JP2005523710A - Leukocyte migration monitoring device and method - Google Patents

Abstract

An apparatus for monitoring leukocyte migration is provided. The present invention provides a method of using a device that monitors leukocyte migration in the presence of physiological shear flow and thus simulates the physiological conditions of blood vessels in vivo. The present invention further provides a method of using the device for high throughput screening of a plurality of test agents. The present invention further provides a number of assays and systems that can be used to further test flexibility assay systems and biological interactions. Laminar gradients are used to mimic gradient situations that exist in vivo.

Description

  The present invention relates to an apparatus and a method for monitoring an interaction between a cell or a group of cells and a base layer. In particular, the present invention relates to an apparatus and method for monitoring leukocyte migration. The present invention relates generally to biological assays performed within a gradient formed in a microsolution system.

  Inflammatory responses are attempts by the body to restore and maintain homeostasis after infection or trauma and are an integral part of body defense. Many body defense elements are located in the blood, and inflammation is the means by which they leave the blood and enter the tissue surrounding the trauma or infection site. The initial goal of inflammation is to localize and eradicate the source of trauma or infection and repair the tissue surrounding the trauma or infection.

  As a result of the initial innate immune response to infection, phagocytic cells such as mast cells in the injured tissue release various cytokines and inflammatory mediators such as histamine, leukotrienes, bradykinins, and prostaglandins. These inflammatory mediators are thin delicate cells on the inner surface of blood vessels known as the endothelium, which reversibly open the junction between cells surrounding the injured tissue. Inflammatory mediators cause increased vascular permeability and decreased blood flow rate. Another consequence of these changes in blood vessels is that leukocytes, which normally move in the middle of the blood vessel, move around the inner surface of the blood vessel and interact with the endothelium. Cytokines and inflammatory mediators released by phagocytic cells also cause adhesion molecule expression on the surface of the endothelium, resulting in an “activated” endothelium.

The initial contact between leukocytes and activated endothelium is known as “trapping” and is thought to involve the adhesion molecules P-selectin and L-selectin. The P-selectin and L-selectin are upregulated on the endothelium after exposure to inflammatory mediators. P-selectin and L-selectin belong to a family of adhesion molecules called selectins. Selectins are a group of monomeric essential membrane glycoproteins that are expressed on the surface of activated endothelium and leukocytes. Selectin is an N-terminal extracellular domain with structural homology to calcium-dependent lectins, followed by a domain homologous to epidermal growth factor, and nine consensus repeats (CR) similar to sequences found in complement control proteins including. There are three early selectins that are thought to be contained within the inflammatory response; P-selectin; E-selectin; and L-selectin. P-selectin, also known as CD62P, GMP-140, and PADGEM, is the largest selectin and is expressed on activated endothelium. E-selectin, also known as ELAM-1, is expressed on the endothelium with chemical or cytokine-induced inflammation. L-selectin, known as LECAM-1, LAM-1, Mel-14 antigen, gp90 ^mel and Leu8 / TQ-1 antigen, is the smallest selectin and is found on many leukocytes. All three selectins are thought to bind through a selectin binding ligand, at least in part, a hydrocarbon element.

During capture, P-selectin is thought to bind to its major leukocyte ligand, P-selectin glycoprotein ligand-1 (PSGL-1). Other ligands for P-selectin include CD24 and ligands that have not yet been identified. Structure of functional PSGL-1 is sialyl - including Lewis ^X (sialyl-Lewis ^X) element. Furthermore, during capture, L-selectin interacts with three well-known counter receptors or ligands, MAdCAM-1, GlyCAM-1, and CD34, but the exact ligand or counter receptor involved in capture is known. It is not done.

  Once leukocytes are captured, the leukocytes temporarily adhere to the endothelium and begin to “roll” along the endothelium. The term “rolling” refers to the literal rolling of leukocytes along the activated endothelium in the presence of fluid resistance resulting from relative movement between the endothelium and leukocytes. Rolling is thought to include P-selectin, L-selectin, and E-selectin. The binding between P-selectin and PSGL-1 appears to initially mediate “rolling” of leukocytes across the endothelium.

Anti-inflammatory cytokines produced by cells at the site of trauma or infection, such as interleukin (IL-1) and tumor necrosis factor-a (TNF-a), stimulate the endothelium to counter endothelial cells against basal lamina On the surface, it produces ligands that bind chemokines such as interleukin-8 (IL-8) and integrin such as cell adhesion factors (ICAMs) and vascular cell adhesion molecules (VCAMs). Chemokines are retained on the surface of endothelial cells opposite the basal lamina, where chemokines interact with chemokine receptors on the surface of rolling leukocytes. The interaction then causes activation of a molecule called an integrin on the leukocyte surface. Integrins are a family of heterodimeric transmembrane glycoproteins that bind cells to basement membrane extracellular matrix proteins or ligands on other cells. Integrins are composed of large α and small β subunits. Mammalian integrins form several subfamilies that share a common β subunit that binds to different α subunits. β ₂ integrin (“CD-18 family”) is composed of four different heterodimers: CD11a / CD18 (Lymphocyte function associated antigen-1 (LFA-1)); CD11b / CD18 (Mac-1); CD11c / CD18 (p150, 95), CD11d / CD18 are included. The most important β integrin subfamily on leukocytes is the latest antigen 4 (VLA-4, CD49d / CD29, α ₄ β ₁ ). Activation of these integrins by chemokines allows slowly rolling leukocytes to “stop” and endothelial ICAMs, VCAMs, and other integrin-binding ligands of endothelial cells such as collagen, fibronectin, and fibrinogen Makes it possible to bind strongly to. Once bound to the endothelial cells, the white blood cells then flatten, press between the endothelial cells, leave the blood vessels, and enter the damaged tissue through a process termed “migration”. Emigration is thought to be mediated by platelets, endothelial cell adhesion molecule (PECAM-1), junctional adhesion molecule, and CD99, a transmembrane protein.

  Despite its importance in combating infection and trauma, leukocytes can themselves promote tissue damage. During an abnormal inflammatory reaction, white blood cells can cause significant tissue damage by releasing toxic substances in the vessel wall or non-traumatic tissue. Alternatively, to the extent that the endothelium is packed with those cells, the leukocytes can adhere to the capillary wall or aggregate within the venule. Such a phenomenon is termed “Pavementing” and relates to the development of arteriosclerosis and related diseases. Such abnormal inflammatory responses include acute respiratory distress syndrome (ARDS), myocardial infarction, heart shock, stroke, or ischemia-reperfusion injury after tissue transplantation; acute and chronic allograft rejection; pulse hepatitis; sepsis, rheumatism It is linked to the pathogenesis of a variety of other clinical diseases including rheumatoid arthritis and inflammatory skin diseases.

  Several methods and devices exist in the art to study the process of leukocyte migration that is linked to various inflammatory diseases. For example, one method seeds isolated endothelial cells with a monolayer on the surface of a microtiter plate, activates the cells with a chemoattractant, and places labeled red blood cells in the plate. A test agent, such as an adhesion inhibitor, can optionally be added to the plate. The number of white blood cells that remain adhered to the endothelial cell monolayer is then measured. A significant drawback of the method is that leukocytes are not exposed to endothelial cells in the presence of shear flow and the method does not simulate physiological conditions in vivo.

  Another method involves contacting a suspension of isolated leukocytes in a suitable medium with a vascular tissue sample mounted on a microscope slide and incubating the tissue with the cell suspension on a rotating table. including. Adhered cells are fixed and counted. Since the cells are fixed, such methods eliminate the simultaneous observation of leukocyte migration. Furthermore, such methods require human vascular tissue that is difficult to obtain and expensive.

  Another method well known in the art for studying leukocyte migration involves a device consisting of two glass tubes called microslides, where one microslide is inserted into the other microslide. Can do. The small microslide is inserted into the large microslide to create a flow channel with a flat surface on which the selected adhesion molecules are present. The leukocyte suspension is perfused through the flow channel using a syringe pump over the surface to which the adhesion molecules have been immobilized. Leukocyte rolling and adhesion is then observed. Due to the size and arrangement of the device, considerable handling and manipulation is required.

  Another apparatus for studying leukocyte migration during an inflammatory response is US Pat. No. 5,460,945 to Springer et al., Entitled “Device and Method for Analysis of Blood Components and Identifying Inhibitorsand Promoters of the Inflammatory Response.” It is described in. The device consists of several different components that are bulky. For example, the device requires external manipulation and placement, creating a risk of contaminating or damaging the endothelial monolayer. The device also requires the use of a large number of cells and consequently a large amount of reagents.

  Therefore, there is a need for an improved device for studying leukocyte migration along the endothelium that simulates the physiological conditions of blood vessels. Also, a device that allows high-throughput screening of test agents that potentially affect the interaction between leukocytes and endothelium, and for each assay required by devices well known in the art There is a need for devices that do not require numbers. The present invention fulfills these needs.

SUMMARY OF THE INVENTION The present invention provides an apparatus for monitoring leukocyte migration that includes a housing defining a plurality of chambers within the apparatus. Each of the plurality of chambers includes a first well region including at least one first well, a second well region including at least one second well; and at least one connecting the first well region and the second well region to each other A channel region including a channel is included. At least one channel includes at least one leukocyte migration mediator disposed therein. At least one of the plurality of chambers on the one hand, and the first well region and the second well region in each one of the plurality of chambers on the other are arranged relative to each other to match the pitch of the standard microtiter plate. .

  The present invention provides an apparatus for monitoring leukocyte migration including a housing defining a plurality of chambers therein. Each of the plurality of chambers includes a first well region including at least one first well; a second well region including at least one second well; and at least interconnecting the first well region and the second well region A channel region including one channel. At least one channel includes an endothelial cell disposed therein. At least one of the plurality of chambers on one side and the first well and second well regions in each one of the plurality of chambers on the other side are arranged relative to each other to fit the pitch of a standard microtiter plate. .

  The present invention further provides an apparatus for monitoring a leukocyte migration mediator comprising: a support member; and a housing including an upper member, wherein the upper member is disposed on the support member by being placed in conformal contact with the support member. And wherein the support member and the top member are formed together to define at least one chamber. The at least one chamber includes a first well region including at least a first well; a second well region including at least one second well; and a channel including at least one channel connected to the first well region and the second well region. Includes area. The at least one channel includes at least one leukocyte migration mediator disposed within the channel or endothelial cells disposed within the channel.

  The present invention further provides a kit for monitoring leukocyte migration. The kit includes a device that includes a housing defining a plurality of chambers in the kit. Each of the plurality of chambers includes a first well region including at least one first well: a second well region including at least one second well; and at least one interconnecting the first well region and the second well region. A channel region including a plurality of channels. At least one of the plurality of chambers on the one hand, and the first well region and the second well region in each one of the plurality of chambers on the other are arranged relative to each other to match the pitch of the standard microtiter plate. The kit also includes a first leukocyte migration mediator.

  The present invention further provides an apparatus for monitoring leukocyte migration, wherein the apparatus includes a housing and means associated with the housing defining a plurality of chambers within the housing. Each of the plurality of chambers has an inlet means for receiving a sample containing leukocytes, an outlet means in flow communication with the inlet means for receiving a sample containing leukocytes from the inlet means; and a connection connecting the inlet means and the outlet means to each other Including means. The connecting means includes at least one leukocyte migration mediator or endothelial cell disposed therein. At least one of the plurality of chambers on one side and the inlet means and outlet means on the other are arranged relative to each other to fit the pitch of a standard microtiter plate.

  The present invention further provides a method of monitoring leukocyte migration. The method includes providing an apparatus including a housing defining a plurality of chambers therein, wherein each of the plurality of chambers includes a first well region including at least one first well, at least one second well. A second well region; and a channel region including at least one channel connecting the first well region and the second well region to each other. At least one of the plurality of chambers on the one hand and the first well region and the second well region in each one of the plurality of chambers on the other are arranged relative to each other to fit the pitch of a standard microtiter plate. . The method further comprises placing at least one leukocyte migration mediator in at least one channel or placing endothelial cells in at least one channel, and providing a sample comprising leukocytes in at least one channel. In certain embodiments, the method further comprises disposing at least one test agent in at least one channel. The method includes observing an interaction between leukocytes and at least one leukocyte migration mediator or endothelial cell. In embodiments where the test agent is placed in at least one channel, the method comprises observing the interaction of leukocytes with at least one leukocyte migration mediator or endothelial cell in the presence of the test agent.

