “at least one of A, B, and C” should be construed to mean a logical (A or B or C), using a non-exclusive logical OR. It should be understood that one or more steps within a method may be executed in different order (or concurrently) without altering the principles of the invention.

[0084] As used herein, the term “porous” generally refers to a material that is permeable or selectively permeable. The term “permeable” as used herein means a material that permits passage of a fluid (e.g., liquid or gas), a molecule, a whole living cell and/or at least a portion of a whole living cell, e.g., for formation of cell-cell contacts. The term “selectively permeable” as used herein refers to a material that permits passage of one or more target group or species, but act as a barrier to non-target groups or species. For example, a selectively-permeable membrane can allow passage of a fluid (e.g., liquid and/or gas), nutrients, wastes, or compounds from one side of the membrane to another side of the membrane, but does not allow whole living cells to pass therethrough. In some embodiments, a selectively-permeable membrane can allow certain cell types to pass therethrough but not other cell types.

[0085] As used herein, the terms “fluidic path” and “fluidic channel” are exchangeable, and refer to a passage, a conduit, a groove, a furrow, or the like that allow a fluid to flow through it. Similarly, “bus line,” “bus,” and “line” can be used interchangeably and refer to a common fluidic supply line or a set of common fluidic supply lines.

[0086] The “blood-brain barrier” or “BBB” refers to the physiological barrier between the peripheral circulation and the brain and spinal cord which is formed by tight junctions within the brain capillary endothelial plasma membranes, creating a tight barrier that restricts the transport of molecules into the brain, even very small molecules. The BBB within the brain, the blood-spinal cord barrier within the spinal cord, and the blood-retinal barrier within the retina are contiguous capillary barriers within the CNS, and are herein collectively referred to as the blood-brain barrier or BBB. The BBB also encompasses the blood-CSF barrier (choroid plexus) where the barrier is comprised of ependymal cells rather than capillary endothelial cells.

[0087] As used herein, “agent” or “biologically active agent” refers to a biological, pharmaceutical, or chemical compound or other moiety. Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, or a chemotherapeutic compound. Various compounds can be synthesized, for example, small molecules and oligomers (e.g., oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.

[0088] As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, palliating the pain resulting from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals. Treatment includes preventing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition prior to the induction of the disease; suppressing the disease, that is, causing the clinical symptoms of the disease not to develop by administration of a protective composition after the inductive event but prior to the clinical appearance or reappearance of the disease; inhibiting the disease, that is, arresting the development of clinical symptoms by administration of a protective composition after their initial appearance; preventing re-occurring of the disease and/or relieving the disease, that is, causing the regression of clinical symptoms by administration of a protective composition after their initial appearance.

General Description

[0089] Referring generally to the figures, a transwell system for a multi-well plate is shown, according to various implementations. The modified transwell system described herein can be used with a variety of types of well plates. In some implementations, the microwell plates comprise various metal materials such as stainless steel 304/316, titanium, and/or aluminum. Microwell plates generally include a plurality of sample wells used as small test tubes, the wells being arranged in a 2:3 ratio rectangular matrix. Examples include but are not limited to, 6, 12, 24, 36, 48, 96, 384, and 1536 well plates. 96-well plates are common in chemical and biological testing (e.g., in a laboratory setting). The microwell plates are also compatible with corresponding testing equipment matching the geometry of the microwell. For example, automatic pipetting tools may include a full row (e.g., a row of 8 pipettes) or a full array (e.g., 96 pipettes) aligned with the wells of a 96-well plate.

[0090] Many assays in biology and drug development use transwell plates. These are cell culture plates with a porous membrane that allow the passage of compounds and/or cells between upper and lower chambers. These plates are commonly used to examine the migration of cells through the membranes or layers of cells grown on the membranes. For example, transwell assays are used to measure inflammatory cells migrating through an endothelial monolayer, cancer cell invasion, and to perform permeability assays for in vitro tests of blood brain barrier or intestinal barrier function.

[0091] Described herein is an adapted flow system for performing transwell assay with controlled shear stress over each well (e.g., 96-well plate). In many cases, shear stress is important to model in vivo biological functions. Shear stress is a key aspect regulating inflammatory cell adhesion, blood clotting, and blood brain barrier function, as well as many other biological functions. Without it, assays can be limited in their accuracy to predict in vivo function. The systems and devices disclosed herein are drawn to a transwell system which is formed by joining two adjacent wells through the conduit plate. Because the well plate (e.g., 96-well plate) is modified to join adjacent pairs of wells, the modified transwell system essentially has half of the total testing units (e.g., 48 transwell chambers). However, the modified transwell system may still operate with standard equipment (e.g., an automatic pipetting system having 96 pipetting devices).

[0092] In some implementations, a customizable membrane or mesh is inserted and utilized to culture cells by inserting it into the device supported by multiple layers of gaskets, polycarbonate plates, and a stainless-steel base to improve the structural integrity. Once the membrane is on the device, two compartments are created. The upper compartment serves as the luminal compartment to culture cells while the lower compartment constitutes the abluminal compartment where additional cell types can be cultured. The device is compatible with the high-throughput cone-and-plate device and can be mounted onto the high-throughput cone-and-plate device to introduce flow into the system therefore creating dynamic cell culture conditions either with steady or pulsatile flow.

[0093] Example Apparatus and Application #1

[0094] FIG. 1 shows a high-throughput cone-and-plate (HT-CAP) device 100, according to one implementation. Device 100 includes multiple drive shafts 102 each having a cone- shaped tip 104. Each drive shaft 102 also includes at least one bearing 106 and a gear 108 that can be rotated in either direction or in an oscillatory manner. The tip 104 may be disposed in a well 118 of a plate 116, the well 118 having a media 120 and cells 122.

[0095] An array of 96 drive shafts 102 are attached to a gear box 110 formed with and/or coupled to the array of gears 108 of the drive shafts 102. A motor 112 is attached to and drives the gear box 110. A lift table 114 (e.g., a micrometer lifting table) lowers and raises the plate 116 having the multiple wells 118 (shown as a 96-well plate) such that the tips 104 of the drive shafts 102 can enter into at least one (e.g., one row, one column, a portion, or all) of the wells 118 of the plate 116.

[0096] In use, the lift table 114 raises the plate 116 up to the drive shafts 102. Each of the tips 104 enters the wells 118 and are disposed adjacent to the cells 122. The motor 112 activates to rotate each of the gears 108 of the gear box 110, thus rotating each of the drive shafts 102. The rotating tip 104 applies flow in close proximity to the cells 122 in culture.

[0097] FIG. 2 shows a detailed diagram of the tip 104 of a drive shaft 102 of FIG. 1 including example dimensions. FIG. 3 shows an image of a prototype device wherein the gearbox and drive shafts are visible with the tips disposed within a plate, according to one implementation.

[0098] Example Apparatus and Application #2

[0099] FIG. 4 shows a high-throughput apparatus 200, similar to device 100 of FIGS. 1-3. The apparatus 200 includes a multi-well plate 202 defining a plurality of wells including, for example, a first well 204 and a second well 206. The multi-well plate 202 includes a top plate 208, a bottom plate 210, and at least one support plate disposed between the top plate 208 and the bottom plate 210. For example, apparatus 200 includes a first support plate 212 and a second support plate 214 each disposed between the top plate 208 and the bottom plate 210. The top plate 208, the bottom plate 210, the first support plate 212, and the second support plate 214 comprise a plastic material (e.g., acrylic or polycarbonate); however, in other implementations, the various plates may comprise a metallic material instead of or in addition to the plastic. In some implementations, an additional steel base plate is disposed underneath all of the plates for support.

