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EP1502108A1 - Dispositif et procede destines a controler la migration des leucocytes - Google Patents

Dispositif et procede destines a controler la migration des leucocytes

Info

Publication number
EP1502108A1
EP1502108A1 EP03719923A EP03719923A EP1502108A1 EP 1502108 A1 EP1502108 A1 EP 1502108A1 EP 03719923 A EP03719923 A EP 03719923A EP 03719923 A EP03719923 A EP 03719923A EP 1502108 A1 EP1502108 A1 EP 1502108A1
Authority
EP
European Patent Office
Prior art keywords
mediator
well
leukocyte
channel
leukocyte migration
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03719923A
Other languages
German (de)
English (en)
Other versions
EP1502108A4 (fr
Inventor
Gregory Kirk
Enoch Kim
Emanuele Ostuni
Olivier Schueller
Paul Sweetnam
Matthew Brown
Bernardo Aumond
Brian Benoit
Johanna Cruceta
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Surface Logix Inc
Original Assignee
Surface Logix Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US10/206,112 external-priority patent/US6893851B2/en
Priority claimed from US10/241,445 external-priority patent/US7326563B2/en
Application filed by Surface Logix Inc filed Critical Surface Logix Inc
Publication of EP1502108A1 publication Critical patent/EP1502108A1/fr
Publication of EP1502108A4 publication Critical patent/EP1502108A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing

Definitions

  • the present invention relates to devices and methods for monitoring the interaction of a cell or group of cells with a substratum.
  • the present invention relates to devices and methods for monitoring leukocyte migration.
  • the present invention also relates generally to biological assays performed in gradients formed in microfluidic systems. BACKGROUND OF THE INVENTION
  • the inflammatory response is an attempt by the body to restore and maintain homeostasis after infection or injury, and is an integral part of body defense. Most of the body defense elements are located in the blood and inflammation is the means by which these elements leave the blood and enter the tissue around the injured or infected site. The primary objective of inflammation is to localize and eradicate the source of injury or infection and repair tissue surrounding the site of injury or infection.
  • phagocytes such as mast cells in the damaged tissue release a variety of cytokines and inflammatory mediators, such as histamines, leukotrienes, bradykinins, and prostaglandins.
  • cytokines such as histamines, leukotrienes, bradykinins, and prostaglandins.
  • the inflammatory mediators reversibly open the junctional zones between the thin delicate cells of the inner surface of the blood vessels, known as the endothelium, that surround the damaged tissue.
  • the inflammatory mediators also cause increased blood vessel permeability and decreased blood flow velocity.
  • leukocytes which normally travel in the center of the blood vessel, move out to the periphery of the inner surface of the blood vessel to interact with the endothelium.
  • the cytokines and inflammatory mediators released by the phagocytes also induce the expression of adhesion molecules on the surface of the endothelium, resulting in an "activated" endothelium.
  • the first contact of leukocytes with the activated endothelium is known as
  • selectins are a group of monomeric, integral membrane glycoproteins expressed on the surface of activated endothelium and leukocytes. Selectins contain 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 regulatory proteins.
  • P-selectin also known as CD62P, GMP-140, and PADGEM
  • E-selectin also known as ELAM-1
  • L-selectin also known as LECAM-1, LAM-1, Mel-14 antigen, gp90 mel , and Leu8/TQ-1 antigen
  • All three selectins are thought to bind to selectin binding ligands, at least in part through a carbohydrate component.
  • PSGL-1 main leukocyte ligand P- selectin glycoprotein ligand- 1
  • Other ligands of P-selectin include CD24 and yet uncharacterized ligands.
  • the structure of functional PSGL-1 includes a sialyl-Lewis x component.
  • L-selectin is thought to bind to its ligand on endothelial cells. L-selectin interacts with three known counter receptors or ligands, MAdCAM-1, GlyCAM-1, and CD34, although the precise ligand or counter receptor involved in capture is unknown.
  • leukocytes Once leukocytes are captured, they may transiently adhere to the endothelium and begin to "roll” along the endothelium.
  • rolling refers to the literal rolling of leukocytes along the activated endothelium in the presence of fluid drag forces arising from the relative movement between the endothelium and the leukocytes. Rolling is thought to involve P-selectin, L-selectin, and E-selectin. Bonds between P-selectin and PSGL-1 are thought to primarily mediate the "rolling" of leukocytes across the endothelium.
  • Proinflammatory cytokines such as interleukin-1 (IL-1), and tumor necrosis factor- ⁇ (TNF- ⁇ ) produced by cells at the injured or infected site stimulate the endothelium to produce chemokines such as interleukin-8 (IL-8) and integrin binding ligands such as intercellular adhesion molecules (ICAMs) and vascular cell adhesion molecules (VCAMs) on the the surface of the endothelial cells opposite the basal lamina.
  • chemokines are held on the surface of the endothelial cells opposite the basal lamina where the chemokines interact with chemokine receptors on the surface of the rolling leukocytes.
  • Integrins are a family of heterodimeric transmembrane glycoproteins that attach cells to extracellular matrix proteins of the basement membrane or to ligands on other cells. Integrins are composed of large ⁇ and small ⁇ subunits. Mammalian integrins form several subfamilies sharing common ⁇ subunits that associate with difference ⁇ subunits.
  • ⁇ 2 integrins include four different heterodimers: CD 11 a/CD 18 (Lymphocyte Function- Associated Antigen- 1 (LFA-1)); CDl lb/CD18 (Mac-l); CDl lc/CD18 (pl50,95), and CDl ld/CD18.
  • LFA-1 Lymphocyte Function- Associated Antigen- 1
  • Mac-l CDl lb/CD18
  • CDl lc/CD18 pl50,95
  • CDl ld/CD18 CDl ld/CD18.
  • the most important member of the ⁇ i integrin subfamily on leukocytes is Very Late Antigen 4 (NLA-4, CD49d/CD29, ⁇ 4 ⁇ ).
  • Transmigration Activation of these integrins by chemokines enables the slowly rolling leukocytes to "arrest” and strongly bind to the endothelium' s ICAMs, VCAMs, and other integrin binding ligands of the endothelial cells, such as collagen, fibronectin, and fibrinogen. Once bound to the endothelial cells, the leukocytes then flatten and squeeze between the endothelial cells to leave the blood vessels and enter the damaged tissue through a process termed "transmigration.” Transmigration is thought to be mediated by platelets, endothelial cell adhesion molecule-1 (PECAM-1), junctional adhesion molecule (JAM), and possibly CD99, a transmembrane protein.
  • PECAM-1 endothelial cell adhesion molecule-1
  • JAM junctional adhesion molecule
  • CD99 a transmembrane protein
  • leukocytes themselves can promote tissue damage.
  • leukocytes can cause significant tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue.
  • leukocytes may stick to the capillary wall or clump in venules to such a degree that the endothelium becomes lined with these cells.
  • Such a phenomenon referred to as "pavementing,” may be related to the development of arteriosclerosis and associated diseases.
  • ARDS adult respiratory distress syndrome
  • ischemia-reperfusion injury following myocardial infarction, shock, stroke, or organ transplantation
  • acute and chronic allograft rejection vasculitis
  • sepsis sepsis
  • rheumotoid arthritis rheumotoid arthritis
  • inflammatory skin diseases Several methods and devices exist in the art to study the processes of leukocyte migration implicated in these various inflammatory diseases. For example, one method involves plating a monolayer of isolated endothelial cells on the surface of microtiter plates, activating the cells with a chemoattractant and then placing labeled leukocytes in the plate.
  • a test agent such as an adhesion inhibitor, may be optionally added to the plate.
  • the number of leukocytes that remain adherent to the endothelial cell monolayer is then determined.
  • a significant disadvantage of this method is that the leukocytes are not exposed to the endothelial cells in the presence of shear flow and thus this method does not simulate physiological conditions in vivo.