  The present invention further provides a method for screening a plurality of test agents, comprising providing a device including a housing defining a plurality of chambers in the device, wherein each chamber comprises at least A first well region including one first well; a second well region including at least one second well; and a channel region including at least one channel connecting the first well region and the second well region to each other. Preferably, each of the plurality of test agents is different from each other. The at least one channel includes at least one leukocyte migration mediator disposed within the channel or endothelial cells disposed within the channel. At least one of the plurality of chambers on one side and the first well region and the second well region in each one of the other plurality of chambers are positioned relative to each other to match the pitch of a standard microtiter plate. . The method provides white blood cells to each channel in each one of a plurality of chambers, and places at least one plurality of test agents and a plurality of test agents in each channel in each one of a plurality of timbres. And further observing the interaction between leukocytes and at least one leukocyte migration mediator or endothelial cells in the presence of the test agent.

  The present invention further provides a method of stimulating vascular physiological conditions in vivo. The method further includes providing an apparatus comprising a chamber, the chamber comprising a first well region comprising at least one first well, a second well region comprising at least one second well, and a first well. A channel region including at least one channel connecting the region and the second well region to each other. The method further comprises placing a first leukocyte mailing mediator capable of mediating leukocyte rolling in at least one channel; a second leukocyte migration mediator capable of mediating leukocyte arrest in at least one channel. Providing a suspension containing leukocytes in about 10 μl to about 100 μl of liquid in at least one channel; and allowing the leukocyte-containing suspension to flow along at least one channel To further include.

  The present invention provides a biological assay performed using a micro-solution device to establish a dynamic gradient that can be used in combination with a static gradient of immobilized biomolecules.

Detailed Description of the Invention
Definitions The terms and definitions used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

  As used herein, the term “white blood cell” refers to neutrophils, eosinophils, basophils, monocytes, and lymphocytes including B cells and T cells, and unless otherwise specified, of platelets. Refers to that. The term “white blood cells” includes white blood cells that are usually obtained from blood samples and pathological blood samples.

  The term “leukocyte migration cascade” refers to a cascade of events that involves the migration of leukocytes along the endothelium lining the blood vessels. The leukocyte migration cascade involves the capture, rolling, arrest and migration of leukocytes on, along or through the endothelium.

  The term “mediates” as used in the context of “leukocyte migration mediator” means to affect leukocyte migration, eg, by binding to a ligand or counter-receptor of the leukocyte migration mediator. In particular, “leukocyte migration mediator” refers to any molecule involved in the leukocyte migration cascade. For example, leukocyte migration mediators include leukocyte capture mediators, leukocyte rolling mediators, leukocyte arrest mediators, leukocyte migration mediators, or any combination thereof.

  As used herein, the term “capture” refers to the tethering or initial contact between leukocytes and the vascular endothelium, and the movement of leukocytes along the endothelium is compared to the flow rate of a liquid containing free flowing leukocytes. Refers to a step in the leukocyte migration cascade characterized by being temporarily delayed.

  The term “leukocyte capture mediator” as used herein refers to a leukocyte migration mediator that mediates the capture of leukocytes on the vascular endothelium. Non-limiting examples of leukocyte capture mediators are P-selectin and L-selectin binding ligands.

  The term “capture mediator binding partner” refers to a ligand or counter-receptor that binds a leukocyte capture mediator. Non-limiting examples of capture mediator binding partners are PSGL-1 and L-selectin.

  The term “rolling” as used herein refers to a step in the leukocyte migration cascade and is characterized by the rolling of leukocytes along the vascular endothelium from receptor to receptor on the endothelium, In addition, leukocytes are characterized by forming and breaking adjacent bonds with endothelial ligands or counter receptors.

  The term “leukocyte rolling mediator” as used herein refers to any leukocyte migration mediator that mediates the rolling of leukocytes along the vascular endothelium. Non-limiting examples of leukocyte rolling mediators are P-selectin, E-selectin, and L-selectin binding ligands.

  As used herein, the term “rolling mediator binding partner” refers to any ligand or counter-receptor that binds to a leukocyte rolling mediator. Non-limiting examples of rolling mediator binding partners are PSGL-1, E-selectin binding ligand, and L-selectin.

  The term “arrest” as used herein refers to a step in the leukocyte migration cascade that is characterized by leukocytes adhering to the vascular endothelium.

  As used herein, the term “leukocyte arrest mediator” refers to any leukocyte migration mediator that mediates arrest of leukocytes on the vascular endothelium. Non-limiting examples of stop mediators are integrin-binding ligands that bind to integrins expressed on the surface of leukocytes, such as ICAM-1, ICAM-2, and VCAM-1.

  The term “stop mediator binding partner” as used herein refers to any ligand or counter-receptor that binds to a leukocyte arrest mediator. Non-limiting examples of stop mediator binding partners are integrins including LFA-1, Mac1, pI50,95, VLA-4, and VLA-5.

  The term “migration” as used herein refers to a step in the leukocyte migration cascade characterized by the exit of leukocytes from the blood vessels through the vascular endothelial cells and into the surrounding tissue.

  The term “leukocyte migration mediator” as used herein refers to any leukocyte migration mediator that mediates leukocyte migration through the vascular endothelium. Non-limiting examples of leukocyte translocation mediators are PECAM-1 and JAM.

  As used herein, the term “migration binding partner” refers to any ligand or counter-receptor that binds to a leukocyte migration mediator.

The term “physiological shear flow” includes shear flow under normal and pathological conditions. The physiological shear flow rate under normal conditions is about 0.1 to about 20 dynes / cm ² .

  As used herein, the term “test agent” refers to any substance that inhibits or promotes leukocyte migration, eg, by inhibiting or promoting capture, rolling, arrest, or migration.

  The term “pitch” as used herein refers to the distance between each vertical centerline between adjacent wells in the test orientation of the device.

  As used herein, the term “well region” refers to a region that includes one or more wells.

  As used herein, the term “well” means any hole in which a liquid can be placed.

  As used herein, the term “channel region” refers to a region that includes one or more channels, while “channel” refers to any passage.

  In the context of the present invention, the term “conformal contact” is meant to indicate a substantially liquid intimate contact on a planar or non-planar surface, indicating a mold-matching contact, and “reversible shape” “Matched contact” is meant to indicate a matched contact that can be disturbed without guaranteeing the structural integrity of the part making the matched contact.

  In the context of the present invention, the “test orientation” of the device is meant to refer to the spatial orientation of the device while monitoring leukocyte migration, and the terms “top”, “bottom”, “top”, “side” Defined against the test orientation of the device. In certain embodiments, the test orientation of the device for use in a method of monitoring leukocyte migration contemplates device orientation such that the path of migration along the channel region of any cell occurs in a substantially horizontal plane. In another aspect, the test orientation of the device for use in monitoring leukocyte migration contemplates device orientation such that the migration path along the channel region in any cell occurs in a substantially vertical plane. To do.

  The present invention generally provides devices and methods for in vitro monitoring of cell-substrate interactions. Non-limiting examples of cell types monitored by the devices and methods of the present invention include leukocytes, erythrocytes, platelets, non-blood cells, and tumor cells. Non-limiting examples of substrates that can interact with cells containing endothelium include endothelium, immobilized ligands, physisorbed adhesion and rolling molecules, and basal lamina or basal lamina mimics. In particular, the present invention provides devices and methods for in vitro monitoring of leukocyte migration in the presence of shear flow to study the cascade of events involved in inflammatory responses in vivo. The present invention provides an apparatus and method for high-throughput screening of test agents that potentially target those events. In particular, the present invention is directed to studying and targeting capture, rolling, arrest, and migration on, along, or through the endothelium (such an event is correctly called the leukocyte migration cascade).

  As schematically described in FIGS. 1-6, the apparatus 10 includes a housing 12 that defines a plurality of chambers 14 therein, for example, embodiments of the chambers 14 are described in FIGS. Each chamber 14 includes a first well region 16 that includes at least one well 18 and a second well region 20 that includes at least one second well 22. The chamber 14 further includes a channel region 24 including at least one channel 26 that connects the first well region 16 and the second well region 20 to each other. As illustrated in FIGS. 5 and 6, the first well region 16 and the second well region 20 in each one of the plurality of chambers are arranged with respect to each other to fit the pitch of a standard microtiter plate. The The plurality of chambers can be arranged with respect to each other to match the pitch of a standard microtiter plate. Generally, the first well 18 and the second well 22 are applied to receive a sample containing leukocytes, and the channel 26 is applied with an endothelial cell or leukocyte migration mediator thereon along with physiological shear. It is formed to support the flow.

  In one aspect of the invention, the channel 26 includes endothelial cells disposed therein. Endothelial cells can be placed on any surface of channel 26. In a preferred embodiment, endothelial cells are present on the bottom and side surfaces of channel 26. Endothelial cells can be arranged uniformly within the channel 26, arranged in a distributed patch, or arranged according to a concentration gradient such that the concentration decreases from the first well 18 to the second well 22. Endothelial cells may have chemokines that are activated prior to being exposed to channel 26 when placed in channel 26 or immobilized on the opposite surface of the basal lamina. A variety of cytophilic substrates can be placed in the channel 26 to aid endothelial cell adhesion. A cytophilic substrate is a substrate that has an affinity for a cell or substrate that promotes cell adhesion to a surface, including but not limited to gelatin, collagen, fibronectin, fibrin, basal lamina, and without limitation MATRIGEL. (Trademark) or other hydrogels.

  In another aspect of the invention, channel 26 includes at least one leukocyte migration mediator disposed therein. Preferably, the at least one leukocyte migration mediator comprises a plurality of leukocyte migration mediators. More preferably, the plurality of leukocyte migration mediators comprises at least one first leukocyte migration mediator and at least one second leukocyte migration mediator, wherein the at least one first and at least one second leukocyte migration mediator are different from each other. . The leukocyte migration mediator can be placed in the channel 26 to form a surface concentration gradient along the longitudinal axis of the chamber 14 at increasing concentrations from the first well 20 to the second well 22.

  In yet another aspect of the invention, channel 26 includes a chemokine disposed therein and interacts with chemokine receptors on the rolling leukocyte surface.

  The present invention further contemplates a method for monitoring leukocyte migration. In some embodiments, channel 26 includes endothelial cells disposed therein, and a sample including leukocytes is disposed in first well 18 (or second well 22), the sample being along channel 26. And the interaction between the leukocytes and the endothelial cells (such interaction, including their lack) is observed, and the sample containing leukocytes is the second well 22 (or first) as leukocytes emerging from the channel 26. Collected in one well 18). A chemoattractant is added to channel 26 to activate the endothelial cells before a sample containing leukocytes is added to first well 18 (or second well 22). In certain embodiments, a test agent is placed in channel 26 and an interaction between leukocytes and endothelial cells in the presence of the test agent is observed.

  In some embodiments, where the channel 26 includes a leukocyte migration mediator disposed therein, a sample containing leukocytes is disposed in the first well 18 (or second well 22), and the sample is along the channel 26. Allowed to flow, interactions between leukocytes and leukocyte migration mediators (such interactions, including their lack) are observed, and samples containing leukocytes are in the second well 22 (or first well 18). ) And collected as white blood cells that exit channel 26. In some embodiments, in the presence of a test agent, the interaction between the test agent and leukocytes and leukocyte migration mediators placed in channel 26 is observed.

Since the device 10 or elements of the device 10 can be adapted to an industry standard microtiter plate foot shape, the advantages of the device 10 are that the device 10 can perform multiple assays simultaneously in the same device and various test agents. It can be used to do high throughput screening. In some embodiments, as illustrated in FIG. 5, each one of the plurality of chambers 14 has a first well region 16 and a second well region 20 relative to each other to fit a standard microtiter plate pitch. Arranged. P is used to name the pitch between each of the wells 18/22, which is a 24-well microtiter plate, a 96-well microtiter plate, a 384-well microtiter plate, a 768-well microtiter plate. Titer plates, 1536 well microtiter plates can be positioned relative to each other to match one pitch. As an example, in the arrangement of the chamber 14 as shown in FIG. 5, the pitch P will be set to about 9 mm. Preferably, the device 10 itself is compatible with industry standard microtiter plate footprints. For example, the device 10 preferably has the same external dimensions and overall size as an industry standard microplate. By way of example, in the arrangement of apparatus 10 shown in FIG. 6, apparatus 10 includes 48 chambers designed in a standard 96 well plate format, each well 18/22 having a standard 96 well microtiter pitch. Each well fits into the space of each well of the plate. In this embodiment, 48 experiments can be performed. Alternatively, as seen in FIG. 6A, the chambers 14 can be positioned relative to each other to fit the pitch of a standard microtiter plate. In this alternative embodiment, the chamber 14 is sized so that the chamber 14 fits the area normally required for one well of a standard microtiter plate. For example, in this embodiment, the device 10 designed in a foot shape with a 96 well microtiter plate arrangement has 96 chambers and therefore allows 96 experiments to be performed. By adapting to the exact dimensions and standards of the standard microtiter plate, the embodiment of the device 10 will advantageously adapt to the foundation in which liquid handling, storage, registration and detection exists. The apparatus 10 facilitates high-throughput screening as it allows robotic fluid handling and automated detection and data-analysis. The use of robotic and automated systems reduces the time to prepare and perform the assay and to analyze the results of the assay. Furthermore, by using an automated system, the use of device 10 reduces the occurrence of human error in assay preparation and execution and data-analysis. Beyond that, the size of the 18/22 wells, or the size of the entire chamber 12, device 10, will fit the size of the wells of the microtiter plate and are required to perform individual assays. The white blood cell count ranges from about 10 ³ to about 10 ⁶ . This makes it possible to study a small leukocyte population, such as basophils or certain lymphocyte subsets. Furthermore, large amounts of test agents, such as leukocyte migration inhibitors and promoters, are not required to perform assays that monitor the effects of these agents on leukocyte migration.