[0100] The distribution and layering of the plates are also shown in FIG. 5 as a crosssection of the multi-well plate 202. Silicon gaskets 230 are disposed between each of the plates, forming a water-tight seal. [0101] The apparatus 200 further includes a cell culture mesh 216 supported in between the first support plate 212 and the second support plate 214. The cell culture mesh 216 is a porous membrane capable of being seeded with cells. For example, the cells may be endothelial cells or epithelial cells corresponding to lung, intestine, or mucosa cells. In some implementations, the cells migrate through the cell culture mesh (e.g., for transmigration assays, models for the immune system, viral/bacterial infection, or cancer metastasis). In other implementations, the cell culture mesh is any flat material (porous or non-porous). The cell culture mesh 216 is applied to the multi -well plate 202 (e.g., a 96-well plate) in strips such that every other column of 8 wells is covered by the cell culture mesh 216, leaving the other 8 columns open.

[0102] The apparatus 200 further includes one or more shafts, cones, or fins that fit within at least one of the first well 204 and the second well 206 of the multi-well plate 202. For example, apparatus 200 includes multiple shafts 218 each having a cone-shaped tip 220. Each tip 220 of each of the shafts 218 can fit within the first well 204 and/or the second well 206 in close proximity to the cell culture mesh 216.

[0103] The apparatus 200 further includes a rotational motor 222 connected to drive the shafts 218 via a gearbox 232 coupled to each of the drive shafts 218. The rotational motor 222 causes the shafts 218 to rotate such that each of the tips 220 of the shafts 218 creates fluid flow and/or shear stress within the corresponding well (e.g., the first well 204 or the second well 206). The fluid flow or shear stress may be constant (via constant rotational velocity co of the shaft 218) or oscillatory (via dynamic rotational velocity co of the shaft 218).

[0104] Apparatus 200 differs from device 100 in that the first well 204 and the second well 206 of apparatus 200 may be connected to each other. For example, the bottom plate 210 defines a lower conduit 224 (e.g., a lower chamber) such that the first well 204 is in fluid communication with the second well 206.

[0105] As shown in FIG. 4, the cell culture mesh 216 is disposed in the first well 204 but not in the second well 206, leaving the second well 206 open on the top plate 208 side. Within the first well 204, the cell culture mesh 216 separates the first well 204 into an upper chamber 226 and a lower chamber 228. The lower chamber 228 is adjacent to and in fluid communication with the lower conduit 224 such that the lower chamber 228 of the first well 204 is in fluid communication with the second well 206. [0106] The tip 220 of the shaft 218 in the first well 204 is in close proximity to the cell culture mesh 216 such that, when rotated, the cell culture mesh 216 experiences a fluid flow and resultant shear stress. On the other side, in the second well 206, the corresponding tip 220 of the shaft 218 also applies shear stress to the fluid in the second well 206. In other implementations, only one of the shafts and tips is used to apply shear stress to only one side of the cell culture mesh.

[0107] Each of the upper chamber 226 of the first well 204 and the lower chamber 228 via the second well 206 are accessible from the top plate 208. For example, a standard robotic pipetting apparatus may be configured to access the upper chamber 226 and the lower chamber 228 (e.g., automatically or via preconfigured instructions). The standard robotic pipetting apparatus may have a standard well geometry and may access and interact with either side of the cell culture mesh 216 (e.g., for sampling the fluid in one of the chambers, for delivery of material into the chamber, or for detecting the cells or other attributes of the chamber).

[0108] In the apparatus 200, the cell culture mesh 216 is seeded with cells that may model the blood brain barrier. In some implementations, the cells are grown (i) on both sides of the cell culture mesh, (ii) in the bottom of the first well or the second well, (iii) on the sides of the first well or the second well, and/or (iv) in gels placed within the first well or the second well.

[0109] In some implementations, the apparatus 200 further includes one or more electrodes configured to measure transendothelial electrical resistance (TEER) across the cell culture mesh. For example, the one or more electrodes may include a first electrode placed in the first well and a second electrode placed in the second well.

[0110] In some implementations, the apparatus 200 further includes a detector capable of measuring cells within at least one of the first well and the second well during at least one of before, during, or after rotation of the one or more shafts, cones, or fins to cause fluid flow within the first well or the second well.

[OHl] Various methods to measure cell response are known in the art, including, but not limited to, cell labeling, immunostaining, optical or microscopic imaging (e.g., immunofluorescence microscopy and/or scanning electron microscopy), spectroscopy, gene expression analysis, cytokine/chemokine secretion analysis, metabolite analysis, polymerase chain reaction (PCR), immunoassays, ELISA, gene arrays, spectroscopy, immunostaining, electrochemical detection, polynucleotide detection, fluorescence anisotropy, fluorescence resonance energy transfer, electron transfer, enzyme assay, magnetism, electrical conductivity (e.g., trans-epithelial electrical resistance (TEER)), isoelectric focusing, chromatography, immunoprecipitation, immunoseparation, aptamer binding, filtration, electrophoresis, use of a CCD camera, mass spectroscopy, or any combination thereof. Detection, such as cell detection, can be carried out using light microscopy with phase contrast imaging and/or fluorescence microscopy based on the characteristic size, shape, and refractile characteristics of specific cell types. Greater specificity can be obtained using optical imaging with fluorescent or cytochemical stains that are specific for individual cell types.

[0112] In some embodiments, cells can be removed from the device and evaluated, or can be evaluated and analyzed using on-chip detection methods, e.g., immunohistochemical detection and/or microscopy. In some embodiments, the entire device including the cell culture mesh can be evaluated and analyzed, e.g., under a microscope.

[0113] FIG. 6 shows a perspective view of a prototype multi-well plate 202, according to one implementation. FIG. 7 provides a side view and FIG. 8 provides a top view of the same prototype implementation. FIG. 9 shows the same prototype implementation within a prototype apparatus having a gearbox, a motor, and a plurality of cone-tipped drive shafts inserted into the wells of the multi-well plate.

[0114] FIGS. 10A, 10B, and 10C provide example applications of the apparatus 200. Each of FIGS. 10A - 10C shows a first well 204 having a cone-tipped shaft 218 therein (e.g., first well 204 of the multi-well plate 202) and a second well 206 having an open top. A cell culture mesh 216 can be disposed within the first well with endothelial cells grown thereon (e.g., a porous mesh) to mimic the blood brain barrier. The cone-tipped shaft 218 is rotated to apply shear stress (e.g., constant or oscillatory) to the fluid in the first well, mimicking in vivo characteristics.

[0115] FIG. 10A shows an example of blood brain barrier metastasis where cells or soluble molecules (e.g., cancer cells 290) are introduced into the first well. Some of the cancer cells 290 pass through the blood brain barrier model (cell culture mesh 216 with endothelial cells), through the lower conduit 224, and into the second well 206 where they are measured, for example, by taking a sample from a pipette 292 as shown.

[0116] FIG. 10B shows an example of chemotherapy screening. The lower conduit 224 includes several healthy cells 280 with a few cancerous cells 282. A chemotherapy drug 284 is placed in the first well 204 to test how well it can pass through the blood brain barrier model created by the cell culture mesh 216. A subset of the chemotherapy drug 284 passes through the BBB model to engage with the cancerous cells 282 in the lower conduit 224. The number, concentration, or ratio of chemotherapy drug 284 passing through the BBB model can then be measured by conventional means or detectors (e.g., pipetting a sample or investigating the number of cancerous cells 282 engaged with the chemotherapy drug 284).

[0117] FIG. 10C shows an example of a model of a blood tumor barrier. In this example, the endothelial cells are grown on the cell culture mesh 216. Additionally, a number of tumor cells 288 (e.g., barrier cells of a tumor) are grown on the opposite side of the cell culture mesh 216. The result is a model of a tumor barrier to test how well certain therapeutics can penetrate into a tumor by passing through the tumor cells 288 and into the second well 206.