  • Another method involves contacting a suspension of isolated leukocytes in a suitable medium with a human vascular tissue sample mounted on a microscope slide and then incubating the tissue with a cell suspension on a rotating table. The adhered cells are fixed and counted. Because cells are fixed, such a method precludes the observation of leukocyte migration in real time. In addition, such a method requires human vascular tissue, which can be difficult and costly to obtain.
  • Another method known in the art to study leukocyte migration involves a device consisting of two glass tubes called microslides, one microslide capable of being inserted into the other.
  • the smaller microslide is inserted into the larger one to create a flow channel with a flat surface on which selected adhesion molecules are present.
  • a suspension of leukocytes is then perfused through the flow channel over the adhesion molecule immobilized surface using a syringe pump. The rolling and adhesion of the leukocytes is then observed. Because of the size and configuration of this device, it requires considerable handling and manipulation.
  • Another device to study leukocyte migration during the inflammatory response is described in U.S. Patent No. 5,460,945 to Springer et al.
  • This device consists of several different components that are bulky in size. As such, it requires extra handling and positioning, creating the risk of contaminating or damaging the endothelial monolayer. This device also requires the use of a large number of cells and consequently a large amount of reagents.
  • the present invention provides a device 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 a channel region including at least one channel connecting the first well region and the second well region with one another.
  • the 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 regions and the second well regions of respective ones of the plurality of chambers on the other hand, are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the present invention also provides a device 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 a channel region including at least one channel connecting the first well region and the second well region with one another.
  • the at least one channel includes endothelial cells disposed therein.
  • At least one of the plurality of chambers on the one hand, and the first well regions and the second well regions of respective ones of the plurality of chambers on the other hand, are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the present invention furthermore provides a device for monitoring leukocyte migration including a housing comprising: a support member; and a top member, the top member mounted to the support member by being placed in conformal contact with the support member, wherein the support member and the top member are configured such that they together define at least one chamber.
  • the at least one chamber includes 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 with one another.
  • the at least one channel includes at least one leukocyte migration mediator disposed therein or endothelial cells disposed therein.
  • the present invention further provides a kit for monitoring leukocyte migration.
  • the kit comprises a device 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 a channel region including at least one channel connecting the first well region and the second well region with one another.
  • At least one of the plurality of chambers on the one hand, and the first well regions and the second well regions of the respective ones of the plurality of chambers on the other hand, are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the kit also comprises a first leukocyte migration mediator.
  • the present invention additionally provides a device for monitoring leukocyte migration comprising a housing and means associated with the housing defining a plurality of chambers in the housing.
  • Each of the plurality of chambers includes an inlet means for receiving a sample comprising leukocytes; an outlet means in flow communication with the inlet means for receiving the sample comprising leukocytes from the inlet means; and connection means connecting the inlet means and the outlet means to one another.
  • the connection means includes at least one leukocyte migration mediator disposed therein or endothelial cells disposed therein.
  • At least one of the plurality of chambers on the one hand, and the inlet means and the outlet means on the other hand are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the invention further provides a method of monitoring leukocyte migration.
  • the method comprises providing a device including a housing defining a plurality of chambers therein, each of the plurality of chambers including: 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 with one another.
  • At least one of the plurality of chambers on the one hand, and the first well regions and the second well regions of the respective ones of the plurality of chambers on the other hand, are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the method further comprises placing at least one leukocyte migration mediator in the at least one channel or placing endothelial cells in the at least one channel and providing a sample comprising leukocytes in the at least one channel.
  • the method additionally includes placing at least one test agent in the at least one channel.
  • the method further includes observing the interaction between the leukocytes and the at least one leukocyte migration mediator or the endothelial cells.
  • a test agent is placed in the at least one channel
  • the method includes observing the interaction between the leukocytes and the at least one leukocyte migration mediator or the endothelial cells in the presence of the test agent.
  • the present invention furthermore provides a method of screening a plurality of test agents comprising: providing a device comprising a housing defining a plurality of chambers therein, each of the chambers including: 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 with one another.
  • each of the plurality of test agents are different from one another.
  • the at least one channel includes at least one leukocyte migration mediator disposed therein or endothelial cells disposed therein.
  • At least one of the plurality of chambers on the one hand, and the first well regions and the second well regions of respective ones of the plurality of chambers on the other hand, are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the method further includes providing leukocytes in each of the channels of the respective ones of the plurality of chambers; placing at least one of the plurality of test agents in each of the channels of respective ones of the plurality of chambers; and observing the interaction between the leukocytes and the at least one leukocyte migration mediator or endothelial cells in the presence of the test agent.
  • the present invention additionally provides a method of simulating physiological conditions of a blood vessel in vivo.
  • the method comprises providing a device comprising a chamber, the chamber including: 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 with one another.
  • the method further comprises placing a first leukocyte migration mediator capable of mediating rolling of a leukocyte in the at least one channel; placing a second leukocyte migration mediator capable of mediating arrest of a leukocyte in the at least one channel; providing a suspension comprising leukocytes in about 10 microliters to about 100 micro liters of fluid in the at least one channel; and allowing the suspension comprising leukocytes to flow along the at least one channel.
  • the present invention also provides biological assays performed using microfluidic devices to establish dynamic gradients that may be used in conjunction with static gradients of immobilized biomolecules.
  • Figure 1 is a perspective view of an embodiment of a device adapted to be used in a method for monitoring leukocyte migration according to the present invention.
  • Figure 2 is a cross-sectional view of the device of Fig. 1 along lines 11-13.
  • Figure 3 is a top plan view of the device of Fig. 1.
  • Figure 4 is a top plan view of an alternative embodiment of a chamber defined in a housing of a device adapted to be used in a method for monitoring leukocyte migration according to the present invention.
  • Figure 5 is a top plan view of a plurality of chambers such as the chamber of Fig. 4 disposed in a predetermined relationship with respect to one another.
  • Figure 6 is a top, perspective view of an alternative embodiment of a device adapted to be used in a method for monitoring leukocyte migration according to the present invention, where the device displays the dimensions and pitch of a standard 96-well microtiter plate.
  • Figure 6 A is a top enlarged view of an individual well of an alternative embodiment of the device according to the present invention.
  • Figure 7 is a bar graph comparing the velocity of shear flow under different cell suspension volumes.
  • Figure 8 is a graph comparing the number of cells rolling under different dilutions of P-selectin antibody.
  • Figure 9 is a graph and time-lapsed still photographs of cells rolling and adhering under different dilutions of P-selectin antibody.
  • FIG 10 is a graph and time-lapsed still photographs of cells rolling and adhering under different dilutions of E-selectin antibody.
  • Figure 11 is a graph and time-lapsed still photographs of cells adhering to endothelium in the presence of antibodies to E-selectin, P-selectin, and VCAM-1.
  • Figure 12 depicts the results of an experiment involving the creation of a concentration gradient of TNF- ⁇ via laminar flow.
  • the TNF- ⁇ was delivered to a confluent "lawn" of endothelial cells.
  • the endothelial cells that were contacted by the TNF- ⁇ were activated and thus are able to bind the leukocytes.
  • Leukocytes were then delivered to the endothelial cells.
  • the leukocytes bound to the area of the endothelial cells that received high concentrations of TNF- ⁇ whereas those areas not exposed to TNF- ⁇ or exposed to very little TNF- ⁇ did not bind leuk
  • Figure 13 depicts an exemplary microfluidic device for creating a laminar flow gradient.
  • leukocytes refers to granulocytes including neutrophils, eosinophils, basophils, monocytes, and lymphocytes including B cells and T cells and unless otherwise specified, platelets.
  • leukocytes includes leukocytes obtained from both normal blood samples and pathological blood samples.
  • leukocyte migration cascade refers to the cascade of sequential events involving a leukocyte's migration along the endothelium lining a blood vessel.
  • the leukocyte migration cascade includes the capture, rolling, arrest, and transmigration of a leukocyte on, along, or through the endothelium.
  • leukocyte migration mediator refers to any molecule that mediates the migration of leukocytes along the endothelium lining a blood vessel.
  • the term “mediates” as used in the context of a “leukocyte migration mediator” means influencing the migration of a leukocyte by, for example, binding to the ligand or counter-receptor of the leukocyte migration mediator.