  Based on the arrangement of the device 10, the present invention contemplates a method for screening a plurality of test agents. In this aspect, a method for screening a test agent includes providing a device that includes a housing defining a plurality of chambers. Each chamber includes: a first well region including at least one first well; a second well region including at least one second well; and at least one channel connecting the first well region and the second well region to each other. Including channel region. In certain embodiments, at least one channel comprises at least one leukocyte migration mediator disposed therein. In another aspect, the at least one channel includes endothelial cells disposed therein.

  In both embodiments, the first well region and the second well region for one of the plurality of chambers on one side and one of the plurality of chambers on the other side are relative to each other to fit the pitch of a standard microtiter plate. Arranged.

  A method for screening a test agent provides white blood cells to each channel in each one of the plurality of chambers, and places at least one of the plurality of test agents in each channel in each one of the plurality of chambers; In the presence of the test agent, the interaction between leukocytes and endothelial cells or the interaction between leukocytes and at least one leukocyte migration mediator is observed.

  For example, it is determined whether the test agent affects the number of leukocytes that have been captured, stopped, or migrated, or whether the test agent affects the number and rate of leukocytes that roll along the channel 26. sell. The test agent includes any desired biological, chemical, or electrical substance, including but not limited to an inhibitor of leukocyte migration, an enhancer of leukocyte migration, or any other agent. Further examples of test agents include proteins, nucleic acids, peptides, polypeptides, carbohydrates, lipids, hormones, enzymes, small molecules or pharmaceutical agents. The method is particularly useful in the area of drug discovery where multiple test agents can be screened on a single device 10.

  Accordingly, it is desirable that each of the test agents is different from each other and a single test agent is placed in each channel. Of course, if it is desired to test the effect of a combination of test agents, for example, to determine if there is a synergistic effect of two or more test agents, two or more test agents of multiple test agents are It can be arranged in each channel of each of the plurality of chambers.

The device of the present invention can be used to monitor leukocyte migration cascade steps under normal or pathological physiological shear flow conditions. Normal physiological flow conditions refer to the shear flow rate during non-pathological conditions and are in the range of about 0.1 dynes / cm ² to about 20 dynes / cm ² . Pathological physiological flow conditions refer to the shear flow rate during an inflammatory reaction and generally vary depending on the disease state. Physiological shear flow is preferably produced by hydrostatic pressure or by capillary action, and the flow can be made by methods well known in the art. For example, if a sample containing leukocytes is introduced into the channel 26 through the first well 18, then physiological shear flow is applied by applying pressure through aspiration adjacent to the second well 22, or the first It is made by applying pressure through a syringe pump adjacent to the well 18. Shear flow can be manipulated by changing the size of the channel or adjusting the degree of pressure applied through a suction or syringe pump. Alternatively, white blood cells can be introduced into channel 16 in a pulsatile manner.

  With respect to certain aspects of the device 10 and methods of using these aspects as described above, the channel 26 can have endothelial cells disposed therein or leukocyte migration mediators disposed therein. Endothelial cells can be grown on channel 26 in the presence or absence of shear flow. In some embodiments, when the channel 26 has an endothelial cell disposed therein, a number of different assays were performed to observe the interaction between leukocytes and endothelial cells during the leukocyte migration cascade. . For example, to study the rolling process, a sample containing white blood cells is introduced into the channel 26 through the first well 18 or the second well 22. The number of white blood cells to roll as well as the white blood cell rolling rate can then be measured. An assay that measures rolling inhibition can be performed by adding to channel 26 an inhibitor that blocks, for example, the interaction between leukocytes and endothelial cells. Similarly, assays that measure increased rolling can be performed by adding to channel 26, for example, a promoter that promotes the interaction between leukocytes and endothelial cells. A test agent can be added to channel 26 and the effect of the test agent on the interaction between leukocytes and endothelial cells can be measured.

  In order to study the process of arrest, preferably a chemoattractant is introduced into the channel 26 to “activate” the endothelium. The chemoattractant can be any molecule suitable for stimulating the endothelium to express integrin binding ligands such as ICAMs and VCAMs. Non-limiting examples of chemoattractants include cytokines such as IL-1 and TNF-α. A sample containing leukocytes is then introduced into channel 26 through first well 18 or second well 22. Preferably, the sample containing leukocytes is pre-incubated with a chemoattractant that can cause activation of a stop mediator binding partner on the leukocyte surface, such as an integrin. The chemoattractant is any suitable substance capable of causing integrin expression by leukocytes, such as formyl peptide, intercrine, IL-8, IL-8, GRO / MGSA, NAP2, ENA-78, MCP- 1 / MCAF, RANTES, I-309, another peptide, platelet activating factor (PAF), lymphokine, collagen, fibrin, and histamine. The number of cells stopped can then be measured. Assays that measure inhibition of arrest can be performed, for example, by adding inhibitors that block the interaction between chemoattractants and chemoattractant receptors on the surface of leukocytes or endothelium. A test agent can be added to channel 26 to measure the interaction between leukocytes and endothelial cells in the presence of the test agent.

  In another embodiment directed to test the migration process, a layer of endothelial cells is placed in the channel 26. In a preferred embodiment that more closely simulates in vivo conditions, the channel 26 may be first coated with a layer of fibronectin or any other basement membrane mimic prior to adding endothelial cells to the channel 26. Preferably, the endothelial cells are exposed to an eotaxin or chemokine comprising RANTES or monocyte chemoattractant protein (MCP-3 or MCP-4) prior to introduction of a sample containing leukocytes. A sample containing white blood cells is introduced into the channel 26 through the first well 18 or the second well 22. Preferably, the leukocytes are preincubated with a chemoattractant that can cause activation of a stop mediator binding partner on the leukocyte surface, such as an integrin. After the white blood cells are allowed to flow along the channel 26, the number of cells that migrate through the endothelium is counted. The migrating cells are characterized by showing a flat and dark phase appearance under the microscope. Flat, dark-phase cells can be confirmed to be present under the endothelial cell monolayer by observing the focal planes of leukocytes and endothelial cells using a microscope. If, for example, more than 50% of the cells are present below the endothelial cell monolayer in terms of quantification, the cells can be considered migrated. Emigration is expressed as the number of migrated cells divided by the total number of cells counted. Inhibition of migration is tested, for example, by blocking receptors on the endothelial cells that bind to the chemoattractant that is responsible for activating the endothelium, and determining the number of cells that migrate the endothelium. A test agent can be introduced in channel 26 to measure the interaction between leukocytes and endothelial cells in the presence of the test agent.

  In another embodiment, the endothelial cells placed in channel 26 have been altered or modified through well-known techniques in molecular biology. For example, the cells can be modified to overexpress or not express any particular gene encoding various leukocyte migration mediators responsible for the leukocyte migration cascade. Such embodiments allow precise control of leukocyte migration mediator expression and allow further manipulation of mediators involved in the leukocyte migration cascade.

  For example, endothelial cells are genetically engineered to reduce or inhibit gene expression believed to encode proteins contained in the leukocyte migration cascade to help elucidate proteins contained in the leukocyte migration cascade. Methods for genetically modifying cells are well known in the art. One such method is disclosed in US Pat. No. 6,025,192 to Beach et al., And in combination with replication deficient retroviral vectors, libraries containing such vectors, cell lines that incorporate retroviruses. Including retroviral particles produced by such vectors, proviral sequences incorporating genes from the retrovirus particles of the present invention, and circularized proviral sequences excised from the proviral sequences of the present invention incorporating the genes. .

  In another non-limiting example, endothelial cells were transformed with the vector to genetically modify the protein expressed by the endothelial cells. For a variety of techniques for transforming mammalian cells, see Keown et al. (1990) Methods in Enzymology 185: 527-537. For example, endothelial cells can be modified to express variants of the protein being tested. For example, if a protein is believed to be included in the cascade, a gene that expresses the particular protein can be modified to express the variant. When using the devices and assays of the invention, the effects of variants on various parts of the cascade can be monitored.

  By making deletions, additions, or substitutions in the sequence encoding the protein, it can be made using techniques well known in the art. A “variant” of a polypeptide is defined as an amino acid sequence that is altered by one or more amino acids. Similar minor variants may contain amino acid deletions or insertions, or both. Computer programs well known in the art to guide how and how many amino acid residues can be substituted, inserted, or deleted without sacrificing biological or immunological activity Can be found, for example, using DNAStar software. A “deletion” is defined as a change in amino acid or nucleic acid sequence that, when compared to the amino acid sequence or nucleic acid sequence of the native polypeptide, is absent of one or more amino acids or nucleic acid residues, respectively. An “insertion” or “addition” is an amino acid or nucleic acid sequence change that results in the addition of one or more amino acids or nucleic acid residues when compared to the amino acid sequence or nucleic acid sequence of the native polypeptide. A “substitution” is due to the replacement of one or more amino acids or nucleic acids by a different amino acid or nucleic acid when compared to the amino acid sequence or nucleic acid sequence of a natural polypeptide. Variants have “conservative” changes, wherein the substituted amino acid has a similar structure or chemical property. For example, replacing leucine with isoleucine. More rarely, a variant has a “non-conservative” change, where the substituted amino acid does not have a similar structure or chemical property, eg, replacement of glycine with triprofan.

  In addition to creating variants of the protein of interest, reducing or inhibiting the expression of proteins expressed by endothelial cells can be accomplished using well-known genetic modification methods. For example, endothelial cells that express leukocyte rolling mediators, such as P-selectin, may be expressed in homologous recombinant genetic “knockout” methods (eg, Capecchi, Nature, 344: 105 (1990) and within them). Cited references, Koller et al., Science, 248: 1227-1230 (1990); Zijlstra et al., Nature, 342: 435-438 (1989), Zijlstra et al., Nature, 342: 435-438 (1989), each of which is incorporated herein by reference, and also see Sena and Zarling, Nat. Genet., 3: 365-372 (1993), which is also incorporated herein. Can be genetically modified to be reduced or inhibited using. A “knockout” of a target gene means a change in the sequence of the gene that results in a decrease in the function of the target gene, resulting in an undetectable or insignificant amount of target gene expression. “Gene knockout means that the function of the gene is substantially reduced and protein expression becomes undetectable or present only in negligible amounts. The“ knock-in ”of the target gene is A host cell that results in altered or increased expression of the target gene, eg, by introducing additional additional copies of the target gene or by operatively inserting a control sequence that provides enhanced endogenous expression of the target gene Means a change in the genome.

  Expression of leukocyte migration mediators by endothelial cells can be reduced or inhibited by providing the antisense cells with an antisense nucleic acid sequence. The antisense nucleic acid sequence is complementary to a nucleic acid sequence encoding a leukocyte migration mediator or a partial nucleic acid sequence. Methods of using antisense nucleic acid sequences to inhibit expression of nucleic acid sequences are well known in the art and are described, for example, by Godson et al., J. Biol. Chem., 268: 11946-11950 (1993). Which are described and incorporated herein by reference.

  Another embodiment that creates control of the exact leukocyte migration mediator being studied, including control of the type and amount of expressed leukocyte migration mediator, is a device in which at least one leukocyte migration mediator is placed in channel 26 in the device. 10 aspects are included. For clarity, the term “leukocyte migration mediator” as used herein refers to at least one leukocyte migration mediator unless otherwise specified. By placing a leukocyte migration mediator in channel 26, it is possible to examine the ligand / receptor interactions underlying the leukocyte migration cascade, which interactions include individual events of the cascade and further You can get an understanding. Placing leukocyte transport mediators in channel 26 allows for precise targeting of the ligand / receptor interactions that underlie individual events in the leukocyte migration cascade.