[0118] Methods

[0119] Disclosed herein are methods of using cells with the apparatus described herein to carry out various assays. These assays can be used, for example, to model cells either in a normal or a diseased state. The apparatus can be used to determine how cells interact with each other, or how cells interact in various environments, or how cells interact when exposed to various compounds.

[0120] For example, cells used with the apparatus can be monitored over a period of time. This can be done to determine their lifespan, how they interact with each other, or what cellular signals are generated. These cells can be monitored for minutes, hours, days, or even years. The cells can be exposed to varying environmental conditions or physiological in order to determine how they react. Such conditions include, but are not limited to, change in nutrients (such as cell media), change in pH, change in temperature, etc.

[0121] The cells used with the apparatus can also be used to determine interaction with various compounds. For example, the cells can be exposed to a test agent and then monitored to see how they respond. In a particular embodiment, this can be done to determine whether the test agent can penetrate the BBB (for example, by entering the cells of the assay). One or more test agents can be used in this assay. For example, one could monitor whether one test agent is able to increase the penetrance of the BBB by another test agent. A first test agent can be administered to the cells before, after, or during monitoring. An additional test agent can be administered before, after, or during the administration of the first test agent. [0122] Specifically, disclosed herein is a method of determining that a test agent interacts with, exerts an effect upon, or is able to enter cells found within the blood brain barrier, the method comprising: a) providing the apparatus described herein, wherein the cell culture mesh of the apparatus has been seeded with cells found in the blood brain barrier; b) applying the test agent to the cells; c) applying, by the one or more shafts, cones, or fins, shear stress to the cells; and d) detecting a change in the cells compared to a control, wherein said change indicates that test agent interacts with, has an effect on, or is able to enter cells within the blood brain barrier.

[0123] The results of detection of cellular response to a test agent can be compared to a control. This control could be a measurement of the cells taken before, after, or during exposure to the test agent. The control can also be cells that are not exposed to the test agent in the same or in a different apparatus. The control can also be an artificially generated standard or a compilation of results from previous assays.

[0124] The cells used within the assay can be “normal,” or standard, non-diseased, healthy, non-engineered cells. Alternatively, the cells can be diseased cells, such as cancer cells, or can be engineered to have certain characteristics, either genotypically or phenotypically.

[0125] As described above, the apparatus described herein can be used to create an in vitro model that mimics a specific condition. As used herein, the term “specific condition” refers to any condition that can be diagnosed in a cell, tissue, or organ in vivo. The condition can occur naturally in the tissue in vivo (including, e.g., a normal healthy condition, or a condition induced or caused by a congenital defect), or induced or caused by a condition-inducing agent or stimulant (e.g., including, but not limited to an environmental agent). Examples of specific conditions include, but are not limited to, a normal state, a disease-specific state, a pre-disease state, a disease remission state, a distressed state, an inflamed state, an infected state, and a stimulated state.

[0126] In these embodiments, the cells used with the apparatus can be adapted to display at least one characteristic associated with a specific condition. For example, in some embodiments, patient- and disease-specific endothelial cells, such as those found within the BBB, can be cultured in the cell culture mesh, for example, to model diseases such as cancer.

[0127] In other embodiments, normal cells can be contacted with a condition-inducing agent (also referred to herein as a “test agent”) that is capable of inducing the normal cells to acquire at least one characteristic associated with the specific condition. [0128] In some embodiments, a disease-specific condition can be created by genetically modifying normal healthy cells, e.g., by silencing one or more genes or over-expressing one or more genes. Methods of gene silencing include, but are not limited to, RNA interference (e.g., but not limited to small interfering RNA (siRNA), microRNA (miRNA), and/or short hairpin RNA (shRNA)), antisense oligonucleotides, ribozymes, triplex forming oligonucleotides, and the like.

[0129] As discussed above, the apparatus described herein can be used to determine an efficacy of a test agent upon exposure of the cells. As used herein, the term “efficacy” generally refers to the ability of a test agent to produce a desired effect or outcome. Depending on the nature and/or type of the test agents, examples of desired effects or outcomes include, but are not limited to, therapeutic effect, cytotoxicity, cell growth, cell differentiation, improved or reduced cell function or phenotype (e.g., but not limited to, ciliary clearance, permeability of a cell layer, cell migration, expression and/or secretion of a protein or cytokine that can be affected by cell exposure to the test agent), and any combinations thereof. The term “therapeutic effect” as used herein refers to a consequence of treatment, the results of which are judged to be desirable and beneficial.

[0130] In accordance with some embodiments of the invention, the devices described herein can be used to determine toxicity of a test agent upon exposure of the cells on one or both surfaces of the membrane to the test agent. As used herein, the term “toxicity” refers to ability of a test agent to induce or cause any adverse and/or side effect on a cell and/or even cell death. For example, the toxicity of a test agent can be characterized by its ability to induce or cause an adverse effect on cell function and/or phenotype, including, but not limited to, alteration in cell metabolism, mutagenicity, carcinogenicity, teratogenicity, DNA damage, protein or membrane damage, cell energy depletion, mitochondrial damage, genotoxicity, apoptosis, cell death, cell rupture, and any combinations thereof.

[0131] In accordance with some embodiments of the invention, the devices described herein can be used to determine a mechanism of action upon exposure of the cells on one or both surfaces of the membrane to the test agent. As used herein, the term “mechanism of action” refers generally to a cellular pathway or biological interaction through which an agent exerts its biological effect on a cell. For example, when an agent is a drug substance, mechanism of action can refer to the biochemical interaction through which a drug substance produces its pharmacological effect. Depending on the nature and/or type of test agents, the mechanism of action can be associated with any art-recognized cellular pathways or biological interaction, e.g., including, but not limited to, protein synthesis, cell migration, chromatin regulation/epigenetics or acetylation, MAPK signaling, apoptosis, autophagy, PI3K/Akt signaling, translation control, cell cycle/checkpoint, Jak/ Stat Pathway, NF-B signaling, TGF- /Smad signaling, lymphocyte signaling, angiogenesis, cytoskeletal signaling, cell adhesion, cell metabolism, cell development and/or differentiation, tyrosine kinase/adaptors, protein stability, protein folding, nuclear receptor signaling, and any combinations thereof. Accordingly, in some embodiments, a mechanism of action can encompass a mechanism of efficacy and/or toxicity of a test agent.

[0132] Any test agent can be introduced into the device described herein to determine its effect on the cells. Examples of the test agent can include, but are not limited to, proteins, peptides, antigens, nanoparticles, environmental toxins or pollutant, cigarette smoke, chemicals or particles used in cosmetic products, small molecules, drugs or drug candidates, vaccine or vaccine candidates, aerosols, inflammatory molecules, naturally occurring particles including pollen, chemical weapons, single or double-stranded nucleic acids, viruses, bacteria, and unicellular organisms.

[0133] In some embodiments, the devices described herein can be used for target identification/validation. For example, the devices described herein can be used to mimic a tissue-specific condition as described herein (e.g., a disease or disorder) in order to elucidate the molecular mechanism underlying a disease or a condition, the identification of candidate target molecules and the evaluation of said target molecules. In some embodiments, use of genetically modified cells, e.g., by silencing or over-expressing a specific gene, in the devices described herein can be used to identify target molecules for a specific disease. Once such a validated target molecule, e.g., ligand, receptor, transcription factor, and/or enzyme, which is herein referred to also as target, is identified, drug candidates directed to the target (e.g., suppression or activation) can be tested. The drug candidate can be introduced to the diseasespecific cells in the devices described herein and cell response to the drug candidate can be measured to validate the identified target. This can also promote drug discovery for a specific disease or condition. In many cases such drug candidates can be members of a compound library which can comprise synthetic and/or natural compounds. Combinatorial libraries can also be used.