  • the term “leukocyte migration mediator” refers to any molecule involved in the leukocyte migration cascade.
  • a leukocyte migration mediator includes a leukocyte capture mediator, a leukocyte rolling mediator, a leukocyte arrest mediator, a leukocyte transmigration mediator, or any combination thereof.
  • capture refers to a step in the leukocyte migration cascade characterized by the tethering or first contact of leukocyte with the endothelium of a blood vessel so that the motion of the leukocyte along the endothelium is temporarily delayed relative to the flow of fluid containing free flowing leukocytes.
  • leukocyte capture mediator refers to a leukocyte migration mediator that mediates the capture of a leukocyte on the endothelium of a blood vessel.
  • leukocyte capture mediators are P-selectin and L-selectin binding ligands.
  • capture mediator binding partner refers to any ligand or counter- receptor that binds a leukocyte capture mediator.
  • capture mediator binding partners are PSGL-1 and L-selectin.
  • rolling refers to a step in the leukocyte migration cascade, and is characterized by the rolling of a leukocyte along the endothelium of a blood vessel from receptor to receptor on the endothelium further characterized by leukocytes forming and breaking adhesive bonds with endothelial ligands or counter-receptors.
  • leukocyte rolling mediator refers to any leukocyte migration mediator that mediates the rolling of a leukocyte along the endothelium of a blood vessel.
  • leukocyte rolling mediators are P-selectin, E-selectin, and L-selectin binding ligands.
  • rolling mediator binding partner refers to any ligand or counter-receptor that binds to a leukocyte rolling mediator.
  • rolling mediator binding partners are PSGL-1, E-selectin binding ligand, and L-selectin.
  • arest refers to a step in the leukocyte migration cascade characterized by the adherence of leukocytes to the endothelium of a blood vessel.
  • leukocyte arrest mediator refers to any leukocyte migration mediator that mediates the arrest of a leukocyte on the endothelium of a blood vessel.
  • arrest mediators are integrin binding ligands, such as ICAM-1, ICAM-2, and VCAM-1 that bind integrins expressed on the surface of leukocytes.
  • arrest mediator binding partner refers to any ligand or counter- receptor that binds to a leukocyte arcest mediator.
  • Non-limiting examples of arrest mediator binding partners are integrins including LF A- 1 , Mac- 1 , pl50,95, VLA-4, and VLA-5.
  • transmigration refers to a step in the leukocyte migration cascade characterized by the exit of leukocytes from a blood vessel to surrounding tissue through passage between cells of the endothelium of the blood vessel.
  • leukocyte transmigration mediator refers to any leukocyte migration mediator that mediates the transmigration of a leukocyte through the endothelium of a blood vessel.
  • leukocyte transmigration mediators are PEC AM- 1 and JAM.
  • transmigration binding partner refers to any ligand or counter-receptor that binds to a leukocyte transmigration mediator.
  • physiological shear flow includes shear flow under normal and pathological conditions. Physiological shear flow rate under normal conditions is about 0.1 to about 20 dynes/cm. 2
  • test agent refers to any substance that inhibits or promotes leukocyte migration, for example, by inhibiting or promoting capture, rolling, arrest, or transmigration.
  • pitch refers to the distance between respective vertical centerlines between adjacent wells in the test orientation of the device.
  • well region as used herein is meant to refer to a region that comprises one or a plurality of wells.
  • well as used herein is meant to indicate any cavity that is able to receive a fluid therein.
  • channel region refers to a region that comprises one or a plurality of channels therein, while “channel” refers to any passageway.
  • conformal contact is meant to designate a substantially fluid-tight, form-fitting contact with a planar or non-planar surface
  • reversible conformal contact is meant to designate a conformal contact that may be interrupted without compromising a structural integrity of the members making the conformal contact.
  • test orientation of the device is meant to refer to a spatial orientation of the device during the monitoring of leukocyte migration and the terms “top,” “bottom,” “upper” and “side” are defined relative to the test orientation of the device.
  • the test orientation of the device for use in a method of monitoring leukocyte migration contemplates the orientation of the device such that a migration path along the channel region of any cells occurs in a substantially horizontal plane.
  • the test orientation of the device for use in monitoring leukocyte migration contemplates the orientation of the device such that a migration path along the channel region of any cells occurs in a substantially vertical plane.
  • the present invention generally provides devices and methods for in vitro monitoring the interaction of cells with a substratum.
  • cell types that may be monitored by the devices and methods of the present invention include leukocytes, red blood cells, platelets, non-blood cells, and tumor cells.
  • types of substratum that may interact with the cells include the endothelium, immobilized ligands, physisorbed adhesion and rolling molecules and basal lamina or basal lamina mimic.
  • the present invention provides a device and method for in vitro monitoring of leukocyte migration in the presence of shear flow in order to study the cascade of events involved in the inflammatory response in vivo.
  • the present invention also provides a device and method for the high- throughput screening of test agents that potentially target these events.
  • the present invention is directed to study and target the capture, rolling, arrest, and transmigration of a leukocyte on, along, or through the endothelium (such events collectively referred to as the "leukocyte migration cascade").
  • device 10 generally includes a housing 12 defining a plurality of chambers 14 therein, such as, by way of example, embodiments of chamber 14 depicted in Figs. 1-6.
  • Each chamber 14 includes: a first well region 16 including at least one first well 18 and a second well region 20 including at least one second well 22.
  • the chamber 14 further includes a channel region 24 including at least one channel 26 connecting the first well region 16 and the second well region 20 with one another.
  • the first well regions 16 and the second well regions 20 of the respective ones of the plurality of chambers are disposed relative to one another to match a pitch of a standard microtiter plate.
  • first well 18 and second well 22 are adapted to receive a sample comprising leukocytes and channel 26 is adapted to receive endothelial cells or leukocyte migration mediators thereon and is configured to support physiological shear flow therealong.
  • channel 26 contains endothelial cells disposed therein.
  • the endothelial cells may be disposed on any surface or surfaces of channel 26. In a preferred embodiment, the endothelial cells are on the bottom and side surfaces of channel 26.
  • the endothelial cells may be disposed uniformly in channel 26, may be disposed in discrete patches, or may be disposed along a concentration gradient such that the concentration of endothelial cells decreases from first well 18 to second well 22.
  • the endothelial cells may be activated prior to exposure to channel 26 or may have chemokines immobilized on the surface opposite the basal lamina therein upon exposure to channel 26.
  • Cytophilic substances may be disposed in channel 26 to assist in the attachment of endothelial cells.
  • Cytophilic substances are generally substances that have an affinity for cells or substances that promote cell attachment to the surface and include, for example, gelatin, collagen, fibronectin, fibrin, basal lamina, including, but not limited to MATRIGELTM or other hydrogels.
  • channel 26 includes at least one leukocyte migration mediator disposed therein.
  • 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 the at least one second leukocyte migration mediators are different from one another.
  • the leukocyte migration mediators may also be disposed in channel 26 so as to form a surface concentration gradient along a longitudinal axis of chamber 14 in increasing concentration from first well 20 to second well 22.
  • channel 26 includes chemokines disposed therein to interact with chemokine receptors on the surface of rolling leukocytes.
  • the present invention also contemplates a method of monitoring leukocyte migration.
  • a sample including leukocytes is placed in first well 18 (or second well 22), the sample is allowed to flow along channel 26, the interaction (such interaction including a lack thereof) between the leukocytes and the endothelial cells is observed, and the sample including leukocytes is collected in second well 22 (or the first well 18) as the leukocytes exit channel 26.
  • a chemoattractant may be added to channel 26 to activate the endothelial cells before a sample containing leukocytes is added to first well 18 (or second well 22).
  • a test agent is placed in channel 26 and the interaction between the leukocytes and endothelial cells in the presence of the test agent is observed.
  • channel 26 contains a leukocyte migration mediator disposed therein
  • a sample including leukocytes is placed in first well 18 (or second well 22), the sample is allowed to flow along channel 26, the interaction (such interaction including a lack thereof) between the leukocytes and the leukocyte migration mediator is observed, and the sample including leukocytes is collected in second well 22 (or the first well 18) as the leukocytes exit channel 26.