  For example, in certain embodiments, the device 10 is used to test leukocyte capture, wherein the leukocyte migration mediator disposed in the channel 26 may include a leukocyte capture mediator. As a result of the initial immune response to infection, inflammatory mediators induce the expression of adhesion molecules on the surface of the endothelium, resulting in an “activated endothelium”. The initial contact between leukocytes and activated endothelium is known as “capture” and is thought to involve the capture mediator P-selectin and the capture mediator binding partner L-selectin. P-selectin is considered as the initial adhesion molecule involved in the capture process, and the binding of P-selectin to its main capture mediator binding partner, PSGL-1, is strongly linked in this process. Although the exact ligand of L-selectin on endothelial cells is unknown, L-selectin has been linked to capture. Thus, in an embodiment of the invention, the leukocyte capture mediator arranged in channel 26 may comprise, for example, P-selectin and / or L-selectin binding ligand.

  In another aspect of the invention, the device 10 can be used to test white blood cell rolling. Here, the leukocyte migration mediator may include a leukocyte rolling mediator. Once leukocytes are captured, the leukocytes temporarily adhere to the endothelium and roll along the endothelium. Leukocyte rolling includes a rolling mediator, P-selectin; a rolling mediator binding partner, L-selectin, and a rolling mediator, E-selectin, which is associated with the initial adhesion substances involved in this process. Conceivable. Thus, in this embodiment of the invention, the leukocyte rolling mediator disposed within channel 26 may comprise, for example, P-selectin, E-selectin, and / or L-selectin ligand.

  In another aspect of the invention, the device 10 is used to test leukocyte arrest, wherein the leukocyte migration mediator can comprise a leukocyte arrest mediator. Many, but not all, white blood cells are thought to adhere to the endothelium only after rolling along the endothelium. The adhesion or “arrest” of leukocytes on the outermost surface of the endothelium is initiated by chemoattractants such as IL-1 and TNF-α produced by cells at the trauma site. These chemoattractants stimulate the endothelium to produce chemokines and arrest mediators on the endothelial surface opposite the basal lamina. Stop mediators include, for example, integrin binding ligands such as ICAMs including ICAM-1, ICAM-2, and / or ICAM-3, and VCAMs including VCAM-1 and / or VCAM-2. Chemokines interact with chemokine receptors on the surface of rolling leukocytes, which causes activation of a stop mediator binding partner on the leukocyte surface. Partners to which stop mediators bind include integrins such as LFA-1, Mac-1, and p150,95, and VLA-4. Activation of the arrest mediator binding partner causes leukocytes that slowly roll, arrest and strongly inhibit arrest mediators such as ICAM-1, VCAM1, and other integrin binding ligands on the endothelium such as collagen, fibronectin, and fibrinogen. Join. Thus, in aspects of the invention, the leukocyte arrest mediator disposed in channel 26 may comprise at least one integrin binding ligand.

  In yet another aspect of the invention, the device 10 is used to test leukocyte emigration, wherein the leukocyte migration mediator disposed in the channel 26 comprises a leukocyte emigration mediator. Once bound to the endothelium, the leukocytes are flattened and squeezed between the endothelium to leave the blood vessel and enter the damaged tissue site. Leukocytes follow a chemotactic gradient of chemoattractants released by cells at the site of injured tissue. Although much remains unknown about emigration, emigration is thought to be mediated by platelets and endothelial cell adhesion factor 1 (PECAM-1). Other promising emigration mediators may be junctional adhesion molecule (JAM), ICAM-1, VE-cadherin, LFA-1, IAP, VLA-4, and possibly CD99, a transmembrane protein. Thus, in this aspect of the invention, the leukocyte translocation mediator disposed in channel 26 may comprise at least one of the adhesion molecules or another molecule determined to be associated in emigration.

  The device 10 of the present invention can be used to study the above steps of the leukocyte migration cascade in isolation, a combination of two or more steps in the leukocyte migration cascade, or all of the leukocyte migration cascade. For example, if it is desired to understand and target rolling, preferably a white blood cell rolling mediator can be placed in channel 26. If it is desired that both leukocyte rolling and arrest be studied, a rolling and arrest mediator can be placed in the channel 26. If the entire leukocyte migration cascade is tested, capture mediators, rolling mediators, stop mediators, and migration mediators can be placed in channel 26. Because a molecule belongs to one or more migrating mediator categories (eg, L-selectin that binds to P-selectin and endothelial ligand functions as a capture mediator and rolling mediator), as well as multiple mediators present together (E.g., rolling and stop mediators may be present in channel 26 to study arrest because direct adhesion from free-flowing leukocytes is considered extremely rare, and can be present in channel 26) Knowledge understands that certain steps are not isolatedly monitored. Since much remains unknown about the specific details of the cascade of events that occur during the inflammatory response, the present invention monitors leukocyte migration to gain a further understanding of the underlying mechanisms that control these events Several methods are contemplated.

  For example, a leukocyte migration mediator containing a capture mediator is placed in channel 26 to study the capture process. A sample containing white blood cells is introduced into the channel 26 through the first well 18 or the second well 22. The capture event is defined as the adhesive interaction of leukocytes that flow freely near the surface of the channel 26 containing the capture mediator, which is then the only white blood cell that can potentially interact with the capture mediator of the channel 26. is there. Different types of initial leukocyte capture can be characterized, observed and monitored. For example, transient capture that includes leukocytes that simply bind to channel 26 without initiating a rolling action, and rolling capture that includes leukocytes that continue to roll on channel 26 may be measured. The number of each type of leukocyte capture is divided by the total number of freely flowing white blood cells to determine the initial leukocyte capture frequency.

  Leukocytes can be observed through any method well known in the art and through the method disclosed in the co-pending application entitled “Test Device and Method of Making Same”. The application is hereby incorporated by reference in its entirety. Briefly, microscopes including phase contrast microscopes, fluorescence microscopes, emission microscopes, differential interference microscopes, dark field microscopes, confocal laser microscopes, digital deconvolution microscopes, and video microscopes, high-speed video cameras; and individual sensors It can be observed by using a sequence. For example, a digital movie camera can be used to monitor leukocyte activity under continuous flow conditions, or can be used to obtain a photograph at a specific time point. Such observations reveal an interaction between the capture mediator binding partner expressed by leukocytes and the capture mediator expressed by the endothelium. In order to detect such interactions, the cells are incubated with staining reagents and detected based on color or intensity contrast using appropriate microscopy techniques. Alternatively, the fluorescent label can be used to detect whether the capture mediator binding partner binds to the capture mediator.

  In another embodiment, unlabeled cells can be used to detect migration. For example, a heterogeneous mixture of multiple cell types can be introduced into channel 26 and only a single cell type can interact with capture mediators in channel 26. After cells are introduced into channel 26, antibodies specific for any antigen on the surface of the cell type can be used to label this cell type. If multiple cell types can interact with capture mediators, antibodies labeled with specific fluorophores can be used to distinguish different cell types.

In another embodiment directed to test the rolling process, a leukocyte migration mediator comprising a rolling mediator is placed in the channel 26. A sample containing leukocytes is introduced into the channel 26 through the first well or the second well. The number of leukocytes to roll and the rolling rate of leukocytes can be measured. In one aspect, the camera is coupled to the device 10 to acquire an image of white blood cell rolling along the channel 26 for a predetermined interval over a predetermined time. In this embodiment, the rolling speed of the cells is determined by measuring the length of movement of the cells (1 _frame ) in the image obtained by the camera and measuring the exposure time (t _exposure ) of the image. . To determine the rolling speed (V), the following formula:
V = c (1 _frame / t _exposure )
[Where c is a conversion factor for measuring the actual distance traveled by the cell. c can vary between images. ]
Is used.

  In another embodiment, several different assays utilizing different types of leukocytes can be performed to characterize and compare rolling rates for different cell types. In another embodiment, a number of different assays utilizing different rolling mediators are performed to characterize and compare cell rolling rates for different rolling mediators. In another embodiment directed to testing the process of arrest, a leukocyte migration mediator comprising a first leukocyte migration mediator and a second leukocyte migration mediator, wherein the first leukocyte migration mediator and the second leukocyte migration mediator are different from each other, Used. For clarity, the term “first leukocyte mediator” as used herein refers to at least one leukocyte mediator and “second leukocyte mediator” as used herein. The term necessarily refers to at least one second leukocyte migration mediator. In this embodiment, a first leukocyte migration mediator comprising a rolling mediator and a second leukocyte migration mediator comprising a stop mediator are disposed in channel 26. A liquid sample containing leukocytes is pre-incubated with a chemoattractant that can cause activation of a stop mediator binding partner on the leukocyte surface, such as an integrin. The chemoattractant is any suitable molecule capable of causing integrin expression by leukocytes, such as formyl peptide, intercrine, IL-8GRO / MGSA, NAP-2, ENA-78, MCP-1 / MCAF, RANTES, I-309, another peptide, platelet activating factor (PAF), lymphokine, collagen, fibrin and histamine. The number of cells to be arrested can then be determined, and an assay similar to that performed with the rolling mediator alone can be performed.

  To further understand the biological effects underlying the leukocyte migration cascade, particularly the interaction between leukocytes and their counter-receptors on the endothelium, the device 10 analyzes the effects of various test agents on the leukocyte migration cascade. Can be used for The test agent comprises a potential inhibitor of leukocyte migration mediator or a potential promoter of migration mediated by leukocyte migration mediator. Further examples of such test agents include proteins, peptides, polypeptides, enzymes, hormones, lipids, hydrocarbons, small molecules, and pharmaceuticals. For example, the device may include an interaction between a capture mediator and a capture mediator binding partner; an interaction between a rolling mediator and a rolling mediator binding partner; an interaction between a stop mediator and a stop mediator binding partner; and / or a translocation mediator Can be used to identify inhibitors or promoters that competitively or non-competitively inhibit or promote the interaction of a mobilizing mediator with a binding partner. As previously mentioned, preferred leukocyte migration mediators include a first leukocyte migration mediator and a second leukocyte migration mediator, wherein the first and second leukocyte migration mediators are different from each other. As such, in certain embodiments, the test agent comprises a first leukocyte migration mediator, a second leukocyte migration mediator, or both. In another embodiment, the test agent comprises a potential migration promoter mediated by a first leukocyte migration mediator, a second leukocyte migration mediator, or both. After identifying inhibitors and enhancers of the leukocyte migration cascade, these inhibitors and enhancers can be tested for efficacy in vivo and ultimately utilized as therapeutic agents.

  A leukocyte migration mediator, including a capture mediator, is placed in the channel 26 to screen for test agents that are potential inhibitors of capture. After the potential inhibitory test agent is incubated with a liquid sample containing leukocytes, the sample is introduced into the channel 26 through the first well 18 or the second well 22. A capture event is defined as the only leukocyte adhesive interaction that flows freely near the surface of the channel 26 containing the capture mediator, and thus can potentially interact with the capture mediator on the channel 26. The For example, transient capture that includes leukocytes that simply bind to channel 26 without initiating a rolling motion, and rolling capture that includes leukocytes that continue to roll on channel 26 may be determined. The number of each type of leukocyte captured is divided by the total number of freely flowing white blood cells to determine the initial capture frequency of leukocytes incubated with the potential inhibitory test agent, and the frequency is a potential inhibition It can be compared to the initial leukocyte capture frequency in the absence of the test agent. If the frequency of initial leukocyte capture is low in the presence of the test agent relative to the initial leukocyte capture frequency in the absence of the test agent, the test agent will be an inhibitor of leukocyte capture.

  A leukocyte migration mediator, including a rolling mediator, is placed in channel 26 to screen for test agents that are potential inhibitors of rolling. After the potential inhibition test agent is incubated with a liquid sample containing white blood cells, the sample is introduced into channel 26 through first well 18 or second well 22. Alternatively, the potential inhibition test agent is introduced into the liquid sample during passage of the liquid sample in channel 26 when the white blood cells begin to roll. Compared to rolling observed in the absence of the test agent, the reduction in rolling (e.g., measured by a decrease in the rate or the number of rolling white blood cells per volume) in the presence of the test agent is: It may refer to the molecule as an inhibitor of capture and / or rolling.

  To test potential inhibition test agents for arrest, leukocyte migration mediators including a first leukocyte migration mediator and a second leukocyte migration mediator are utilized, wherein the first and second leukocyte migration mediators are different from each other. In this embodiment, a first leukocyte migration mediator comprising a rolling mediator and a second leukocyte migration mediator comprising a stop mediator are disposed in the channel 26. A liquid sample containing leukocytes is pre-incubated with a chemoattractant that can cause activation of a stop mediator binding partner on the leukocyte surface, such as an integrin. After the liquid sample has been preincubated with the potential inhibition test agent, the sample is introduced into the channel 26 through the first well 18 or the second well 22. Alternatively, the potential inhibition test agent is introduced into the liquid sample during the passage of the liquid sample in channel 26 when the white blood cells begin to roll. A decrease in leukocyte arrest in the presence of the test drug compared to the observed leukocyte arrest in the absence of the test drug (e.g., a decrease in the percentage of leukocytes that are stopped, or a stop per volume) (Measured by a decrease in white blood cell count) indicates that the test agent can be an inhibitor of leukocyte arrest.