[0134] Experimental Tests and Results [0135] Blood brain barrier assay using the system and identification of clinically approved compounds that can facilitate trastuzumab entry into the brain for treating brain metastasis.

[0136] A study was conducted using the novel transwell system (e.g., apparatus 200) according to one implementation. In the study, an in vitro model of the blood brain barrier was created and optimized for performing a short term assay for the ability of compounds to open or close the BBB or to penetrate the BBB. This model had a monolayer of human brain endothelial cells grown on a transwell membrane. After conditioning with shear stress, compounds could be added to open or close the BBB and tracers such as fluorescently labeled dextran or therapeutic molecules can be added to assay their ability to cross the BBB.

[0137] Materials and Methods

[0138] Cell Culture. Primary human brain microvascular endothelial cells (BMEC; Cell Systems) were cultured in MCDB-131 media supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin-streptomycin, and endothelial cell growth supplement (R&D Systems). Cells were grown in 5% CO2 at 37°C and the experiments were conducted with cells of passages four to nine. Human brain vascular pericytes (HBVP, ScienCell) were cultured in DMEM/F12 media supplemented with 10% FBS, penicillin-streptomycin, and GlutaMAX. All experiments conducted with HBVPs were performed on cells of passages three to seven.

[0139] Immunostaining. Following the treatments, the cells were washed with phosphate- buffered saline (PBS) and then incubated with 4% paraformaldehyde for 10 mins at room temperature. Samples were then rinsed three times with PBS before incubating with 0.2% Triton X-100 for 5 mins. After permeabilizing the cells, they were blocked with 5% FBS with 1% bovine serum albumin (BSA) in PBS for 40 mins. Cells were then labeled with primary antibodies diluted as described in Supplemental Table 1, diluted in 1% BSA in PBS overnight at 4°C. The next day, cells were washed three times with 1% BSA PBS and then treated with fluorophore-conjugated secondary antibodies (1 : 1000) for 75 minutes at room temperature. Samples were then mounted using antifade media (Vector Labs, Inc.). The cells were then imaged utilizing a Fluoview F VI 000 Confocal Laser Scanning Microscope (Olympus).

[0140] High Throughput Blood Brain Barrier Device. The computer-aided design (CAD) for the device was performed using commercially available CAD software (SolidWorks). The device components were created through conventional machining techniques. Polycarbonate framed filters with 3 -pm pores (NeuroProbe, Inc.) were cut to size and coated overnight with fibronectin (8 pg/ml) or collagen I (10 pg/ml). Cells were trypsinized, counted, and seeded onto the wells at a concentration of 100,000 cells/well. For monolayer culturing, BMECs or HBVPs were grown to confluence for two days before introducing flow. For the co-culture model, HBVPs were grown to confluence for two days then BMEC were layered on top the HBVP and grown to confluence for an additional two days. The cone-tipped rods of the HT- CAP system were aligned and sterilized as previously described. The system is constructed of stainless steel (Alloy 316L).

[0141] Measurement of Transendothelial Electrical Resistance. Transendothelial electrical resistance (TEER) measurements were taken daily during the seven days of shear stress treatment. The cells were equilibrated at room temperature for 15 minutes and then the measurements were done utilizing a commercially available electrode system (EV0M2, World Precision Instruments) with a chopstick configuration (STX3). Chopsticks were carefully inserted into the transwell without scratching the cell monolayer. The measurements were recorded after the signal had stabilized. The endothelial TEER (TE) reported was determined with the following formula:

Eqn. (1): T_E = (T_M ~ T_B) X Area

[0142] The measured TEER from an endothelial monolayer (TM) was subtracted by the measured TEER from a blank well containing the polycarbonate membrane but no cells (TB). This quantity was then multiplied by the surface area of the membrane to determine TE.

[0143] Permeability Assay . Fluorescently labeled BSA (Thermo Fischer Scientific), 10 kDa dextran (Thermo Fisher Scientific) and 20 kDa dextran (Sigma-Aldrich) were diluted to a final concentration of 100 pg/ml in phenol red free DMEM supplemented with 5% FBS, L- glutamine, penicillin-streptomycin, and endothelial cell growth supplements (R&D Systems). To measure monolayer permeability, BMECs were rinsed with PBS twice and media was changed to phenol red free DMEM media supplemented as previously described. No shear stress was applied during the permeability assay. The luminal compartment (side exposed to flow) of the device was filled with 200 pl of the media containing the conjugated molecules. The abluminal device compartment (side not exposed to flow) was then sampled for 100 pl of media and replaced immediately with an equal volume of media without the conjugated molecules at each time point (0, 60, 120, and 240 mins). The harvested media was then placed in a glass bottom multi-well plate and read in a fluorescent plate reader (Varioskan Flash; Thermo Fischer Scientific) to determine molecule concentration. [0144] Drug Screening Assay. Endothelial cells were grown as previously mentioned and seeded onto the HT-BBB device. Cells were acclimated to static or shear stress conditions (10 dyn/cm²) for two continuous days while being treated for with one of the forty kinase inhibitors, shown in Table 1 below, at 1 pM using DMSO as a carrier. Throughout the acclimation process, resistance measurements were collected. Once cells finished their two days of treatment and acclimation, permeability assays were conducted as previously described.

Table 1. Kinase Inhibitors Used in Drug Screening

[0145] Statistical Analysis. All results are shown as mean ± standard error of the mean. Comparisons between only two groups were performed using a two-tailed Student’s t-test. Multiple comparisons between groups were analyzed by two-way ANOVA followed by a Tukey posthoc test. A two-tailed probability value p < 0.05 was considered statistically significant.

[0146] Results

[0147] Designing and Testing a High-Throughput Transwell System. A custom transwell system was created, substantially similar to apparatus 200 as shown in FIGS. 2-9. The system included of two thin support plates with gaskets that hold a porous cell culture membrane. The culture membrane can be any flat material (porous or nonporous) that applied to the wells of the 96 well plate in strips such that every other column of 8 wells is covered by the membrane. In this case, porous polycarbonate membranes were used, similar to conventional transwell membranes. A top plate is placed on top of the support plates/membrane to create an upper chamber on top of the membrane. A bottom plate creates a chamber between two adjacent wells in the 96-well plate to create the bottom chamber of the transwell. Shear stress is applied to the model using a high throughput cone-and-plate (HT-CAP) system. The system applies shear stress by rotating steel rods that have a low-angle cone tip, which are held in close approximation to the cells on the culture surface to create uniform shear stress. In the HT-CAP system, the rods are rotated by a single rotational motor that applies torque to all the rods through a 96-shaft gear assembly. The system can apply both steady and pulsatile shear stresses by controlling the angular velocity profile of the motor.

[0148] The assembled BBB plate is held together with screws that compress silicone gaskets to form the multi-well transwell plate. The upper chamber is used as the luminal side of the model with shear stress while the lower chamber is used as the abluminal side. From the abluminal side, media can be collected to assess permeability to compounds or labeled molecules (eg. FITC dextran). Measurements of transendothelial electrical resistance (TEER) can be taken using conventional chopstick electrodes. Different gasket configurations were tested to eliminate leaking. With the optimized gasket configuration, there were very high resistance values between non-linked neighboring wells. A permeability assay was conducted on the device itself to assess the barrier contributions of the device to the model, as shown in the graph in FIG. 11. Specifically, FIG. 11 shows the device contribution to barrier properties of the high-throughput blood brain barrier model with the permeability coefficient of lOkDa dextran, 20kDa dextran, and BSA. The assembled device allows cell media for both the luminal and abluminal compartments to be exchanged utilizing a multi-channel micropipette for ease of use without leaking. Overall, the system provides a total of 48 BBB assays with shear stress simultaneously.