  • a test agent is placed in channel 26 and the interaction between leukocytes and the leukocyte migration mediator in the presence of the test agent is observed.
  • device 10 may match the footprint of an industry standard microtiter plate
  • an advantage of device 10 is that device 10 may be used to conduct multiple assays simultaneously in the same device, and to high throughput screen various test agents.
  • the first well regions 16 and the second well regions 20 of the respective ones of the plurality of chambers 14 are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the wells may be disposed relative to one another to match a pitch of one of a 24-well microtiter plate, a 96-well microtiter plate, a 384-well microtiter plate, 768- well microtiter plate and a 1536-well microtiter plate.
  • pitch P will be set to about 9 mm.
  • device 10 itself fits in the footprint of an industry standard microtiter plate. As such, device 10 preferably has the same outer dimensions and overall size of an industry standard microtiter plate.
  • device 10 comprises 48 chambers designed in the format of a standard 96-well plate, such that the respective wells 18/22 are disposed relative to one another to match a pitch of a standard 96-well microtiter plate with each well fitting in the space of each well of the plate.
  • 48 experiments can be conducted.
  • chambers 14 may be disposed relative to one another to match a pitch of a standard microtiter plate.
  • chambers 14 are sized so that a chamber 14 fits in the area normally required for a single well of a standard microtiter plate.
  • device 10 designed in the footprint of a 96-well microtiter plate configuration, has 96 chambers and therefore allows 96 experiments to be performed.
  • embodiments of device 10 would advantageously fit into existing infrastructures of fluid handling, storage, registration and detection.
  • Device 10 is also conducive to high throughput screening as it allows robotic fluid handling and automated detection and data analysis.
  • the use of robotic and automated systems also decreases the amount of time to prepare and perform the assays and analyze the results of the assays.
  • the use of device 10 decreases the occurrence of human error in preparing and performing assays and analyzing data.
  • the present invention also contemplates a method of screening a plurality of test agents.
  • the method of screening test agents includes providing a device comprising 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 a channel region including at least one channel connecting the first well region and the second well region with one another.
  • the at least one channel includes at least one leukocyte migration mediator disposed therein.
  • the at least one channel includes endothelial cells disposed therein.
  • At least one of the plurality of chambers on the one hand, and the first well regions and the second well regions of respective ones of the plurality of chambers on the other hand, are disposed relative to one another to match a pitch of a standard microtiter plate.
  • the method of screening test agents further includes providing leukocytes in each of the channels of respective ones of the plurality of chambers; placing at least one of a plurality of test agents in each of the channels of respective ones of the plurality of chambers; and observing the interaction between the leukocytes and the endothelial cells or the interaction between the leukocytes and the at least one leukocyte migration mediator in the presence of the test agents.
  • test agents it can be determined whether the test agents have an effect on the number of leukocytes that are captured, arrested, or have transmigrated as well as whether the test agents have an effect on velocity and number of leukocytes that roll along channel 26.
  • the test agent may include any desired biological, chemical, or electrical substance, including but not limited to, an inhibitor of leukocyte migration, a promoter of leukocyte migration, or any other therapeutic agent.
  • Further examples of test agents include proteins, nucleic acids, peptides, polypeptides, carbohydrates, lipids, hormones, enzymes, small molecules or pharmaceutical agents. This method is particularly useful in the area of drug discovery where a plurality of test agents may be screened in a single device 10.
  • each of the test agents is different from one another and a single test agent is placed in each channel.
  • a single test agent is placed in each channel.
  • the device of the present invention may also be used to monitor the steps of the leukocyte migration cascade under a normal or pathological physiological shear flow condition.
  • a normal physiological flow condition refers to the shear flow rate during a non-pathological state and is in the range of about 0.1 dynes/cm 2 to about 20 dynes/cm.
  • a pathological physiological flow condition refers to the shear flow rate during the inflammatory response and is generally varied depending on the disease state.
  • the physiological shear flow is preferably produced by hydrostatic pressure, or microcapillary action, the flow can be produced by any means known in the art.
  • channel 26 may have endothelial cells disposed therein or leukocyte migration mediators disposed therein. The endothelial cells may be grown on channel 26 in the presence or absence of shear flow.
  • channel 26 has endothelial cells disposed therein
  • several different assays may be performed to observe the interaction between leukocytes and the endothelial cells during the leukocyte migration cascade.
  • a sample containing leukocytes is introduced into channel 26 via first well 18 or second well 22.
  • the number of leukocytes rolling as well as the rolling velocity of the leukocytes may then be determined.
  • Assays measuring the inhibition of rolling may also be performed by adding to channel 26, for example, inhibitors that block the interaction between leukocytes and endothelial cells.
  • assays measuring the enhancement of rolling may be performed by adding to channel 26, for example, promoters that promote the interaction between leukocytes and endothelial cells.
  • a test agent could also be added to channel 26 to determine the effect of the test agent on the interaction between leukocytes and endothelial cells.
  • a chemoattractant is introduced into channel 26 in order to "activate" the endothelium.
  • the chemoattractants may be any molecule suitable to stimulate 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- ⁇ .
  • a sample including leukocytes is then introduced in channel 26 via first well 18 or second well 22.
  • the sample including leukocytes is preincubated with a chemoattractant capable of triggering the activation of arrest mediator binding partners, for example integrins, on the surface of leukocytes.
  • the chemoattractant is any suitable substance capable of triggering integrin expression by leukocytes and includes, for example, formyl peptides, intercrines, IL-8, GRO/MGSA, NAP2, ENA-78, MCP-1/MCAF, RANTES, 1-309, other peptides, platelet activating factor (PAF), lymphokines, collagen, fibrin, and histamines.
  • the number of arrested cells may then be determined.
  • Assays measuring the inhibition of arrest may also be performed, for example, by adding inhibitors that block the interaction between chemoattractants and chemoattractant receptors on the surface of the leukocytes or the endothelium, or that block the interaction between leukocyte arrest mediators and arrest mediator binding partners.
  • a test agent could also be added to channel 26 to determine the interaction between the leukocytes and the endothelial cells in the presence of the test agent.
  • a layer of endothelial cells is placed in channel 26.
  • channel 26 may first be coated with a layer of fibronectin or any other basement membrane mimic before adding the endothelial cells to channel 26.
  • the endothelial cells are exposed to eotaxins or chemokines, including RANTES or monocyte chemoattractant protein (MCP-3 or MCP-4) prior to introduction of the sample containing leukocytes.
  • MCP-3 or MCP-4 monocyte chemoattractant protein
  • the leukocytes are preincubated with a chemoattractant capable of triggering the activation of arrest mediator binding partners, for example integrins, on the surface of leukocytes.
  • a chemoattractant capable of triggering the activation of arrest mediator binding partners, for example integrins, on the surface of leukocytes.
  • the leukocytes are allowed to flow along channel 26, the number of cells that transmigrated through the endothelium are counted.
  • Transmigrated cells may be characterized by appearing flattened and phase-dark under a microscope. Flattened, phase-dark cells may be confirmed as being under the endothelial cell monolayer by observing the focal plane of the leukocytes and the endothelial cells using a microscope.
  • a cell may be considered transmigrated if, for example, greater than 50 % of the cell is under the endothelial cell monolayer at the point of quantification.
  • Transmigration may be expressed as the number of transmigrated cells divided by the total cells counted. Inhibition of transmigration may also be examined by blocking, for example, the receptor on endothelium cells that binds the chemoattractant responsible for activating the endothelium and then determining the number of cells that transmigrate across the endothelium.
  • a test agent may also be introduced in channel 26 to determine the interaction between the leukocytes and the endothelial cells in the presence of the test agent.
  • the endothelial cells disposed in channel 26 have been altered or modified through known techniques in molecular biology.
  • the cells may be modified to overexpress particular genes or to not express particular genes coding for the various leukocyte migration mediators responsible for the leukocyte migration cascade.