  The device 10 can be activated by the test agent acting by increasing the efficiency of the leukocyte migration cascade or by acting as a functional component thereof (eg, capture mediator, rolling mediator, stop mediator, or transmigration mediator). Can be used to identify whether it acts as a promoter of sexual response. Such functional components are detected by their ability to capture, roll, stop, or promote leukocyte emigration, where such action (of rolling mediator or stop mediator pre-existing in channel 26 of chamber 14). Loss in advance (due to lack of proper cell specificity or due to lack of rolling mediator or stop mediator). For example, the device 10 comprising a first leukocyte migration mediator comprising a rolling mediator disposed in the channel 26 and a second leukocyte migration mediator comprising a stop mediator can be used to identify a stop mediator functional in leukocyte migration. . Furthermore, the device 10 comprising a stop mediator and / or rolling mediator placed in the channel 26 can be used to identify a rolling mediator that functions in leukocyte migration.

  In order to identify the stop mediator, the rolling mediator is placed in the channel 26 with the rolling binding partner present on the surface of the leukocytes in the liquid sample and into the channel 26 through the first well 18 or the second well 22. be introduced. One or more chemoattractants that can activate leukocytes to express a stop mediator binding partner are pre-incubated with a liquid sample containing leukocytes. A test agent for testing the function that mediates arrest is placed in the channel 26. A liquid sample containing white blood cells is introduced into the channel 26 through the first well 18 or the second well 22, and after the sample has passed through the channel 26, it is determined whether the white blood cells have been stopped on the channel 26. Leukocyte arrest indicates that the test agent can be a stop mediator that recognizes the stop mediator binding partner on the surface of the same leukocyte that expresses the rolling mediator binding partner.

  To identify a rolling mediator, a test agent to be tested for rolling mediator function is placed in channel 26 and a liquid sample containing leukocytes is introduced into channel 26 through first well 18 or second well 22, The sample is then allowed to flow along the channel 26. The rolling of leukocytes along channel 26 indicates that the test agent has a rolling mediator function and that the leukocytes express a binding partner for the rolling mediator. A rolling mediator is identified by placing a test agent to be tested for rolling mediator function in channel 26 and placing a stop mediator in channel 26. One or more chemoattractants that enable the leukocytes to activate a leukocyte expressing a stop mediator binding partner, such as an integrin, are pre-incubated with a liquid sample containing leukocytes. A liquid sample containing white blood cells is then introduced into the channel 26 through the first well 18 or the second well 22 and the sample is allowed to flow along the channel 26. Leukocyte arrest indicates that the test agent has a rolling mediator function and that leukocytes expressing the arrest mediator binding partner and chemoattractant receptor also express a binding partner for the test agent.

  According to the method described above in which an increase in the number or percentage of white blood cells is detected compared to the number or percentage of white blood cells that are rolling or arrested in the absence of the test agent, the test agent is subject to leukocyte rolling or rolling and arrest. Identified as a functional element in the process or as its enhancer. The migration of leukocytes is observed and monitored by methods well known in the art and through the methods disclosed in co-pending applications, ie, “Test Device and Method of Making Same” referenced above. , Recorded and analyzed.

  The present invention provides kits for performing the aforementioned assays. For example, the kit includes a device that includes a housing 12 that defines a plurality of chambers 14. Each of the plurality of chambers 14 includes a first well region 16 including at least one first well region 18; a second well region 20 including at least one second well 22; and a first well region 16 and a second well region. 20 includes a channel region 24 that includes at least one channel 26 that interconnects the two. The first well region 16 and the second well region 20 in each one of the plurality of chambers 14 are positioned relative to each other to match the pitch of a standard microtiter plate to allow high throughput screening of test agents. The kit further includes a first leukocyte migration mediator. The kit can include a sample containing leukocytes. The sample comprising the first leukocyte migration mediator and leukocytes can be packaged in a kit in any manner well known in the art. For example, the first leukocyte migration mediator can be contained in a vial or container. A sample containing leukocytes can then be included in a separate vial or container as well. The kit may further comprise a second leukocyte migration mediator different from the first leukocyte migration mediator. The kit can include an inhibitor adapted to inhibit the first leukocyte migration mediator, the second leukocyte migration mediator, or both. The kit can include any media and buffers required for use in the device and the particular assay.

  With respect to certain details of the device 10, preferably, as illustrated by the examples in FIGS. 1 and 6, the housing 12 of the device 10 includes a support member 28 and an upper member 30 on which the support member 28 is placed. Here, the support member 28 and the upper member 30 are formed so as to define the plurality of chambers 14 together. Preferably, the housing is the size of a standard microtiter plate, such as a 24 well microtiter plate, a 96 well microtiter plate, a 384 well microtiter plate, a 768 well microtiter plate, and a 1536 well microtiter plate. The size can be adjusted to the dimensions of The top member can be made from any suitable material known in the art, including glass, plastic, or elastomeric materials such as polydimethylsiloxane. The support member can be made of glass, polyester, polycarbonate, polyacrylate, polymethyl methacrylate (PMMA), PDMS and another plastic. Preferably, the upper member 30 contacts the support member 28 in a shape-matching manner.

  In another aspect of the present invention, the device 10 includes a support member 28; an upper member 30. Here, the upper member 30 is placed on the support member 28 by coming into contact with the support member 28 in a shape-matching manner. Support member 28 and upper member 30 are formed to define at least one chamber 14 together. The at least one chamber 14 includes a first well region 16 including at least one first well 18; a second well region 20 including at least one second well 22; and the first well region 16 and the second well region 20; Including a channel region 24 including at least one channel 26 that connects to each other. In certain embodiments, at least one channel 26 includes at least one leukocyte migration mediator disposed therein. In another embodiment, at least one channel 26 includes endothelial cells disposed therein. Preferably, the upper member 30 is reversible and is configured to be placed in contact with the support member 28 in conformity with the shape. Thus, the top member 30 is preferably made from a material that is adapted to provide the support member 28 with conformal contact, preferably reversible conformal contact. In accordance with the embodiment, the flexibility of such materials among other things allows the top member 30 to closely adhere to the support member 28 in a manner that forms a substantially tight bond with the liquid therebetween. To do. The conformal contact should preferably be strong enough to prevent slippage of the top member 30 on the support member 28. If the conformal contact is reversible, the top member 30 can be made of a material that has structural integrity and allows the top member 30 to be removed by a simple peeling process. This allows the upper member 30 to be removed from the support member 28 after the experiment, properly washed, and reused for later assays. Preferably, the peeling process does not disturb any treated surface on the support member 28, such as leukocyte migration mediators or endothelial cells. Further, due to the effect of the conformal contact between the upper member 30 and the support member 28, the substantial strong liquid adhesion between the upper member 30 and the support member 28 may cause the liquid to move from the chamber to the adjacent chamber. And prevents contaminants from entering the well. Adhesion preferably occurs substantially immediately without the need to maintain external pressure. The matching of the shapes eliminates the need for the use of an adhesive agent that closely attaches the upper member 30 to the support member 28. However, although aspects of the present invention include the use of reagent-contacting reagents, the fact that such use is eliminated according to the preferred embodiment provides cost savings, time savings, and each The risk of contamination of the chamber 14 is eliminated. Preferably, the top member 30 is made of a material that does not decompose and is not easily damaged by the efficacy used in multiple tests, and a material that provides the possible versatility of the top member profile while manufacturing the same. . More preferably, the material may be chosen to allow the top member 30 to be made using photolithography. In a preferred embodiment, the material comprises an elastomer such as silicon, natural or synthetic rubber, or polyurethane. In a more preferred embodiment, the material is PDMS. The support member 28 provides support for the top member 30 to rest on and can be of any material suitable for the function. Suitable materials are well known in the art, such as glass, polystyrene, polycarbonate, PMMA, polyacrylate, PDMS, and other plastics.

  In certain embodiments, with respect to the portion of the chamber 14, the well regions 16 and 20 are juxtaposed vertically with respect to each other in the test orientation of the device 10. In a preferred embodiment, well regions 16 and 20 are positioned horizontally with respect to each other in the test orientation of device 10. Each well 18 and 22 in each well region 16 and 20 of each chamber 14 is not limited in its outer shape to a specific three-dimensional contour. They only need to be applied to receive a liquid, preferably a sample containing leukocytes therein. Preferably, the wells 18 and 22 are formed such that they are substantially defined as circles in the top view, as shown in the form of examples in FIGS. However, other contours of the top view of a given well are within the scope of the present invention and will be readily recognized by those skilled in the art. If the wells define a circle in the top view, and if the well regions are positioned with respect to each other and conform to the pitch of a standard 96 well microtiter plate, then the pitch P is set to be equal to about 9 mm, and The diameter Dw of the upper surface contour of the well is set to be equal to about 6 mm. In such a case, the length L of each channel 26 is equal to about 3 mm. As shown particularly in FIG. 2, the well 18 and well 22 are defined in part by respective through holes 18 and 22 in the upper member 30 and in part by the upper surface U of the support member 28. It is prescribed. In particular, the side of each well 18 and 22 is defined by the respective walls of the through holes 18 and 22 in the upper member 30 respectively, and the bottom surfaces of the wells 18 and 22 correspond to corresponding portions of the upper surface U of the support member 28. It is prescribed by.

  With respect to the channel region 24 of the chamber 12, the length L of the channel 26 is defined in the direction of its longitudinal axis, as seen collectively in FIGS. Further, the depth D of the channel 26 is usually defined in a direction relative to the upper surface of the housing 12; the width W of the channel 26 defined in the channel 26 is generally defined in a direction relative to the length L and the depth D. Preferably, the channel region 24 includes a plurality of straight, balanced channels 26 that extend between the well regions 16 and 20. Preferably, the channels 26 have a length L that is substantially the same, as schematically illustrated by the mode of the example of FIG. More preferably, the plurality of channels 26 includes eight channels. By using multiple channels, multiple assays can be performed simultaneously using a single sample containing white blood cells. In such embodiments, all assays are performed under unified and consistent conditions, and therefore provide statistically more accurate results. The channels 26 preferably each have a width W of 50 μm to 5 mm; a length L of about 1 to 10 mm; and a height H of about 10 to about 100 μm. More preferably, the channel 26 has a width W of about 100 microns, a length L of about 3 mm, and a height of about 50 μm to about 80 μm. The dimensions of channel 26 in channel region 24 should be configured to support leukocyte migration under conditions that stimulate such migration during the inflammatory response. Thus, the channel region should be applied to support leukocyte migration under shear flow and to support at least one leukocyte migration mediator disposed therein. It should be noted that the embodiment of the apparatus 10 described with respect to FIGS. 1-6 is merely an example, and various other forms are within the scope of the present invention. Another example of the form of device 10 is provided in the above-referenced co-pending patent application, named “Test Device and Method of Making Same”.

  The apparatus 10 of the present invention can be fabricated according to a preferred embodiment of the method of the present invention by standard photolithographic procedures. Photolithographic procedures can be used to produce a master that is a negative image of the desired profile of the top member 30. For example, the dimensions of the chamber 14, such as the size of the well regions 16 and 18, or the length of the channel region 24 can be varied to suit any advantageous design. Once an appropriate design for the original plate was chosen, and the original plate was made according to such a design, the top member material was cast, injected, poured into the original plate, and cured. Once the mold is created, the process can be repeated as long as necessary. This process not only provides flexibility in the design of the upper member, but also allows the upper member to be replicated in large quantities.

Once the device is fabricated, the leukocyte migration mediator can be placed in the channel 26. The leukocyte migration mediator can be used by directly immobilizing or physiologically adsorbing them on the upper surface U of the support member 28 or as long as the mediator can access the flow of leukocytes by the upper surface U. The solution or suspension can be placed in the channel 26 by coating the upper surface U of the support member 28. In certain embodiments, leukocyte migration mediators are immobilized directly to the top surface U, either covalently or non-covalently, such as by covalent bonding through amide, ester or lysine side chain linkages, or by adsorption techniques. Another method of placing the leukocyte migration mediator, including immobilizing the leukocyte migration mediator on the upper surface U of the support member 28, is in co-pending termed “Test Device and Method of Making Same” referred to above. In the US patent application.
The present invention provides an apparatus that includes a housing 12. The housing 12 is meant to be associated with a housing that defines a plurality of chambers 14 in the housing 12. Each of the plurality of chambers 14 includes: an inlet means for receiving a sample containing leukocytes; an outlet means in flow communication with the inlet means for receiving a sample containing leukocytes from the inlet means; and a connection connecting the inlet means and the outlet means to each other Means and the connecting means comprises at least one leukocyte migration mediator disposed therein. As shown in FIG. 6, an example of means associated with the housing defining a plurality of chambers within the housing includes a top member positioned on the support member. Such means have been substantially shown and described with respect to the embodiment of FIGS. Other such means are within the knowledge of one of ordinary skill in the art.