[0149] Validation of High Throughput Blood Brain Barrier Device for BBB Development and Transendothelial Electrical Resistance. Human primary BMECs were seeded into the system and grown to confluence. The plate was then mounted in a modified version of the HT-CAP and the cells were cultivated under flow (6.2 dyn/cm²) for 8 hours per day for seven days. The endothelial cells were treated with physiological shear stress (6.2 dyn/cm²) to mimic the flow in brain vascular conditions. The TEER value increased over the first 48 hours and then was relatively constant over seven days, as shown in the graph of FIG. 12 which shows a daily measurement of TEER for the cells grown in the system with or without shear stress (*p<0.05; n=10). The device was able to recreate the functionality of conventional transwell plates by reliably providing TEER measurements comparable for this specific cell line cultured in polycarbonate membrane in static conditions. The cells treated with flow developed actin and paxillin stress fibers oriented on the direction of the shear stress, as shown in FIG. 13. Specifically, FIG. 13 shows images from immunostaining for actin and paxillin for both the shear stress treated and static groups. Stress fiber formation is a classic response in endothelial cells after exposure to flow. Relative total actin immunofluorescent intensity remained unchanged after flow treatment, as shown in FIG. 14. Specifically, FIG. 14 shows a graph of Actin relative intensity measurements from 6.2 dyn/cm² for 8 hours per day for seven days.

[0150] Co-localization of actin/paxillin, as well as paxillin relative intensity, were significantly upregulated in the shear-stress-treated group, as shown in FIGS. 15 and 16. Specifically, FIG. 15 shows a graph of quantification of actin/paxillin colocalization shown as the average Pearson correlation coefficient (*p<0.05; n = 50), and FIG. 16 shows a graph of relative intensity values for paxillin immunostaining (*p<0.05; n = 10). The exposure BMECs to flow also led to the reorganization of the actin cytoskeleton as well as the alignment of stress fibers that were not present in static conditions, see FIG. 13 and FIG. 17 which shows higher-resolution images of actin and paxillin staining for static and shear stress acclimated BMECs. The formation of stress fibers along the endothelial junctions promotes the formation of tight junctions such as ZO-1 to increase the barrier function of the BBB.

[0151] Expression of transporter protein essential to maintaining BBB homeostasis and selectivity in transport across the BBB. After seven days of treatment with shear stress, there was a significant upregulation in the relative intensity of multidrug resistance-associated protein 1 (MRP-1), as shown in FIGS. 18 and 19, and breast cancer-resistant protein (BCRP/ABCG2), as shown in FIGS. 18 and 20, in BMECs treated with shear stress. Specifically, FIG. 18 shows images of the MRP-1 and BCRP immunofluorescent staining after seven days of shear stress or static culture (Scale bar = 40 pm). FIG. 19 shows a graph of relative intensity values for MRP-1 expression (*p<0.05; n = 40). FIG. 20 shows a graph of the quantified BCRP relative fluorescent intensity values (*p<0.05; n = 40).

[0152] In addition, after the seven days of treatment with shear stress, immunofluorescent staining of several tight junction markers was taken to assess the barrier properties of our model. Claudin-5 expression at the cell junctions significantly decreased after the seven days of flow treatment, as shown in FIGS. 21 and 22. Specifically, FIG. 21 shows immunofluorescent pictures of claudin-5 and ZO-1 for flow-treated and static HBMECs (Scale bar = 40 pm). FIG. 22 shows a graph of luantification of claudin-5 positive cell border shown as a percentage of total cell border (*p<0.05; n = 20). Claudin-5 is essential for barrier function and integrity of the BBB. However, the primary human BMEC line has been found to have very low Claudin- 5 expression in previous studies and did not affect TEER in culture models. The application of flow significantly increased the expression of zona occludens-1 (ZO-1), as shown in FIG. 21 and 23, and junctional adhesion molecule-A (JAM- A) at the cell junctions, as shown in FIGS 24 and 25. Specifically, FIG. 23 shows a graph of quantification of ZO-1 positive cell border shown as a percentage of total cell border (*p<0.05; n = 20). FIG. 24 shows images of JAM-A and occludin immunofluorescent staining after 7-day shear stress treatment alongside static group (scale bar = 40 pm). FIG. 25 shows a graph of percentage of JAM-A positive cell border staining (*p<0.05; n = 10).

[0153] Zona occludens-1 forms a link between occludin, JAM-A and Claudin-5 to bind to the actin cytoskeleton, making it an indispensable protein for tight junction formation. An increase in expression of JAM-A is associated with restricting free diffusion of proteins and stabilizing existing tight junctions, therefore improving the barrier function of our model. Occludin expression for the shear stress-treated BMECs was significantly lower compared to its static control, as shown in FIG. 24 and FIG. 26 which shows a graph of quantification of positive cell border percentage from occludin staining (*p<0.05; n = 10). However, it has been noted that in occludin knockout animal studies, occludin is not essential for tight junction formation and the barrier function of the BBB. The study demonstrated that the device is able to apply flow and upregulate the expression of two key tight junction proteins (ZO-1 and JAM-A) to enhance the barrier properties of this BBB model utilizing primary human BMECs.

[0154] Sequential Layering of Pericytes and Endothelial Cell Co-Culture Disrupt Tight Junctions and Increases Barrier Properties. To improve the barrier function of our BBB model using BMECs, the use of pericyte cultures in direct contact with the endothelial cells was tested. Pericytes have been shown to increase the barrier properties of BMECs when cultured together. Pericytes were cultured on the membrane for 2 days and then seeded endothelial cells on top of these cells until confluence. The cells were treated with shear stress (6.2 dyn/cm²) for 8 hours per day for 7 days. Immunostaining of the cultures showed good separation between the layers of cells for static conditions, as can be seen in FIG. 27 which shows images of cocultured BMECs and pericytes immunostained for PEC AM- 1 (red), PDGFRp (green), and JAG-1 (blue). Scale bar = 40 pm.

[0155] With shear stress application, the cells mixed and the separation between the cell types was lost, as shown in FIG. 28 which shows images of immunostaining for tight junction proteins claudin-5 and ZO-1 in cocultures of BMECs and pericytes (Scale bar = 40 pm). For the static culture, there was a localized region of endothelial pericyte interactions labeled by JAG-1 between the cell layers, as shown in FIG. 27. For the cultures treated with shear stress, there were extensive pericyte endothelial interactions throughout the culture, as shown in FIG. 28. In addition, well-defined tight junctions were formed in the static cultures in the endothelial layer, as shown in FIGS. 29-33. For the shear-treated co-cultures, it was found that there were JAMA containing junctions distributed in the 3D culture, as shown in FIGS. 29-33. Specifically, FIG. 29 shows a graph of quantification of claudin-5 positive cell border shown for co-culture as a percentage of total cell border (*p<0.05; n = 10). FIG. 30 shows a graph of quantification of ZO-1 positive cell border shown as a percentage of total cell border (*p<0.05; n = 10). FIG. 31 shows images of immunostaining for tight junction proteins JAM- A and occuludin in the coculture model (Scale bar = 40 m). FIG. 32 shows a graph of the percentage of JAM-A positive cell border staining (n = 10). FIG. 33 shows a graph of quantification of positive cell border percentage from occludin staining (n = 10).

[0156] Transendothelial electrical resistance (TEER) measurements for the co-cultures were significantly higher for pericyte and endothelial cells that were co-cultured under shear stress in comparison to monoculture of endothelial cells, as shown in FIG. 34 displaying a graph of measurements of TEER on the cultures over the seven days of culture (*p<0.05 vs static endothelial control; fp<0.05 vs respective static control; n=31-32).