  • Such an embodiment affords control over the expression of precise leukocyte migration mediators and allows greater manipulation of the mediator involved in the leukocyte migration cascade.
  • the endothelial cells may be genetically modified to reduce or inhibit the expression of a gene believed to encode a protein involved in the leukocyte migration cascade to assist in the elucidation of the proteins involved in leukocyte migration cascade.
  • Methods for genetically modifying a cell are known in the art.
  • One such method is disclosed in U.S. Patent 6,025,192 to Beach et al. and involves replication-deficient retroviral vectors, libraries comprising such vectors, retroviral particles produced by such vectors in conjunction with retroviral packaging cell lines, integrated provirus sequences derived from the retroviral particles of the invention and circularized provirus sequences which have been excised from the integrated provirus sequences of the invention.
  • the endothelial cells may be transfected with a vector to genetically modify a protein expressed by the endothelial cells.
  • a vector to genetically modify a protein expressed by the endothelial cells.
  • the endothelial cells may be modified to express a variant of the protein to be tested. For example, if it is believed that a certain protein is involved in the cascade, the gene expressing the particular protein can be modified to express a variant. Then using the device and assays of the present invention, the effect of this variant on the various parts of the cascade can be monitored.
  • the variant can be created using techniques known in the art by making deletions, additions or substitutions in the sequence encoding the protein.
  • a "variant" of a polypeptide is defined as an amino acid sequence that is altered by one or more amino acids. Similar minor variations can also include amino acid deletions or insertions, or both. Guidance in determining which and how many amino acid residues may be substituted, inserted or deleted without abolishing biological or immunological activity can be found using computer programs well known in the art, for example, DNAStar software.
  • a “deletion” is defined as a change in either amino acid or nucleotide sequence in which one or more amino acid or nucleotide residues, respectively, are absent as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide.
  • An “insertion” or “addition” is that change in an amino acid or nucleotide sequence which has resulted in the addition of one or more amino acid or nucleotide residues, respectively, as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide.
  • substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a naturally occurring polypeptide.
  • the variant can have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant can have "nonconservative" changes wherein a substituted amino acid does not have similar structural or chemical properties such as replacement of a glycine with a tryptophan.
  • an endothelial cell expressing a leukocyte rolling mediator such as P-selectin can be genetically modified such that the expression of the P-selectin is reduced or inhibited using a homologous recombination gene "knock-out" method (see, for example, Capecchi, Nature, 344:105 (1990) and references cited therein; Koller et al., Science, 248:1227-1230 (1990); Zijlstra et al., Nature, 342:435-438 (1989), each of which is incorporated herein by reference; see, also, Sena and Zarling, Nat.
  • a homologous recombination gene "knock-out” method see, for example, Capecchi, Nature, 344:105 (1990) and references cited therein; Koller et al., Science, 248:1227-1230 (1990); Zijlstra et al., Nature, 342:435-438 (1989), each of which is incorporated herein by reference
  • a “knockout” of a target gene means an alteration in the sequence of the gene that results in a decrease of function of the target gene, preferably such that target gene expression is undetectable or insignificant.
  • a knock-out of a gene means that function of the gene has been substantially decreased so that protein expression is not detectable or only present at insignificant levels.
  • a “knock-in” of a target gene means an alteration in a host cell genome that results in altered expression or increased expression of the target gene, e.g., by introduction of an additional copy of the target gene, or by operatively inserting a regulatory sequence that provides for enhanced expression of an endogenous copy of the target gene.
  • the expression of the leukocyte migration mediator by an endothelial cell also can be reduced or inhibited by providing in the endothelial cell an antisense nucleic acid sequence, which is complementary to a nucleic acid sequence or a portion of a nucleic acid sequence encoding a leukocyte migration mediator.
  • an antisense nucleic acid sequence to inhibit the expression of a nucleic acid sequence are known in the art and described, for example, by Godson et al., J. Biol. Chem., 268:11946-11950 (1993), which is incorporated herein by reference.
  • leukocyte migration mediator used herein necessarily refers to at least one leukocyte migration mediator, unless otherwise specified.
  • a leukocyte migration mediator By disposing a leukocyte migration mediator in channel 26, it is possible to examine the ligand/receptor interactions underlying the leukocyte migration cascade, including the individual events of the cascade to gain a further understanding of this process. Disposing a leukocyte migration mediator in channel 26 also allows for the precise targeting of the ligand/receptor interactions underlying the individual events of the leukocyte migration cascade.
  • device 10 may be used to examine the capture of a leukocyte wherein the leukocyte migration mediator disposed in channel 26 may comprise a leukocyte capture mediator.
  • the leukocyte migration mediator disposed in channel 26 may comprise a leukocyte capture mediator.
  • inflammatory mediators induce the expression of adhesion molecules on the surface of the endothelium, resulting in an "activated endothelium.”
  • the first contact of a leukocyte with the activated endothelium is known as “capture” and is thought to involve a capture mediator P-selectin and a capture mediator binding partner L-selectin.
  • the leukocyte capture mediator disposed in channel 26 may comprise, for example, P-selectin and/or an L-selectin binding ligand.
  • device 10 may be used to examine the rolling of a leukocyte wherein the leukocyte migration mediator may comprise a leukocyte rolling mediator.
  • the leukocyte migration mediator may comprise a leukocyte rolling mediator.
  • the leukocytes Once leukocytes are captured, they may transiently adhere to the endothelium and begin to roll along the endothelium.
  • the rolling of leukocytes is thought to involve: a rolling mediator, P-selectin; a rolling mediator binding partner, L-selectin; and a rolling mediator, E-selectin, although P- selectin is considered the primary adhesion molecule involved in this process.
  • the leukocyte rolling mediator disposed in channel 26 may include, for example P-selectin, E-selectin, and/or an L-selectin ligand.
  • device 10 may be used to examine the arrest of a leukocyte wherein the leukocyte migration mediator may comprise a leukocyte anest mediator. It is thought that most, if not all, leukocytes adhere to the endothelium only after having rolled along the endothelium. This adhesion, or "arrest" of the leukocytes on the top surface of the endothelium is initiated by chemoattractants such as IL-1 and TNF- ⁇ produced by cells at the injured site. These chemoattractants stimulate the endothelium to produce chemokines and arrest mediators on the surface of the endothelium opposite the basal lamina.
  • chemoattractants such as IL-1 and TNF- ⁇
  • the anest mediators comprise, for example, integrin binding ligands such as ICAMs, including ICAM-1, ICAM-2, or/and ICAM-3 and VCAMs, including VCAM-1 and/or VCAM-2.
  • the chemokines interact with chemokine receptors on the surface of the rolling leukocytes, which triggers the activation of anest mediator binding partners on the surface of leukocytes.
  • Arrest mediator binding partners include integrins, such as, for example, LFA-1, Mac-1, and pl50,95, and VLA-4.
  • the leukocyte anest mediator disposed in channel 26 may include at least one integrin binding ligand.
  • device 10 may be used to examine the transmigration of a leukocyte wherein the leukocyte migration mediator disposed in channel 26 comprises a leukocyte transmigration mediator.
  • the leukocytes flatten and squeeze between the endothelium to leave the blood vessel and enter the damaged tissue.
  • the leukocytes follow a chemotactic gradient of chemoattractants released by cells in the damaged tissue area.
  • transmigration is thought to be mediated by platelets and endothelial cell adhesion molecule- 1 (PECAM-1).
  • Other potential transmigration mediators may be junctional adhesion molecule (JAM), ICAM-,1 VE-cadherin, LFA-1, LAP, VLA-4 and possibly CD99, a transmembrane protein.
  • the leukocyte transmigration mediator disposed in channel 26 may include at least one of the aforementioned adhesion molecules or any other molecule determined to be implicated in transmigration.
  • Device 10 of the present invention may be used to study each aforementioned step in the leukocyte migration cascade in isolation, a combination of two or more steps in the leukocyte migration cascade, or the leukocyte migration cascade in its entirety. For example, if understanding and targeting rolling are desired, then preferably only leukocyte rolling mediators may be disposed in channel 26. If both rolling and anest of leukocytes are desired to be studied, then both rolling and anest mediators may be disposed in channel 26.