  Throughout this patent application, reference has been made to various publications, patents, and patent applications. The teachings and disclosure of these publications, patents, and patent applications are all incorporated herein by reference in order to more fully describe the state of the art to which this invention belongs.

  In another aspect, the present invention provides a method for assaying and studying biological phenomena that depend on or react to gradient formation and / or flow conditions. Such biological phenomena include many processes in the body, such as cell surface interactions that occur during leukocyte adhesion and rolling. In addition, studies involving chemotaxis, chemotaxis, and cell migration will provide assays that can study the movement of such cells in the presence of gradients and / or flow conditions.

  A variety of concentration gradient types are useful for studying biological systems. Such useful gradients include static gradients that have a fixed or set or substantially fixed or set concentration. One example of a static gradient is a gradient of molecules immobilized on the surface. Non-limiting examples of static gradients include the use of different concentrations of immobilized biomolecules (proteins, antibodies, nucleic acids, etc.) or immobilized chemical molecules (drugs and small molecules). Another useful gradient includes a dynamic gradient with a variable concentration. One example of a dynamic gradient is a gradient of a liquid flow having molecules at various concentrations. Non-limiting examples of liquid gradients include the use of liquid streams containing biomolecules such as growth factors, toxins, enzymes, proteins, antibodies, hydrocarbons, drugs, or other chemical and small molecules at various concentrations. .

  In certain embodiments of the invention, gradient based dynamics / solutions can be made by laminar flow techniques. Laminar flow techniques typically include two or more liquid streams from two or more different feedstocks. These liquid streams can be combined into one stream and made to flow in equilibrium with each other without turbulent mixing with each other. Liquids with different properties, such as different properties that change the low Reynolds number, flow in equilibrium and do not mix in the absence of turbulence. Since the liquid does not mix, the liquid creates a false channel (called false due to the fact that there is no physical separation between the liquids). Solution creation and surface gradients are disclosed in US Patent Application No. 2002/0113095 and Jeon, Noo Li, et al., Langmuir, 16,8311-8316 (2000). The reference is incorporated herein in its entirety. In these references, the PDMS microsolution device was used to create a gradient through a capillary microsolution network. Solutions containing different chemicals are introduced into three separate inlets and allowed to flow through a network of capillaries. The liquid stream is repeatedly mixed, mixed and divided to yield separate liquid mixtures having a pronounced composition in each of the branched channels. As illustrated in FIG. 13, when all branches were remixed, a concentration gradient was achieved through the outlet channel, perpendicular to the direction of flow.

  By combining the device of the present invention with the formation of a dynamic gradient, a vast number of assay parameters can be created by changing any part of the device. For example, along with dynamic gradient support, various conditions can be created to test many biological interactions by combining the devices described herein with cell patterning techniques. In addition, the devices and assays are useful in drug discovery and drug testing. Because many cells and biological materials are exposed to gradients ex vivo compared to when cells or biological materials exist in vivo and thus are exposed to gradient and flow conditions. Because they act differently.

  Thus, in certain aspects of the invention, the cell may be typed for channel 26. Cell shaping is well known in the art and can be accomplished by methods disclosed in the present invention (eg, but not limited to, using microcontact printing, or the use of elastomeric stencils). A solution containing any desired biomolecule or chemical / drug can be flowed over the molded cell. Furthermore, the cells are first treated with a biomolecule, such as an activator, to more closely reconstruct the biological system and then exposed to chemicals or drugs. By creating a gradient, for example by laminar flow, different amounts of biomolecules or chemicals / drugs are carried to the typed cell, and the effect of the concentration of each biomolecule or chemical / drug is tested simultaneously with each other Is done. In equilibrium with this, simultaneous comparisons reduce the variety of assays for assaying conditions.

  Creating a dynamic gradient with laminar flow provides many assay formats in combination with the device of the present invention. For example, a very large number of assays can be created by varying the combination of cells on the surface, biomolecules in channel 26, and compounds in channel 26.

  Different assay formats are possible for immobilized cells or other immobilized biomolecules such as proteins, antibodies, nucleic acids and the like. In certain embodiments, one type of cell is immobilized throughout the channel region 24. In another embodiment, the mixture of cell types is immobilized in one cell type per region 24. In another aspect, the mixture of cell types is immobilized throughout the channel region 24. This can be an advantage in monitoring cell-cell interactions. In yet another aspect, different cell types are immobilized in each different region 24.

  In addition to various immobilization schemes, further assay design flexibility depends on leukocyte migration mediators or other biomolecules present in channel 24. For example, in certain embodiments, a type of leukocyte migration mediator is present in each channel 24 at the same concentration. In another embodiment, one type of leukocyte migration mediator is present in each channel 24 at different concentrations. In certain embodiments, a different leukocyte migration mediator is present in each channel 24. In certain other embodiments, there is a mixture of leukocyte migration mediators in each channel 24. Each channel 24 may have the same mixture or a different mixture. When the mixture is the same, the ratio or concentration of different leukocyte migration mediators can be different in each channel 24.

  Similarly for compounds such as drugs or test substances, the present invention provides flexibility in assay design. For example, in certain embodiments, a single compound is present at the same concentration throughout all channels 24. In another embodiment, the same compound is present in all channels 24, but each channel 24 has a different concentration of the compound. In another embodiment, each channel 24 has a different compound. In another embodiment, one or more compounds are present. When more than one compound is present, each channel 24 can have the same mixture of compounds or a different mixture of compounds. Furthermore, when the mixture of compounds is the same, each chamber 24 can receive a different concentration of that mixture. In addition, each channel 24 receives a mixture of compounds. Here, each channel 24 may have a different proportion of each other compound.

  Such assay systems can be used to test the effects of a number of biological interactions, chemicals or drugs on cells or other biomolecules. For example, using the devices and assays of the present invention, a compound's IC50 can be measured using a laminar flow gradient of the compound from low to high concentration on the immobilized biomolecule.

Method for producing a device for monitoring leukocyte migration An original plate of a device according to the present invention is prepared using a photolithograph. The silicon substrate is cast based on the master pattern of the top member using a suitable photoresist. Thereafter, polydimethylsiloxane (PDMS) is poured onto the top of the original plate and placed under suction to draw air bubbles therefrom. The PDMS layer thus poured is cured in an oven at about 30 ° C. for about 17 hours. The apparatus is then thoroughly washed with 2% Micro 90 (product of International Products Corp.), rinsed in an “ultrasonic bath” at 70 ° C. for 10 minutes, and rinsed with deionized water followed by 100% ethanol. G The PDMS layer is then dried in nitrogen. Simultaneously pre-washed glass slides, eg, square glass slides having a size of about 12.479 +/− 0.010 cm × about 8.247 +/− 0.10 cm and a thickness of about 1.75 mm with ethanol. Wash 3 times and 2 times with methanol. Preferably, both the surface of the PDMS layer and the glass slide bonded together were plasma oxidized for about 84 seconds. Pressure is applied to the PDMS layer and glass slide using tweezers, and air bubbles in between are squeezed out. In this system, liquid contact and conformal contact is made between the PDMS layer of the upper member, the glass slide and the support member. Furthermore, due to the effectiveness of PDMS used as the material for the top member, the conformal contact between the PDMS layer and the glass slide is reversible.

  It should be noted that the method of manufacturing the device of the present invention described above is merely an example. Another example for a method of manufacturing a device is provided in a co-pending application named “Test Device and Method of Making Same” referenced above.

II. Leukocyte migration assay using a device with a rolling mediator placed in a channel in the device of the invention
Isolation of leukocytes Neutrophils are isolated from 5 ml of human blood from healthy volunteers. 5 ml of blood is diluted 1: 2 with Hank's balanced salt solution (HBSS), thereby increasing the volume of blood to the equivalent of 15 ml. The whole blood dilution is layered on 10 ml Ficoll-Paque Plus (purchased from Amersham Pharmacia Biotech AB, catalog number 17-1440-02). The blood is then centrifuged at 400 g for 30 minutes at room temperature. Remove the supernatant by aspiration without disturbing the pellet. The pellet is resuspended with HBSS and 150 μl of 6% dextran to make a 1% solution. Allow red blood cells to settle for at least 1 hour at room temperature. Many of the red blood cells settle and form a pellet, while neutrophils remain in the supernatant. The supernatant is pipetted off and diluted 1: 2 using HBSS. The suspension is centrifuged for 10 minutes at a speed of 800 g. Aspirate the supernatant and dissolve the pellet with 19 ml of deionized water. After 1 minute, resuspend the pellet with 1 ml of 10 × PBS. The suspension is centrifuged at 400 g for 10 minutes. Red blood cells are lysed in this process, and the remaining cells are mostly neutrophils. To avoid cell aggregation after an extended period at room temperature, the remaining pellet can be lysed in medium containing BSA. Cell density is determined by counting the number of cells using a hemocytometer.

B. Placement of white blood cells and leukocyte migration mediators in the chamber 20 μl of water is pipetted into the first well of the chamber of the device made according to the method disclosed in section 1 and capillary action draws water into the channel. After ensuring that there are no bubbles in the channel, an additional 10 μl of water is pipetted into the second well of the chamber. After 15 minutes and the hydrostatic pressure has been equalized, 10 μl of P-selectin (purchased from R & D Systems, catalog # ADP3) at a concentration of 50 μg / ml is pipetted into both wells. The apparatus was incubated for 2 hours at room temperature in a dish with a cover to protect the wells from drying. After incubation, the channels were washed 4 times using 0.1% bovine serum albumin (BSA) in PBS. After the last wash, all the liquid in the well was removed with a pipette, leaving the liquid only in the channel. 20 μl of 0.1% BSA in PBS was added to the first well and 10 μl of BSA in PBS was added to the second well. After 15 minutes and the hydrostatic pressure has been equalized, the neutrophils obtained from the method described in Part A in 60 μl of medium are collected in the first well of each chamber (approximately 10 ³ per well of a 24-well plate). To 10 ⁶ cells (in a media volume of 60 μl per well). Unlabeled and fluorescently labeled monocyte cell line-U937 (purchased from ATCC, catalog # TIB-202) and THP-1 (purchased from ATCC, catalog # CRL-1593.2) and other early leukocytes may be used . As seen in FIG. 7, 40 μl to 60 μl of medium can be used to create a range of flow rates under normal physiological conditions (about 0.1 dynes / cm ² to about 20 dynes / cm ² ).