[0157] In addition, permeability for the cultures was measured in comparison to monocultured cells, and it was found the co-cultures had significantly reduced permeability, as shown in FIGS. 35-40. Specifically, FIGS. 35 and 36 show graphs after seven days wherein the permeability of all monoculture and co-culture conditions were assessed with lOkDa dextran. FIGS. 37 and 38 show similar graphs for 20kDa dextran, and FIGS. 39 and 40 show similar graphs for 66 kDa BSA (p<0.05; n=15-16). No flow was applied during the permeability assays. Together these studies demonstrate that it is possible to generate a layered construct of endothelial cells over pericytes in static culture. However, the findings support that shear stress causes the two layers of cells to intermingle rather than remain organized into two layers, as they do in static culture. This allows for more interactions between the two cell types cells but is less physiologically relevant since the fluid in the upper chamber is in contact with both cell types and the endothelial monolayer structure is no longer present.

[0158] Optimization of Short-Term Assay with Varying Shear Stress Levels in High- Throughput Blood Brain Barrier Model. To further optimize the timeline of the system for high throughput screening, two days of continuous shear stress on the culture were used with varying levels of shear stress. Under these conditions, the shear stress of 6.2 dyn/cm² induced increased permeability in both monocultures of endothelial cells and co-cultures of pericytes and endothelial cells, as shown in FIG. 41. At 10 dyn/cm², the permeability coefficients for all tracers had significantly decreased for all tracers for both the BMEC monolayer and the HBVP and BMEC co-culture, as shown in FIG. 42. For cultures treated with 20 dyn/cm², there was a decrease in some of the tracers in BMEC monocultures or HBVP and BMEC cocultures, as shown in FIG. 43. Thus, for short-term, continuous shear stress studies, 10 dyn/cm² was optimal to maximize the barrier function of the model. [0159] Effects of Shear Stress on Drug Stimulation of Brain Microvascular Endothelial Cells. Previous studies have identified compounds that improve BBB barrier function in static, in vitro models. To understand whether these compounds could be used to improve our BBB model, these compounds were tested in the short-term, continuous-flow BBB assay. Endothelial cell monocultures were used, conditioned by 10 dyn/cm² for 48 continuous hours, and then treated with one of the following compounds: angiotensin I, cAMP, lexiscan (A2A adenosine receptor agonist), corticosterone, hydrocortisone, and dexamethasone. See Table 2 below. Many of the drugs induced a non-significant reduction in the permeability coefficient under static conditions and there was a significant reduction in the permeability coefficient for dexamethasone, as shown in FIGS. 44-46. Specifically, FIG. 44 shows a graph of permeability coefficients for 10 kDa dextran, FIG. 45 shows a graph of permeability coefficients for 20 kDa dextran, and FIG. 46 shows a bar graph representation of 66 kDa BSA permeability coefficients (*p < 0.05 versus static DMSO. fp < 0.05 versus shear DMSO).

Table 2. Drugs Used to Enhance BBB Function

[0160] However, under shear stress, none of the compounds induced a significant improvement in barrier function, as shown in FIGS. 47-49. Specifically, FIG. 47 shows a plot of the normalized permeability coefficients of 20 kDa dextran vs 66 kDa BSA. FIG. 48 shows a similar plot for 20 kDa dextran vs 10 kDa dextran, and FIG. 49 shows a similar plot for 10 kDa Dextran vs 66 kDa BSA. [0161] Overall, this screening experiment illustrates the importance of shear stress when conducting drugs discovery assays as the biochemical effect of drugs is different when flow is present in cell culture.

[0162] Drug Screening Assay for Compounds to Improve BBB Barrier Function. Next, a library of 40 kinase inhibitors was screened for their effect on the barrier function of the BBB. BMECs were grown to confluence in the system and treated the cells with 48 hours of continuous shear stress (10 dyn/cm²), with combined treatment with DMSO or one of the compounds from the library. At the end of the shear stress treatment, the permeability of the cells was assayed using the three tracers (10 kDa dextran, 20 kDa dextran, or 66 kDa BSA) and calculated permeability coefficients for each.

[0163] Under shear stress, the compound that most reduced the permeability coefficient was an EGFR/ErbB2 inhibitor. Other kinase inhibitors that reduced permeability under shear stress included those targeting EGFR, ROCK1/2, PKCP, y-secretase, and Aurora A, as shown in Table 3 below, wherein the abbreviations are: AD: Alzheimer’s Disease; TJ: Tight Junction; TBI: Traumatic Brain Injury.

Table 3. Summary of Hits from BBB Screening Assay Incorporating Shear Stress

[0164] Under static conditions, there were no compounds that reduced permeability for all three tracers. None of the compounds significantly increase the permeability of the BBB in the model. In addition, the DMSO treatment appeared to alter the effects of shear stress on the cells and there was no decrease in permeability with shear in comparison to the static DMSO control. Scatter plots were created of the permeability for the different tracers, allowing identification of compounds that could allow larger/small molecules to pass through the BBB while maintaining or increasing permeability for molecules of different sizes, as shown in FIGS. 50-52. Specifically, FIG. 50 shows a scatter plot of the normalized permeability coefficients to the static DMSO control of 20 kDa dextran vs 66 kDa BSA, FIG. 51 shows a similar graph for 20 kDa dextran vs 10 kDa dextran, and FIG. 52 shows a similar graph for 10 kDa Dextran vs 66 kDa BSA.

[0165] For the most part, the compounds affected the permeability of all three tracers similarly. However, for three of the compounds, there was differential modulation of permeability based on the type of tracer used, including theophylline, PF-05274857, and ruxolitinib (See Table 4 below). For theophylline, there was decreased permeability for the 20 kDa dextran but increased permeability for BSA and the 10 kDA dextran. For PF- 05274857, permeability for BSA was unchanged while it was decreased for both dextrans. For ruxolitinib, the permeability for the 10 kDa dextran was unchanged while the permeability was decreased for the larger tracers.

Table 4. Relative Permeability of Compounds from Kinase Screen

Kinase Inhibitor BSA (66 kDa) 20 kDa Dextran 10 kDa Dextran

Theophylline 1.24 0.64 1.25

PF-05274857 0.98 0.63 0.75

Ruxolitinib 0.77 0.75 0.98

[0166] To further confirm the activity of the primary hit from the drug screen, the study was repeated with higher n and found consistent results for the EGFR/ErbB2 inhibitor in decreasing the permeability of the BBB and increasing TEER, as shown in FIGS. 53 and 54. Specifically, FIG. 53 shows a plot of permeability coefficients for 10 kDa dextran, 20 kDa dextran, and BSA (*p<0.05 versus static DMSO; n = 9). FIG. 54 shows a plot of measurements of TEER for the model under shear stress and drug treatment (*p<0.05 versus static DMSO; n = 40). Overall, the HT-BBB system was able to perform a high-throughput screening assay and identify potential drug hits in the shear stress incorporating assay. This was in sharp contrast to the static assay, which did not identify compounds with BBB regulating activity.

[0167] Discussion

[0168] Conventional static, transwell BBB models remain the most common model for BBB research as they are easy to use and widely available. However, most multi-well transwell models do not incorporate flow, an important biophysical force in the human body that regulates the behavior of endothelial cells in the BBB. Conversely, there have been many advanced microfluidic models of the BBB that provide increased complexity to match the physiological aspects of the BBB. However, these models are often low throughput and consequently more difficult to utilize for screening and drug development studies requiring many simultaneous assays. In this study, a HT-BBB model was developed that incorporates shear stress and is practical for use in drug discovery assays. Using this model, multiple compounds were identified that regulate the BBB previous in vivo studies. Thus, the system may have practical utility in drug discovery as an initial screen for compounds prior to using more complex animal or in vitro models.