  • capture mediators may be disposed in channel 26.
  • certain molecules belong in more than one category of migration mediators (for example P selectin and an endothelial ligand binding L-selectin function as both capture mediators and rolling mediators) and because certain mediators may be present in conjunction (for example to study arrest, both rolling and anest mediators may be present in channel 26 since direct adhesion from free-flowing leukocytes is thought to be extremely rare), certain steps, with the knowledge cunently available, may not be monitored in isolation.
  • this invention contemplates several methods of monitoring leukocyte migration in order to gain further understanding of the basic mechanisms controlling these events.
  • a leukocyte migration mediator comprising a capture mediator is disposed in channel 26.
  • a sample comprising leukocytes is introduced into channel 26 via first well 18 or second well 22.
  • Capture events are defined as adhesive interactions of those freely flowing leukocytes moving closest to the surface of channel 26 containing the capture mediators and that are therefore the only leukocytes potentially capable of interacting with the capture mediators on channel 26.
  • Different types of initial leukocyte capture can be characterized, observed, and monitored.
  • transient capture involving leukocytes only attaching briefly to channel 26 without initiating rolling motions, and rolling capture involving leukocytes that remain rolling on channel 26, can be determined.
  • the number of each type of captured leukocyte can be divided by the total number of free flowing leukocytes to determine the frequency of initial capture of leukocytes.
  • the leukocytes can also be observed via any method known in the art and via methods disclosed in co-pending application entitled “Test Device and Method of Making Same," which is herein incorporated by reference in its entirety.
  • the leukocytes may be observed by using a microscope, including phase-contrast, fluorescence, luminescence, differential-interference contrast, dark field, confocal laser-scanning, digital deconvolution, and video microscopes; a high-speed video camera; and an array of individual sensors.
  • a digital movie camera may be used to monitor leukocyte activity under continuous flow conditions or a camera may be used to obtain still photographic images at particular points in time.
  • the cells may be incubated with staining agents and then detected based upon color or intensity contrast using any suitable microscopy technique(s).
  • fluorescence-labeling may be used to detect whether capture mediator binding partners bind to capture mediators.
  • non-labeled cells may be used to monitor migration.
  • a heterogeneous mixture of multiple cell types may be introduced into channel 26 with only one cell type capable of interacting with the capture mediators in channel 26. After the cells have been introduced into channel 26, an antibody specific to any antigen on the surface of this cell type may be used to label this cell type. If a multiple number of cell types can interact with the capture mediators, antibodies labeled with specific fluorophores can be used to distinguish different cell types.
  • a leukocyte migration mediator comprising a rolling mediator is placed in channel 26.
  • a sample comprising leukocytes is introduced into channel 26 via first well 18 or second well 22.
  • the number of leukocytes rolling and the rolling velocity of the leukocytes can be determined.
  • a camera is operatively linked to device 10 to obtain images of leukocytes rolling along channel 26 during predetermined intervals over a predetermined period of time.
  • the rolling velocity of the cells is determined by measuring the length the cells traveled (l frame ) in an image obtained by the camera and determining the exposure time of the image (t eX p o sure)-
  • the following formula is used: — C ⁇ lf rame / texposure) where c is a conversion factor for determining the actual distance the cells have traveled. It may vary from image to image.
  • a leukocyte migration mediator comprising a first leukocyte migration mediator and a second leukocyte migration mediator, the first and second leukocyte migration mediators being different from one another is utilized.
  • first leukocyte migration mediator used herein necessarily refers to at least one first leukocyte migration mediator and the term “second leukocyte migration mediator” used herein necessarily refers to at least one second leukocyte migration mediator.
  • first leukocyte migration mediator comprising a rolling mediator
  • second leukocyte migration mediator comprising an anest mediator are placed in channel 26.
  • a fluid sample comprising leukocytes is preincubated with a chemoattractant capable of triggering the activation of anest mediator binding partners, for example, integrins, on the surface of the leukocytes.
  • the chemoattractant is any suitable substance capable of triggering integrin expression by leukocytes and includes, for example, a formyl peptide, intercrines, IL-8 GRO/MGSA,NAP-2, ENA-78, MCP-1/MCAF, RANTES, 1-309, other peptides, platelet activating factor (PAF), lymphokines, collagen, fibrin and histamines.
  • a formyl peptide intercrines
  • IL-8 GRO/MGSA,NAP-2 ENA-78
  • MCP-1/MCAF MCP-1/MCAF
  • RANTES 1-309
  • other peptides peptides
  • PAF platelet activating factor
  • lymphokines collagen, fibrin and histamines.
  • test agents may comprise any biological, chemical or electrical substance that includes, but is not limited to potential inhibitors of the leukocyte migration mediator or potential promoters of migration mediated by the leukocyte migration mediator.
  • test agents include proteins, peptides, polypeptides, enzymes, hormones, lipids, carbohydrates, small molecules, and pharmaceutical agents.
  • the device may be used to identify an inhibitor or promoter that competitively or noncompetitively inhibits or promotes a capture mediator and capture mediator binding partner interaction; rolling mediator and rolling mediator binding partner interaction; anest mediator and anest mediator binding partner interaction; and/or transmigration mediator and transmigration mediator binding partner interaction.
  • the leukocyte migration mediator comprises a first leukocyte migration mediator and a second leukocyte migration mediator, the first and second leukocyte migration mediators beings different from one another.
  • the test agent comprises a potential inhibitor of the first leukocyte migration mediator, the second leukocyte migration mediator, or both.
  • the test agent comprises a potential promoter of migration mediated by the first leukocyte migration mediator, the second leukocyte migration mediator, or both.
  • a potential promoter of migration mediated by the first leukocyte migration mediator, the second leukocyte migration mediator, or both.
  • these inhibitors and promoters can be tested for efficacy in vivo and ultimately utilized as therapeutic agents.
  • a leukocyte migration mediator comprising a capture mediator is disposed in channel 26.
  • the potentially inhibitory test agent is incubated with a fluid sample comprising leukocytes, the sample is introduced into channel 26 via first well 18 or second well 22.
  • Capture events are defined as adhesive interactions of those freely flowing leukocytes moving closest to the surface of channel 26 containing the capture mediators and that are, therefore, the only leukocytes potentially capable of interacting with the capture mediators on channel 26.
  • Different types of initial leukocyte capture can be characterized, observed, and monitored. For example, transient capture involving leukocytes only attaching briefly to channel 26 without initiating rolling motions, and rolling capture involving leukocytes that remain rolling on channel 26 can be determined.
  • the number of each type of captured leukocyte can be divided by the total number of free flowing leukocytes to determine the frequency of initial capture of leukocytes incubated with the potential inhibitory test agent and this frequency can be compared to the frequency of initial leukocyte capture in the absence of the potential inhibitory test agent.
  • the test agent is likely an inhibitor of leukocyte capture.
  • a leukocyte migration mediator comprising a rolling mediator is placed in channel 26.
  • the potentially inhibitory test agent is incubated with a fluid sample comprising leukocytes, the sample is introduced into channel 26 via first well 18 or second well 22.
  • the potentially inhibitory test agent is introduced into the fluid sample during passage of the fluid sample in channel 26 when leukocytes have begun rolling. A decrease in rolling (e.g.
  • a leukocyte migration mediator comprising a first leukocyte migration mediator and a second leukocyte migration mediator, the first and second leukocyte migration mediators being different from one another may be utilized.
  • the first leukocyte migration mediator comprising a rolling mediator and the second leukocyte migration mediator comprising an anest mediator are placed in channel 26.
  • a fluid sample comprising leukocytes is preincubated with a chemoattractant capable of triggering the activation of anest mediator binding partners, for example, integrins, on the surface of the leukocytes.
  • a chemoattractant capable of triggering the activation of anest mediator binding partners, for example, integrins, on the surface of the leukocytes.
  • the sample is introduced into channel 26 via first well 18 or second well 22.