C. Data Acquisition Initially 15 seconds after a sample containing leukocytes was added to the first well, a digital image was taken on a Zeiss inverted microscope using AXIOCAM ™. Data is analyzed on AXIOVISION ™ software. Time-lapse images are taken every 30 seconds for 5 minutes and every 15 seconds. Using a 10 × objective lens, the number of cells rolling along the channel was observed and recorded.
To characterize the rolling speed of leukocytes in determining a time of leukocytes rolling speed, using the images acquired by using the methods described in Part C, and the distance that leukocytes during the exposure time of the image moves Measured. In order to determine the rolling speed (V), the following formula V = c (l _time / t _exposure )
[Where
c: conversion factor for determining the actual distance traveled by the cells l _time : distance traveled by white blood cells _during capture time t _exposure : image exposure time]
Is used.
When the flow rate ratio is about 0.1 dynes / cm ² to about 20 dynes / cm ² , the t _exposure is preferably 100 milliseconds (ms).
III. Leukocyte migration assay utilizing a device having a rolling mediator and a stop mediator placed in a channel within the device of the present invention
A. Leukocyte isolation Neutrophils are isolated according to the methods described in Section II, Part A.
B. Placement of white blood cells and leukocyte migration mediator in the chamber 20 μl of water is pipetted into the first well of the chamber of the device made according to the method disclosed in section 1. Capillary action draws water into the channel. After ensuring that there are no bubbles in the channel, transfer an additional 10 μl of water into the second well of the chamber. After 15 minutes and hydrostatic pressure has been equalized, 10 μl P-selectin (purchased from R & D Systems, catalog # ADP3) with a concentration of 50 μg / ml is pipetted into the first well and 50 μg 10 μl of ICAM-1 (purchased from R & D Systems) with a concentration of / ml was simultaneously pipetted into the second well. The apparatus was incubated for 2 hours at room temperature in a dish with a cover to protect the wells from drying. Following incubation, it is washed 4 times using 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). After the last wash, all the liquid in the well was removed with a pipette, leaving the liquid only in the channel. 20 μl of 0.1% BSA in PBS was added to the first well and 10 μl of BSA in PBS was added to the second well. After 15 minutes and the hydrostatic pressure has been equalized, the neutrophils obtained from the method described in Part A, wherein the neutrophils in 60 μl of medium are placed in the first well (24 (In medium volume of about 10 ³ to 10 ⁶ cells per well of well plate, 60 μl per well) (unlabeled and fluorescently labeled monocyte cell line-U937 and THP-1 and other early leukocytes may be used) Added. As seen in FIG. 7, 40 μl to 60 μl of medium can be used to create a flow rate range (about 0.1 dynes / cm ² to about 20 dynes / cm ² ) under normal physiological conditions.
Initially 15 seconds after the sample containing data acquisition leukocytes was added to the first well, digital images were taken on a Zeiss inverted microscope using Axiocam ™. Data is analyzed on Axiovision ™ software. Time-lapse images are taken every 30 seconds for 5 minutes and every 15 seconds. Using a 10 × objective, the number of cells rolling along the channel and the number of cells adhering to the channel were observed and recorded.
IV. Leukocyte migration assay using a confluent layer of endothelial cells
Leukocyte isolation Neutrophils are isolated according to the methods disclosed in Section I, Part A.
Placement of white blood cells and endothelial cells in the chamber 10 μl of a 10-fold dilution of Matrigel ™ (purchased from BD Biosciences, catalog # 356231) is added to the first well of a device manufactured according to the method placed in Section 1 It is done. 10 μl is added to the first well and capillary action draws the solution into the channel. Matrigel ™ is allowed to gel for 15 minutes at room temperature. Another option is that the channel is fibronectin at a concentration of 1 mg / ml (purchased from GibcoBRL, catalog # 33016-015) by diluting the stock concentration of fibronectin using a 0.1% BSA solution. The channel is coated with the fibronectin obtained. 5 μl of fibronectin at a concentration of 1 mg / ml is pipetted into the first well and capillary action draws the solution into the channel. Once the channel has been coated with Matrigel ™ or fibronectin, endothelial cells are prepared for seeding. Cells are purchased in cryogenic storage vials from Clontics, Bio-Whinttaker. Cells are grown in T75 flasks until ready to split using 0.025% trypsin / EDTA. Cells are seeded onto the channel for approximately two days at 1 × 10 ⁵ cells per 5 μl medium per assay to form a confluent monolayer of endothelial cells. During these 2 days, endothelial cells were supplemented with 40 μl of fresh medium added to each well. After approximately 2 days, the endothelial cells are exposed to a concentration of 1 ng / ml TNF-α (another chemokine can be used instead) at 37 ° C. for 4 hours. At the end of 4 hours, TNF-α is washed twice using 60 μl fresh medium. The volume of internal medium in each well is washed twice with 60 μl fresh medium. The volume of medium in each well is replaced with 15 μl of fresh medium. Neutrophils in 60 μl of medium isolated from Section II, Part A are placed in the first well of the chamber (approximately 10 ³ to 10 ⁶ cells per well of a 24-well plate, in a medium volume of 60 μl per well) (unlabeled and Fluorescently labeled monocyte cell line-U937 and THP-1 and early leukocytes can be used). If a monocytic cell line is used, the cells are fluorescently labeled using cell tracking probes (Molecular Probes, catalog # C-2925 and C-2927). Cells are incubated with 1 μm concentration probe at 37 ° C. for 30 minutes. The medium is changed and the cells are placed in the incubator for an additional 30 minutes.

As seen in FIG. 7, 40 μl to 60 μl of medium is preferably used to create a range of flow rates under normal physiological conditions (about 0.1 dynes / cm ² to about 20 dynes / Cm ² .
Initially 15 seconds after the sample containing data acquisition leukocytes was added to the first well, digital images were taken on a Zeiss inverted microscope using Axiocam ™. Data is analyzed on Axiovision ™ software. Time-lapse images are taken every 30 seconds for 5 minutes and every 15 seconds. Using a 10 × objective lens, the number of cells rolling along the channel was observed and recorded.
Determination of white blood cell rolling speed To characterize the white blood cell rolling speed at a certain time, the image acquired from the method described in Part C was used to measure the distance traveled by the white blood cell during the exposure time of the image. In order to determine the rolling speed (V), the following formula V = c (l _time / t _exposure )
[Where
c: Conversion factor for determining the actual distance traveled by the cell The factor can vary from image to image.
l _time : leukocyte movement distance in captured image t _exposure : image exposure time]
Is used.
When the flow rate ratio is about 0.1 dynes / cm ² to about 20 dynes / cm ² , the t _exposure is preferably 100 ms.

V. Inhibition of leukocyte migration assay utilizing devices having rolling and stop mediators located in channels within the devices of the present invention
Leukocyte isolation Neutrophils are isolated according to the methods disclosed in Section II, Part A.
Placement of leukocytes, P-selectin, and P-selectin antibodies in the chambers For each of the five chambers, pipette 20 μl of 0.1% BSA into each of the devices 1 manufactured according to the method described in Section 1. Transfer to the first well of the chamber. Capillary action draws water into the channel. After ensuring that no bubbles were in the channel, an additional 10 μl of BSA was pipetted into the second well of each chamber. After 15 minutes and the hydrostatic pressure was assimilated, using a multipipette, 10 μl of P-selectin (50 μg / ml) was pipetted into the first well and 10 μl of ICAM-1 (50 μg / ml) ) Was pipetted into the second well. To protect the wells from drying, the apparatus was incubated for 2 hours at room temperature in a dish with a cover. After incubation, the channels in each well are washed 4 times using 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). For 5 different chambers, 100 ng / ml P-selectin antibody is pipetted into the first well of chamber # 1; 10 ng / ml P-selectin antibody is pipetted into the first well of chamber # 2. And 1 ng / ml P-selectin antibody is pipetted into the first well of chamber # 3; 100 μg / ml P-selectin antibody is pipetted into the first well of chamber # 4; and in PBS 0.1% BSA was pipetted into the first well of chamber # 5. To protect the wells from drying, the apparatus is incubated for 30 minutes at room temperature in a dish with a cover. After incubation, the channel was first washed with 20 μl BSA followed by 10 μl BSA followed by 0.1% BSA in PBS. Neutrophils in 20 μl of medium are added to the first well of each chamber (about 10 ³ to about 10 ⁶ cells per well of a 24-well plate, in a medium volume of 20 μl per well) (unlabeled and fluorescently labeled) Monocytic cell line-U937 and THP-1 and early leukocytes may be used). Initially 15 seconds after the sample containing leukocytes was added to the first well, digital images were taken on a Zeiss inverted microscope using Axiocam ™. Data is analyzed on Axiovision ™ software. Time-lapse images are taken every 30 seconds for 5 minutes and every 15 seconds. After treatment with P-selectin antibody, the number of rolling cells was observed and recorded using a 10 × objective. As can be seen from FIGS. 8 and 9, a 100 ng / ml dilution of antibody is the preferred concentration to inhibit cell rolling. As can be seen from the still photo image of FIG. 9, the number of white blood cells that adhere to the rolling and endothelium is reduced in the presence of anti-P-selectin.

Placement of leukocytes, E-selectin, and E-selectin antibodies into the chambers For each of the five chambers, pipette 20 μl of 0.1% BSA into each chamber of the device manufactured according to the method described in Section 1. To the first well. Capillary action pulls BSA into the channel. After ensuring that no air bubbles were in the channel, another 10 μl of 0.1% BSA was pipetted into the second well of each chamber. After 15 minutes and hydrostatic pressure assimilated, using a multipipette, pipet 10 μl E-selectin (50 μg / ml) into the first well and 10 μl ICAM-1 (50 μg / ml) ) Was pipetted into the second well. To protect the wells from drying, the apparatus was incubated for 2 hours at room temperature in a dish with a cover. After incubation, the channels in each well are washed 4 times using 0.1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). For 5 different chambers, 100 ng / ml E-selectin antibody is pipetted into the first well of chamber # 1; 10 ng / ml E-selectin antibody is pipetted into the first well of chamber # 2. And pipet 1 ng / ml E-selectin antibody into the first well of chamber # 3; pipette 100 μg / ml E-selectin antibody into the first well of chamber # 4; and in PBS Of 0.1% BSA was pipetted into the first well of chamber # 5. To protect the wells from drying, the apparatus is incubated for 30 minutes at room temperature in a dish with a cover. After incubation, the channels are washed 4 times with 0.1% BSA in PBS. Neutrophils in 20 μl of medium are added to the first well of each chamber (about 10 ³ to about 10 ⁶ cells per well of a 24-well plate, in a medium volume of 20 μl per well) (unlabeled and fluorescently labeled) Monocytic cell line-U937 and THP-1 and early leukocytes may be used). Initially 15 seconds after the sample containing leukocytes was added to the first well, digital images were taken on a Zeiss inverted microscope using Axiocam ™. Data is analyzed on Axiovision ™ software. Time-lapse images are taken every 30 seconds for 5 minutes and every 15 seconds. After treatment with E-selectin antibody, the number of rolling cells was observed and recorded using a 10 × objective. As can be seen from FIG. 10, a 100 ng / ml dilution of antibody is the preferred concentration to inhibit cell rolling. As can be seen from the still photo image of FIG. 10, the number of rolling and adherent leukocytes is reduced in the presence of anti-E-selectin.

VI. Inhibition of leukocyte migration assay utilizing the device of the present invention having a confluent layer of endothelial cells disposed in a channel within the device of the present invention
Leukocyte isolation Neutrophils are isolated according to the methods disclosed in Section IV, Part A.
Placement of white blood cells and endothelial cells in chambers Endothelial cells are placed in four different channels and active in four chambers (# 1- # 4) according to the method described in Section IV, Part B It becomes. For the fifth chamber (# 5), endothelial cells are placed in the channel but not activated. For these five different chambers, 100 μg / ml P-selectin antibody is pipetted into the first well of chamber # 1; 100 μg / ml E-selectin antibody is pipetted into the first well of chamber # 2. 100 μg / ml VCAM1 antibody was pipetted into the first well of chamber # 3; and 100 μg / ml BSA in PBS was pipetted into the first well of chamber # 4. To protect the wells from drying, the apparatus is incubated for 30 minutes at room temperature in a dish with a cover. After incubation, the channels were washed 4 times with 0.1% BSA in PBS. Neutrophils in 20 μl of medium are added to the first well of each chamber (about 10 ³ to about 10 ⁶ cells per well of a 24-well plate, in a medium volume of 20 μl per well) (unlabeled and fluorescently labeled) Monocytic cell line-U937 and THP-1 and early leukocytes may be used). Initially 15 seconds after the sample containing leukocytes was added to the first well, digital images were taken on a Zeiss inverted microscope using Axiocam ™. Data is analyzed on Axiovision ™ software. Time-lapse images are taken every 30 seconds for 5 minutes and every 15 seconds. After treatment with the antibody seen in FIG. 10, a 10 × objective is used to observe and record the number of rolling cells.

  The foregoing description and examples are presented merely to illustrate the invention and are not intended to be limiting. Since one of ordinary skill in the art may devise modifications of the disclosed embodiments, including the scope and materials of the present invention, the present invention should be construed to include all within the scope of the appended claims and their equivalents. .

Example VII: Selective activation of endothelial cells by transport of TNF-α in a gradient created by laminar flow The device of the present invention is covered with endothelial cells and grows to confluence (cell "cell plexus ( lawn) ". TNF-α is transported through the laminar flow into the endothelial cell flora to “activate” the endothelial cells. Each flow of solution containing TNF-α is at a different concentration, creating a vertical gradient for the channel. The gradient effectively delivers TNFα to the endothelial cell flora at different concentrations at different locations on the cell flora. Leukocytes flow over the activated endothelial cell plexus. When referring to FIG. 12, only endothelial cells activated by TNF-α provide a suitable “binding” site for leukocytes. Leukocytes did not bind equally to the entire cell flora but bound to endothelial cell plexus sites exposed to high concentrations of TNF-α and were exposed to low concentrations of TNF-α or not at all TNF -It did not bind to cell plexus sites that were not exposed to α. These results indicate that there was indeed a creation of a TNF-α concentration gradient by laminar flow.

  The invention will be more fully understood from the detailed description given below and the accompanying drawings. These are given only by way of illustration, but do not limit the invention.