[0169] Overall, the system and drug screening assay were optimized for using the human BMEC cell line in the HT-BBB system. In the HT-BBB model, both intermittent and continuous treatment with shear stress led to enhanced barrier function. In optimizing the short-term continuous flow version of the assay, it was found that the level of shear stress was a key parameter with optimal barrier properties occurring at 10 dyn/cm² of shear stress. Following validation of the system/BBB culture, a novel approach was examined for seeding endothelial cells directly on top of a pericyte layer as a BBB model. Under static conditions, the two layers retained their separation and had localized zone of interaction. However, shear stress stimulated remodeling of the two layers leading to intermingling of the two cell types and extensive endothelial-pericyte interactions. Both models had improved barrier function in comparison to monocultures. Under static conditions, this may be a practical approach for creating a multi-layer BBB model. Under shear stress, the mixed culture is different than the in vivo BBB structure and therefore may only be useful to study pericyte-endothelial interactions rather than mimicking native BBB physiology. Shear stress induces pro- angiogenic signaling and expression of microRNAs that enhance blood vessel stabilization. Thus, a likely cause of the remodeling under shear stress is the induction of signaling in the endothelial cells that lead to migration/sprouting type behavior along with enhanced drive to recruit pericytes to surround the endothelial cells.

[0170] Previous work on in vitro BBB models has examined ways to improve the barrier properties of primary brain microvascular endothelial cells using drugs. In the HT-BBB model, only slight improvements were found in barrier function with these drugs under static conditions and no improvements under shear stress conditions. In addition, a smallscale screening assay was performed for compounds that improve BBB barrier function. This assay identified that inhibitors of EGFR/ErbB2, EGFR, ROCK1/2, PKCP, y-secretase and Aurora A had activity in improving BBB barrier function under flow. However, in the absence of flow there were no compounds that improved barrier function for all of the tracers used to measure permeability. For many of the compounds that were identified in the screen, previous studies have supported their activity in BBB regulation. ROCK activity leads to phosphorylation of tight junction proteins and induces permeability of the BBB in vivo, leading to enhanced extravasation of monocytes and BBB disruption in mouse models of Alzheimer’s disease. Inhibition of ROCK signaling prevents BBB disruption in cerebral ischemia, autoimmune disease, intracerebral hemorrhage and ischemic stroke. Similarly, PKCP can phosphorylate tight junction proteins leading to their disruption and its activity has been linked to BBB permeability in hyperglycemia and diabetes. In addition, inhibition of y- secretase also prevents BBB disruption in an in vivo model of permanent ischemia. Inhibition of EGFR signaling has shown promise for multiple neurodegenerative disorders. Thus, the shear stress incorporating HT-BBB assay was able to identify many promising compounds with in vivo activity on the BBB while the static assay did not, illustrating the essential need for recapitulating the biophysical environment of the BBB in screening assays.

[0171] Prior to this work, microfluidic devices incorporated shear stress to improve the functionality of in vitro BBB models have been created but often have limited throughput capacity, reducing their utility in drug screening applications. One of the first in vitro models to incorporate flow was the DIV-BBB system, which uses porous hollow fibers to culture immortalized endothelial cells. Other work has concentrated on miniaturization and mimicking the complexity of the NVU by incorporating more cell types and complex 3D architecture. These devices have been used to study immune cell trafficking, mechanical/biochemical modulation of the barrier, and the permeability of the blood tumor barrier in the BBB. Recent models have incorporated 3D architecture of the NVU and incorporate shear stress. These models are excellent tools for mimicking the complex physiology of the BBB for scientific studies but may have limitations in performing high throughput studies.

[0172] Recently, several microfluidic platforms have been developed that incorporate flow into cell culture and tissue models. The OrganoPlate platform can accommodate up to 96 independent tissues culture chips. Flow is generated in the culture chips through a gravity- driven mechanism utilizing controlled rocking of the plates. The PREDICT96 system creates flow by using pumps to fill chambers to drive gravity-driven flow in a multiwell format. This system has been used to examine endothelial cell-pericyte interaction under flow as well as perform assays of kidney, liver, and respiratory system-related biology. The model developed in this work is capable of applying higher shear stresses than previous models due to the cone-and-plate mechanism of applying shear stress. This mechanism does not require gravity- driven flow and thus can provide higher levels of shear with lower volume. In addition, the well plate format enables robotic pipetting and other standard methods to be used in assay performance. The complexity of the optimized HT-BBB model we used is lower than that of the 3D BBB chip models that can incorporate multiple cell types. This is advantageous in terms of logistical performance and well-well variability but may also limit some behaviors of the assay in terms of matching BBB function. Thus, the HT-BBB model may provide an effective way to screen a larger number of compounds prior to using more complex in vitro models or animal studies.

[0173] Overall, a novel HT-BBB model was demonstrated that has practical strengths in performing drug screening assays under flow conditions. This model was highly capable of drug screening studies and identified several compounds that have known in vivo activity towards the BBB. While it has been shown that the model is effective using primary BMEC cells, further development of the model would be merited to improve cellular aspects of the screening assay. The system, as designed, can integrate a variety of engineering culture surface or porous membranes through flexible design of the BBB plate. Thus, its capabilities could be expanded through further studies to optimize the incorporation of 3D geometry and additional cell types.

[0174] Example Drug Screen [0175] A drug screen was performed specifically using a fluorescently labeled antibody used to treat breast cancer (trastuzumab). Trastuzumab is very effective in treating HER2+ breast cancer, however, it does not penetrate the BBB to enter the brain. Breast cancer patients are often well treated with trastuzumab until they have a brain metastasis, at which point the therapy is no longer effective. A library of 780 clinically approved compounds were screened for their ability to allow trastuzumab to pass through the BBB (see attached paper). Since these compounds are already approved for clinical use, there is the potential that these drugs could be repurposed to enhance trastuzumab chemotherapy for breast cancer patients with brain metastasis.

[0176] FIG. 55 shows a graph of TEER measurements for a device utilizing various pore size polycarbonate mesh after 48 hours of shear stress exposure, according to one implementation.

[0177] FIG. 56 shows a graph of permeability of trastuzumab of an example device with BMEC seeded on the mesh, according to one implementation.

[0178] FIG. 57 shows a graph of cancer endothelial adhesion assay measuring the effects of different compounds on cell adhesion, according to one implementation.

Configuration of Certain Implementations

[0179] The construction and arrangement of the systems and methods as shown in the various implementations are illustrative only. Although only a few implementations have been described in detail in this disclosure, many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes, and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.). For example, the position of elements may be reversed or otherwise varied, and the nature or number of discrete elements or positions may be altered or varied. Accordingly, all such modifications are intended to be included within the scope of the present disclosure. The order or sequence of any process or method steps may be varied or re-sequenced according to alternative implementations. Other substitutions, modifications, changes, and omissions may be made in the design, operating conditions, and arrangement of the implementations without departing from the scope of the present disclosure.

[0180] The present disclosure contemplates methods, systems, and program products on any machine-readable media for accomplishing various operations. The implementations of the present disclosure may be implemented using existing computer processors, or by a special purpose computer processor for an appropriate system, incorporated for this or another purpose, or by a hardwired system. Implementations within the scope of the present disclosure include program products including machine-readable media for carrying or having machine-executable instructions or data structures stored thereon. Such machine- readable media can be any available media that can be accessed by a general purpose or special purpose computer or other machine with a processor. By way of example, such machine-readable media can comprise RAM, ROM, EPROM, EEPROM, CD-ROM or other optical disk storage, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to carry or store desired program code in the form of machineexecutable instructions or data structures, and which can be accessed by a general purpose or special purpose computer or other machine with a processor.