  • the potentially inhibitory test agent is introduced into the fluid sample during passage of the fluid sample in channel 26 when leukocytes have begun rolling.
  • a decrease in anest of the leukocytes indicates that the test agent may be an inhibitor of leukocyte anest.
  • Device 10 can also be used to identify whether a test agent acts as a promoter of the inflammatory response by increasing the efficiency of the leukocyte migration cascade or by acting as a functional component thereof (e.g. a capture mediator, a rolling mediator, an anest mediator, or a transmigration mediator).
  • a functional component e.g. a capture mediator, a rolling mediator, an anest mediator, or a transmigration mediator.
  • Such a functional component may be detected by its ability to promote capture, rolling, anest or transmigration of a leukocyte where such action was previously lacking (due to lack of appropriate cellular specificity of a rolling mediator or anest mediator previously present in channel 26 of chamber 14 or lack of any rolling mediator or arrest mediator).
  • device 10 comprising a first leukocyte migration mediator comprising a rolling mediator and second leukocyte migration mediator comprising an arrest mediator disposed in channel 26 may be used to identify an arrest mediator functional in leukocyte migration.
  • device 10 comprising an anest mediator and/or a rolling mediator disposed in channel 26 may be used to identify a rolling mediator functional in leukocyte migration.
  • rolling mediators are disposed in channel 26 that have rolling binding partners present on the surface of leukocytes in a fluid sample to be introduced into channel 26 through first well 18 or second well 22.
  • One or more chemoattractants capable of activating the leukocytes to express anest mediator binding partners are preincubated with the fluid sample comprising leukocytes.
  • a test agent to be tested for anest mediating function is disposed in channel 26. After the fluid sample comprising leukocytes is introduced into channel 26 via first well 18 or second well 22 and the sample passes along channel 26, it is determined whether any leukocytes have anested on channel 26.
  • Anest of leukocytes indicates that the test agent may be an anest mediator that recognizes an anest mediator binding partner on the surface of the same leukocytes that express the rolling mediator binding partner.
  • the test agent to be tested for rolling mediator function is disposed in channel 26 and the fluid sample comprising leukocytes is introduced into channel 26 via the first well 18 or second well 22 and the sample is allowed to flow along channel 26. Rolling of the leukocytes along channel 26 indicates that the test agent has rolling mediator function and that the leukocytes express a binding partner for the rolling mediator.
  • a rolling mediator is also identified by disposing the test agent to be tested for rolling mediator function in channel 26 and also disposing an anest mediator in channel 26.
  • One or more chemoattractants capable of activating the leukocytes to express anest mediator binding partners, such as integrins, are preincubated with the fluid sample comprising leukocytes.
  • the fluid sample comprising leukocytes is then introduced into channel 26 via the first well 18 or second well 22 and the sample is allowed to flow along channel 26.
  • Arrest of leukocytes indicates that the test agent has rolling mediator function and that the leukocytes that express the anest mediator binding partner and the chemoattractant receptor also express a binding partner for the test agent.
  • a test agent may also be identified as a functional component in the processes of leukocyte rolling or rolling and anest, or an enhancer thereof, by the aforementioned methods in which an increase in number or percentage of leukocytes rolling or anested is detected relative to the number or percentage of such leukocytes in the absence of the test agent.
  • the migration of the leukocytes may be observed, monitored, recorded, and analyzed by any method known in the art and via the methods disclosed in co-pending application, "Test Device and Method of Making Same" refened to above.
  • the present invention also provides a kit to conduct the aforementioned assays.
  • the kit comprises a device including a housing 12 defining a plurality of chambers 14.
  • Each of the plurality of chambers 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 a channel region 24 including at least one channel 26 connecting the first well region 16 and the second well region 20 with another.
  • the first well regions 16 and the second well regions 20 of the respective ones of the plurality of chambers 14 are disposed relative to one another to match a pitch of a standard microtiter plate, thus advantageously allowing for high through-put screening of tests agents.
  • the kit further includes a first leukocyte migration mediator.
  • the kit may also contain a sample comprising leukocytes.
  • the first leukocyte migration mediator and the sample comprising leukocytes may be packaged in the kit in any manner known in the art.
  • the first leukocyte migration mediator may be contained in a vial or container and the sample comprising leukocytes may similarly be contained in a separate vial or container.
  • the kit may further include a second leukocyte migration mediator different from the first leukocyte migration mediator.
  • the kit may additionally include an inhibitor adapted to inhibit the first leukocyte migration mediator, the second leukocyte migration mediator, or both.
  • the kit may further include a promoter adapted to promote migration mediated by the first leukocyte migration mediator, the second leukocyte migration mediator, or both.
  • the kit may also include any media and buffers necessary for use with the device and particular assays.
  • the housing 12 of device 10 comprises a support member 28, and a top member 30 mounted to the support member 28, wherein the support member 28 and the top member 30 are configured such that they together define the plurality of chambers 14.
  • the housing is also sized to match dimensions of a standard microtiter plate, for example, the dimensions of a 24-well microtiter plate, a 96-well microtiter plate, a 384-well microtiter plate, 768-well microtiter plate and a 1536-well microtiter plate.
  • the top member may be made of any suitable material known in the art including glass, plastic, or an elastomeric material such as polydimethylsiloxane (PDMS).
  • the support member may be made of glass, polystyrene, polycarbonate, polyacrylates, polymethyl methacrylate (PMMA), PDMS and other plastics.
  • top member 30 is in conformal contact with support member 28.
  • device 10 comprises a support member 28; and top member 30, the top member 30 mounted to the support member 28 by being placed in conformal contact with the support member 28.
  • the support member 28 and the top member 30 are configured such that they together define at least one chamber 14.
  • 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 a channel region 24 including at least one channel 26 connecting the first well region 16 and the second well region 20 with one another.
  • the at least one channel 26 includes at least one leukocyte migration mediator disposed therein.
  • the at least one channel 26 includes endothelial cells disposed therein.
  • the top member 30 is configured to be placed in reversible, conformal contact with the support member 28.
  • top member 30 is preferably made of a material that is adapted to effect conformal contact, preferably reversible conformal contact, with support member 28.
  • the flexibility of such a material allows top member 30 to form-fittingly adhere to support member 28 in such a way as to form a substantially fluid-tight seal therewith.
  • the conformal contact should preferably be strong enough to prevent slippage of top member 30 on support member 28.
  • top member 30 may be made of a material having the structural integrity to allow top member 30 to be removed by a simple peeling process.
  • top member 30 to be removed from support member 28 after experimentation, properly cleansed, and then reused for future assays.
  • the peeling process does not disturb any surface treatment, such as leukocyte migration mediators or endothelial cells, on support member 28.
  • the substantially fluid-tight seal effected between top member 30 and support member 28 by virtue of the conformal contact of top member 30 with support member 28 prevents fluid from leaking from one chamber to an adjacent chamber, and also prevents contaminants from entering the wells.
  • the seal preferably occurs essentially instantaneously without the necessity to maintain external pressure.
  • the conformal contact obviates the need to use a sealing agent to seal top member 30 to support member 28.
  • the top member 30 is made of a material that does not degrade and is not easily damaged by virtue of being used in multiple tests, and that affords considerable variability in the top member's configuration during manufacture of the same. More preferably, the material may be selected for allowing the top member 30 to be made using photolithography.
  • the material comprises an elastomer such as silicone, natural or synthetic rubber, or polyurethane.
  • the material is PDMS.
  • Support member 28 provides a support upon which top member 30 rests, and may be made of any material suitable for this function. Suitable materials are known in the art such as glass, polystyrene, polycarbonate, PMMA, polyacrylates, PDMS, and other plastics.
  • well regions 16 and 20 are vertically offset with respect to one another is a test orientation of device 10.
  • well regions 16 and 20 are horizontally offset with respect to one another is a test orientation of device 10.
  • Wells 18 and 22 of respective well regions 16 and 20 of each chamber 14 are not limited in their configuration to any particular three dimensional contour, it being only required that they be adapted to receive a fluid therein, preferably a sample comprising leukocytes.