FIG. 1 is a perspective view of an embodiment of an adapted device used in the method for monitoring leukocyte migration according to the present invention. 2 is a cross-sectional view of the device of FIG. 1 taken along line II-II. FIG. 3 is a top view of the apparatus of FIG. FIG. 4 is a top view of an alternative embodiment of the chamber defined in the housing of the adapted device used in the method of monitoring leukocyte migration according to the present invention. FIG. 5 is a top view of a plurality of chambers such as the chamber of FIG. 4 arranged in a predetermined relationship with respect to each other. FIG. 6 is a top, perspective view of an alternative embodiment of the adapted device used in the method of monitoring leukocyte migration according to the present invention. The apparatus here shows the dimensions and pitch of a conventional 96-well microtiter plate. FIG. 6A is an enlarged top view of individual wells of an alternative embodiment of an apparatus according to the present invention. FIG. 7 is a bar graph comparing shear flow rates under different cell suspension volumes. FIG. 8 is a graph comparing the number of rolling cells in different dilutions of P-selectin antibody. FIG. 9 is a graph and time-lapse still picture of rolling and adhering cells in different dilutions of P-selectin antibody. FIG. 10 is a graph and time-lapse still picture of rolling and adhering cells in different dilutions of E-selectin antibody. FIG. 11 is a graph and time-lapse still photographs of cells that adhere to endothelial cells in the presence of antibodies against E-selectin, P-selectin, and VCAM-1. FIG. 12 describes the experimental results including the creation of a TNF-α concentration gradient through laminar flow. The TNF-α was delivered to the confluent “cell flora” of endothelial cells. Endothelial cells contacted by TNF-α are activated and can therefore bind to leukocytes. The leukocytes were then delivered to endothelial cells. As shown in the figure, leukocytes bound to regions of endothelial cells that received high concentrations of TNF-α. On the other hand, a region not exposed to TNF-α or a region exposed to a very small amount of TNF-α does not bind leukocytes. FIG. 13 describes an exemplary capillary device that creates a laminar flow gradient.

Claims (55)

A device for monitoring leukocyte migration including a housing defining a plurality of chambers therein, each of the plurality of chambers,
A first well region comprising at least one first well;
A second well region comprising at least one second well; and a channel region comprising at least one channel connecting the first well region and the second well region to each other, wherein the at least one channel comprises Comprising at least one leukocyte migration mediator or endothelial cell disposed therein,
Here, at least one of the plurality of chambers on one side and the first well region and the second well region in each one of the plurality of chambers on the other side are arranged relative to each other to fit the pitch of a standard microtiter plate. Said device.   The device of claim 1, wherein the at least one leukocyte migration mediator comprises a leukocyte capture mediator, a leukocyte rolling mediator, a leukocyte arrest mediator, a leukocyte migration mediator, or a combination thereof.   The device of claim 2, wherein the leukocyte capture mediator is a selectin or a selectin binding ligand.   The device of claim 2, wherein the leukocyte rolling mediator is a selectin or a selectin binding ligand.   The device of claim 2, wherein the leukocyte arrest mediator is an integrin binding ligand.   The device according to claim 2, wherein the leukocyte translocation mediator is PECAM-1 or JAM.   The apparatus according to claim 1, wherein the at least one leukocyte migration mediator comprises a first leukocyte migration mediator and a second leukocyte migration mediator, wherein the first leukocyte migration mediator and the second leukocyte migration mediator are different from each other. .   8. The device of claim 7, wherein the first leukocyte migration mediator comprises a leukocyte rolling mediator and the second leukocyte migration mediator comprises a leukocyte arrest mediator.   The apparatus of claim 1, further comprising a chemokine disposed in at least one channel. The housing is:
A support member; and an upper member placed on the support member;
The apparatus of claim 1, wherein the support member and upper member are formed such that they together define a plurality of chambers.   The apparatus of claim 10, wherein the upper member contacts the support member in conformity.   At least one of the plurality of chambers on one side, and the first well region and the second well region in each one of the plurality of chambers on the other side are a 24 well microtiter plate, a 96 well microtiter plate, The apparatus of claim 1, wherein the apparatus is positioned relative to each other to match the pitch of a 384 well microtiter plate, a 768 well microtiter plate, or a 1536 well microtiter plate.   The apparatus of claim 1, wherein the at least one channel comprises a plurality of straight and balanced channels.   The apparatus of claim 1, wherein the second well region is juxtaposed horizontally with respect to the first well region in a test orientation of the apparatus.   The device of claim 1, wherein the channel region is formed to support physiological shear flow along it.   The apparatus of claim 15, wherein the channel region is formed to support physiological shear flow due to hydrostatic pressure.   The system comprising the apparatus of claim 1, further comprising a video camera operatively connected to the apparatus for observing the at least one channel.   The apparatus of claim 1, wherein the at least one leukocyte migration mediator comprises a plurality of leukocyte migration mediators, and the concentration of the plurality of leukocyte migration mediators is different in each of the channel regions in each of the plurality of chambers. A kit for monitoring leukocyte migration,
An apparatus including a housing defining a plurality of chambers therein, wherein each of the plurality of chambers includes:
A first well region comprising at least one first well;
A second well region including at least one second well; and a channel region including at least one channel connecting the first well region and the second well region to each other, wherein at least one of the plurality of chambers 1 and, on the other hand, the first well region and the second well region in each one of the plurality of chambers are positioned relative to each other to match the pitch of a standard microtiter plate; and a first leukocyte migration mediator or The kit comprising endothelial cells.   The kit according to claim 19, further comprising an inhibitor or promoter of leukocyte migration.   20. The kit of claim 19, further comprising a second leukocyte migration mediator, wherein the first leukocyte migration mediator and the second leukocyte migration mediator are different from each other. A device for monitoring leukocyte migration,
housing;
Means associated with the housing defining a plurality of chambers within the housing, wherein each of the plurality of chambers comprises:
Inlet means for receiving a sample containing white blood cells;
Outlet means for communicating a flow with the inlet means for receiving the sample containing the leukocytes from the inlet means; and connecting means for connecting the inlet means and the outlet means to each other;
Wherein the connecting means comprises at least one leukocyte migration mediator or an endothelial cell disposed therein.
Wherein the at least one of the plurality of chambers on one side and the inlet means and the outlet means on the other are arranged relative to each other to fit the pitch of a standard microtiter plate. The support means includes a support member;
23. The apparatus of claim 22, wherein the means for defining includes a support member and a top member, wherein the support member and the top member together define the plurality of chambers. Said device formed. 23. The apparatus of claim 22, wherein the inlet means includes at least one first well; and the outlet means includes at least one second well. A method for monitoring leukocyte migration, comprising a housing defining a plurality of chambers therein, wherein each of the plurality of chambers is:
A first well region including at least one first well; a second well region including at least one second well; and a channel region including at least one channel connecting the first well region and the second well region to each other. Where, on the one hand, at least one of the plurality of chambers, each of the first well region and second well region of each of the plurality of chambers are arranged relative to each other to fit the pitch of a standard microtiter plate Is;
Placing at least one leukocyte migration mediator or endothelial cell in the at least one channel;
Providing a sample comprising leukocytes in the at least one channel; and observing an interaction between the leukocytes and the at least one leukocyte migration mediator, or an interaction between the leukocytes and the endothelial cells. The monitoring method.   26. The method of claim 25, wherein the at least one leukocyte migration mediator comprises a leukocyte capture mediator, a leukocyte rolling mediator, a leukocyte arrest mediator, a leukocyte migration mediator, or any combination thereof.   27. The method of claim 26, wherein the leukocyte capture mediator is a selectin or a selectin binding ligand.   27. The method of claim 26, wherein the leukocyte rolling mediator is a selectin or a selectin binding ligand.   27. The method of claim 26, wherein the leukocyte arrest mediator is an integrin binding ligand.   26. The at least one leukocyte migration mediator comprises a first leukocyte migration mediator and a second leukocyte migration mediator, wherein the first leukocyte migration mediator and the second leukocyte migration mediator are different from each other. Method.   32. The method of claim 30, wherein the first leukocyte migration mediator comprises a leukocyte rolling mediator and the second leukocyte migration mediator comprises a leukocyte arrest mediator.   32. The method of claim 30, further comprising adding at least one inhibitor adapted to inhibit the first leukocyte migration mediator, the second leukocyte migration mediator, or both.   32. The method of claim 30, further comprising adding at least one promoter applied to promote leukocyte migration mediated by the first leukocyte migration mediator, the second leukocyte migration mediator, or both. Method.   26. The method of claim 25, further comprising adding a test agent to the sample containing leukocytes.   35. The method of claim 34, wherein the test agent comprises an inhibitor of leukocyte migration or a promoter of migration.   26. The method of claim 25, wherein providing a sample comprising leukocytes in the at least one channel comprises introducing the sample into the at least one channel in a pulsatile manner.   26. The method of claim 25, further comprising providing physiological shear flow along a longitudinal axis of the at least one channel.   38. The method of claim 37, wherein the shear flow is provided by hydrostatic pressure. The shear flow has a velocity in the range of about 0.1dynes / cm ² ~ about 20dynes / cm ^2, The method of claim 37.   26. The method of claim 25, further comprising providing a video camera operatively coupled to the device for observing the at least one channel.   26. The method of claim 25, wherein the video camera is adapted to capture images for a predetermined interval over a predetermined period of time.   26. The method of claim 25, further comprising determining a white blood cell rolling rate.   26. The method of claim 25, wherein the sample of leukocytes is present in about 10 [mu] l to about 100 [mu] l of medium. 26. The method of claim 25, wherein the white blood cell count in the sample comprising white blood cells is in the range of about 10 < ³ > to 10 < ⁶ >.   26. The method of claim 25, wherein providing the sample comprises delivering leukocytes to the at least one channel by laminar flow.   46. The method of claim 45, wherein the leukocytes are carried through a capillary microsolution network.   The at least one leukocyte migration mediator comprises a plurality of leukocyte migration mediators, and placing at least one leukocyte migration mediator or endothelial cell in the at least one channel comprises the plurality of leukocyte migration mediators or endothelial cells. 26. The method of claim 25, comprising disposing in a channel.   Arranging a plurality of leukocyte migration mediators or endothelial cells in the at least one channel comprises adjusting the concentration of the plurality of leukocyte migration mediators or endothelial cells from the at least one first well to the at least one second well. 48. The method of claim 47, comprising reducing to form a surface concentration gradient along a major axis of at least one of the plurality of chambers. A method for stimulating physiological conditions of blood vessels in vivo,
An apparatus is provided that includes a housing defining a chamber, wherein the chamber includes:
A first well region comprising at least one first well;
A second well region including at least one second well; and a channel region including at least one channel connecting the first well region and the second well region to each other;
Placing a first leukocyte migration mediator adapted to mediate leukocyte rolling in said at least one channel;
Placing a second leukocyte migration mediator that applies leukocyte arrest to the at least one channel;
Providing the at least one channel with a suspension containing leukocytes in about 10 μl to about 100 μl of medium; and allowing the suspension containing leukocytes to flow along the at least one channel. , Said method.   50. The method of claim 49, wherein the first leukocyte migration mediator is a leukocyte rolling mediator and the second leukocyte migration mediator is a leukocyte arrest mediator.   50. The method of claim 49, wherein the white blood cells flow along the at least one channel under hydrostatic pressure. A method for screening a test drug comprising:
An apparatus is provided that includes a housing defining a plurality of chambers, wherein each chamber includes:
A first well region comprising at least one first well;
A second well region comprising at least one second well; and a channel region comprising at least one channel connecting the first well region and the second well region to each other, wherein the at least one channel comprises at least One leukocyte migration mediator, or endothelial cells disposed therein, wherein the first and second well regions in each one of the plurality of chambers are adapted to each other to fit the pitch of a standard microtiter plate. Placed against
Providing white blood cells to each of the first well regions in each one of the plurality of chambers;
Placing at least one plurality of test agents in each of the first well regions in each one of the plurality of chambers;
Observing an interaction between said leukocytes and at least one leukocyte migration mediator in the presence of said at least one plurality of test agents.   53. The method of claim 52, wherein the concentration of the at least one plurality of test agents is different in each one of the first well regions of the plurality of chambers. A device for monitoring leukocyte migration including a housing,
A support member; and an upper member, wherein the upper member is placed in contact with the support member by being placed in conformity with the support member, wherein the support member and the upper member are Are formed such that they together define at least one chamber, wherein the at least one chamber is:
A first well region comprising at least one first well;
A second well region including at least one second well; and a channel region including at least one channel connecting the first well region and the second well region to each other, wherein the at least one channel includes therein The device comprising at least one leukocyte migration mediator disposed in the device.   55. The apparatus of claim 54, wherein the at least one chamber includes a plurality of chambers.

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