[0181] When information is transferred or provided over a network or another communications connection (either hardwired, wireless, or a combination of hardwired or wireless) to a machine, the machine properly views the connection as a machine-readable medium. Thus, any such connection is properly termed a machine-readable medium. Combinations of the above are also included within the scope of machine-readable media. Machine-executable instructions include, for example, instructions and data which cause a general-purpose computer, special purpose computer, or special purpose processing machines to perform a certain function or group of functions.

[0182] Although the figures show a specific order of method steps, the order of the steps may differ from what is depicted. Also, two or more steps may be performed concurrently or with partial concurrence. Such variation will depend on the software and hardware systems chosen and on designer choice. All such variations are within the scope of the disclosure. Likewise, software implementations could be accomplished with standard programming techniques with rule-based logic and other logic to accomplish the various connection steps, processing steps, comparison steps and decision steps.

[0183] It is to be understood that the methods and systems are not limited to specific synthetic methods, specific components, or to particular compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular implementations only and is not intended to be limiting.

[0184] Disclosed are components that can be used to perform the disclosed methods and systems. These and other components are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these components are disclosed that while specific reference of each various individual and collective combinations and permutation of these may not be explicitly disclosed, each is specifically contemplated and described herein, for all methods and systems. This applies to all aspects of this application including, but not limited to, steps in disclosed methods. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific implementation or combination of implementations of the disclosed methods.

Claims

WHAT IS CLAIMED IS:

1. An apparatus comprising: a multi-well plate defining a plurality of wells including a first well and a second well, the multi-well plate comprising a top plate, a bottom plate, and at least one support plate disposed between the top plate and the bottom plate, wherein the at least one support plate comprises a first support plate and a second support plate; a cell culture mesh supported in between the first support plate and the second support plate; one or more shafts, cones, or fins that fit within at least one of the first well and the second well of the multi-well plate and that can be positioned in close proximity to the cell culture mesh; and a rotational motor connected to drive the one or more shafts, cones, or fins to rotate, such that rotation of the shafts, cones, or fins creates fluid flow or shear stress within at least one of the first well and the second well, wherein the bottom plate defines a conduit or a lower chamber such that the first well is in fluid communication with the second well.

2. The apparatus of claim 1, wherein the cell culture mesh is disposed in the first well while the second well is left open, wherein the cell culture mesh separates the first well into an upper chamber and the lower chamber, the lower chamber being in fluid communication with the second well.

3. The apparatus of claim 1, wherein the cell culture mesh is seeded with cells.

4. The apparatus of claim 3, wherein the cells are endothelial cells or epithelial cells including lung, intestine, or mucosa cells.

5. The apparatus of any one of claims 1-4, wherein the cell culture mesh models a blood brain barrier (BBB), and the cells are endothelial cells found within the BBB.

6. The apparatus of any one of claims 1-5, wherein cells are grown (i) on both sides the cell culture mesh, (ii) in the bottom of the first well or the second well, (iii) on the sides of the first well or the second well, and/or (iv) in gels placed within the first well or the second well.

7. The apparatus of any one of claims 1-6, wherein the cells migrate through the cell culture mesh.

8. The apparatus of any one of claims 1-7, wherein both of the upper chamber of the first well and the lower chamber via the second well are accessible from the top plate such that a standard robotic pipetting apparatus having standard well geometry may access and interact with either side of the cell culture mesh.

9. The apparatus of any one of claims 1-8, further comprising one or more electrodes configured to measure transendothelial electrical resistance (TEER) across the cell culture mesh, the one or more electrodes including a first electrode placed in the first well and a second electrode placed in the second well.

10. The apparatus of any of claims 1-9, further comprising a detector capable of measuring cells within at least one of the first well and the second well during at least one of before, during, or after rotation of the one or more shafts, cones, or fins to cause fluid flow within the first well or the second well.

11. The apparatus of claim 10, wherein the detector is a laser speckle imager capable of quantifying the velocity of fluid at or about the bottom of the first well or the second well, or the detector is at least one of plate reader, a cell sorter, a cell counter, a microscope, or a camera.

12. The apparatus of claim any of claims 1-11, wherein the one or more shafts, cones, or fins are capable of fluid flow within the well of between 0.1 dyn/cm² to 20 dyn/cm², 0.5 dyn/cm²to 15 dyn/cm², 1.0 dyn/cm²to 12 dyn/cm², 0.2 dyn/cm²to 12 dyn/cm², and 5.0 dyn/cm² to 12 dyn/cm².

13. The apparatus of any of claims 1-12, wherein the flow is an oscillatory flow.

14. The apparatus of any of claims 1-13 further comprising a computer connected to at least one of the rotational motor, the detector, or both, and that includes one or more code segments that: calculate the fluid flow within each of the one or more wells; that measure cell death on exposure to fluid flow; or that measure cell adhesion.

15. The apparatus of any of claims 1-14, wherein the multi-well plate comprises a 2, 4, 6, 8, 10, 12, 24, 48, 96, 394, or 1536 well plate.

16. The apparatus of any of claims 1-15, wherein the one or more shafts, cones, or fins comprise at least one of biocompatible material, sterile, smooth, rough, trapezoidal, conical, flat, cut, or polygonal.

17. The apparatus of any of claims 1-16, wherein the one or more shafts, cones, or fins comprise at least one of steel, steel alloy, stainless steel, titanium, plastic, polymer, glass, quartz, or wood.

18. The apparatus of any of claims 1-17, wherein the multi-well plate comprises a polyacrylate, a polymethylacrylate, a polycarbonate, a polysulphone, a polyhydroxy acid, a polyanhydride, a polyorthoester, a polypropylene, a polyphosphazene, a polyphosphate, a polyester, a nylon or a mixture thereof.

19. A method of determining that a test agent interacts with, exerts an effect upon, or is able to enter cells found within the blood brain barrier, the method comprising: a) providing the apparatus of claim 1, wherein the cell culture mesh of the apparatus has been seeded with cells found in the blood brain barrier; and b) applying the test agent to the cells; c) applying, by the one or more shafts, cones, or fins, shear stress to the cells; and d) detecting a change in the cells compared to a control, wherein said change indicates that test agent interacts with, has an effect on, or is able to enter cells within the blood brain barrier.

20. The method of claim 19, wherein test agent is capable of entering the cells.

21. The method of claim 19 or 20, further comprising exposing the cells to an additional agent either before, during, or after exposure of the test agent.

22. The method of claim 21, further comprising measuring an effect of the additional agent upon interaction of the cells with the composition of interest.

23. The method of any one of claims 19-22, wherein the control comprises a measurement of the cells prior to exposure of the test agent.

24. The method of any one of claims 19-22, wherein the control comprises a standard baseline.

25. The method of any one of claims 19-24, wherein the cells or membrane have been modified to model other physiological or pathophysiological processes in the body including but not limited to modeling intestinal permeability, vascular permeability, immune cell transmigration, cancer metastasis and invasion, liver function/permeability, kidney function/permeability, lung function/permeability, or any other biological process.

26. The method of any one of claims 19-24, wherein the cells or membrane have been modified to model infection or invasion of a pathogen including bacteria, viruses, parasites, fungi, prion or other pathogen.

27. The method of any one of claims 19-24, wherein the cells or membrane have been modified to model a physiologic or pathophysiological process involving the transmigration, translocation or accumulation of a disease or injury causing agent such as a toxin, inflammatory cytokine, lipids , protein, glycan or glycocprotein.

28. The method of claim 25, wherein said modification represents a disease state.

29. The method of claim 25, wherein the disease state comprises cancer.