  • wells 18 and 22 are configured such that they substantially define circles in top plan views thereof, as shown by way of example in Figs. 1-6.
  • the pitch P is set to be equal to about 9 mm
  • the diameter D w of a top plan contour of the wells is set to be equal to about 6 mm.
  • length L of each channel 26 is equal to about 3 mm.
  • wells 18 and 22 are defined in part by respective through-holes 18 and 22 in top member 30, and in part by an upper surface U of support member 28.
  • the sides of each well 18 and 22 are defined by respective walls of the through holes 18 and 22 in the top member 30, and the bottoms of wells 18 and 22 are defined by a conesponding portion of the upper surface U of support member 28.
  • a length L of a channel 26 is defined in a direction of the longitudinal axis thereof.
  • depth D of a channel 26 is defined in a direction normal to a top surface of housing 12; and a width W of a channel region 26 is defined in a direction normal to length L and depth D.
  • channel region 24 comprises a plurality of rectilinear, parallel channels 26 extending between well regions 16 and 20.
  • channels 26 have lengths L that are substantially identical, as shown schematically by way of example in Fig. 4. More preferably, the plurality of channels 26 comprises eight channels. By using multiple channels, multiple assays can be performed simultaneously using one sample comprising leukocytes.
  • Channels 26 preferably each have a width W of 50 ⁇ m to 5 mm; a length L of about 1-10 mm; and a height H of about 10-100 ⁇ m. More preferably, the channels 26 each have a width W of about 100 microns, a length L of about 3 mm and a height of about 50 ⁇ m to about 80 ⁇ m.
  • the dimensions of the channels 26 of channel region 24 should be configured to support the migration of leukocytes under conditions simulating such migration during an inflammatory response. As such, the channel region should be adapted to support the migration of leukocytes under shear flow and to support at least one leukocyte migration mediator disposed therein.
  • Device 10 of the present invention can be fabricated, according to a preferred embodiment of a method of the present invention, by standard photolithographic procedures. Photolithographic procedures can be used to produce a master that is the negative image of any desired configuration of top member 30. For example, the dimensions of chamber 14, such as the size of well region 16 and 18, or the length of channel region 24, can be altered to fit any advantageous specification.
  • top member 30 is either spin cast, injected, or poured over the master and cured. Once the mold is created, this process can be repeated as often as necessary. This process not only provides great flexibility in the top member's design, it also allows the top members to be massively replicated.
  • leukocyte migration mediators can be disposed in channel 26.
  • the leukocyte migration mediators can be disposed in channel 26 by affixing them or physioadsorbing them directly on the upper surface U of support member 28, or by coating a solution or suspension comprising the leukocyte migration mediators on the upper surface U of support member 28, as long as the mediators are accessible to leukocytes flowing by the upper surface U.
  • the leukocyte migration mediators are either covalently or non- covalently affixed directly to upper surface U by techniques such as covalent bonding via an amide, ester or lysine side chain linkage or adso ⁇ tion.
  • the present invention also provides a device comprising a housing 12; means associated with the housing defining a plurality of chambers 14 in the housing 12.
  • Each of the plurality of chambers 14 includes: an inlet means for receiving a sample comprising leukocytes; an outlet means in flow communication with the inlet means for receiving the sample comprising leukocytes from the inlet means; and connection means connecting the inlet means and the outlet means to one another, the connection means including at least one leukocyte migration mediator disposed therein.
  • An example of means associated with the housing defining a plurality of chambers in the housing comprises a top member mounted to a support member as shown in Fig. 6.
  • the above means have been substantially shown and described in relation to the embodiments of the Fig. 1-6. Other such means would be within the knowledge of persons skilled in the art.
  • the present invention provides methods of assaying and studying biological phenomenon that either depend on or react to gradient formation and/or flow conditions.
  • biological phenomenon include many of the processes in the body such as cell-surface interactions such as that occurring during leukocyte adhesion and rolling.
  • studies involving chemotaxis, haptotaxis and cell migration will be better served with assays that are able to study such cell movement in the presence of gradients and/or flow conditions.
  • Such useful gradients include static gradients, which have concentrations that are fixed, or set or substantially fixed or set.
  • a static gradient is a gradients of immobilized molecules on a surface.
  • static gradients include the use of differing concentrations of immobilized biomolecules (proteins, antibodies, nucleic acids, and the like) or immobilized chemical moieties (drugs and small molecules).
  • Other useful gradients include dynamic gradients, which have concentrations that may be varied.
  • One example of a dynamic gradient is a gradient of fluid streams having molecules in varying concentrations.
  • fluid gradients include the use of fluid streams containing biomolecules such as growth factors, toxins, enzymes, proteins, antibodies, carbohydrates, drugs or other chemical and small molecules in varying concentrations.
  • a dynamic/solution based gradient is created by laminar flow technology.
  • Laminar flow technology typically involves two or more fluid streams from two or more different sources. These fluid streams are brought together into a single stream and are made to flow parallel to each other without turbulent mixing. Fluids with different characteristics such as varying low Reynolds numbers will flow side by side and will not mix in the absence of turbulence. Since the fluids do not mix, they create pseudo-channels (pseudo by the fact that there are no physical separation between the fluids). The generation of solution and surface gradients is discussed in U.S. patent application 2002/0113095 and an article, Jeon, Noo Li, et al., Langmuir, 16, 8311-8316 (2000).
  • the devices of the present invention By combining the devices of the present invention with the formation of a dynamic gradient, a vast number of assay parameters can be generated by altering any portion of the device. For example, by combining the device as disclosed herein with cell patterning techniques, along with the introduction of a dynamic gradient, various conditions can be created to test numerous biological interactions. Further, the device and assays may be useful in drug discovery and drug testing as many cells and biological materials behave differently ex vivo when not exposed to gradients than compared to when the cells or biological materials are present in vivo and thus exposed to gradients and flow conditions.
  • cells can be patterned across channel 26.
  • Cell patterning can be achieved by methods known in the art, as well as disclosed in the present invention (such as, but not limited to, microcontact printing or by the use of elastomeric stencils).
  • a solution containing any desired biomolecule or chemical/drug can then be flowed across the patterned cells.
  • the cells could be first treated by a biomolecule such as an activator to more closely recreate a biological system, and then be subsequently exposed to a chemical or drug.
  • a biomolecule such as an activator
  • a chemical or drug By creating a gradient, such as by laminar flow, different amounts of biomolecules or chemicals/drugs can be delivered to the patterned cells and thus the effect of concentration of each biomolecule or chemical/drug be tested simultaneously against each other.
  • Creating dynamic gradients with laminar flow in combination with the devices of the present invention provides numerous assay configurations. For example, by varying the combinations of the cells on the surface, the biomolecule in channels 26 and the compounds in channel 26, one can create a vast multitude of assays. With respect to immobilized cells or other immobilized biomolecules such as proteins, antibodies, nucleic acids, etc. different assay configurations are possible. In one embodiment, a single type of cell is immobilized throughout the entire channel region 24. In another embodiment, a mixture of cell types are immobilized, one cell type per region 24. In another embodiment, a mixture of cell types is immobilized throughout the entire channel region 24. This may be advantageous in monitoring cell-cell interactions. In yet another embodiment, different cell types are immobilized in each different region 24.
  • further assay design flexibility centers around the leukocyte migration mediators or other biomolecules present in channels 24.
  • one type of leukocyte migration mediator is present in each channel 24 at the same concentration.
  • one type of leukocyte migration mediator is present in each channel 24 at differing concentrations.
  • different leukocyte migration mediators are present in each channel 24.
  • Each channel 24 may have the same mixture or a different mixture. When the mixture is the same, the ratios or concentrations of the different leukocyte migration mediators may be different in each channel 24.
  • compounds, such as drugs or test substances the present invention provides flexibility in assay design.
  • a single compound is present in all channels 24 at the same concentration throughout.
  • the same compound is present in all the channels 24 but each channel 24 has a different concentration of that compound.
  • each channel 24 has a different compound.
  • there is more than one compound there is more than one compound.
  • each channel 24 may have the same mixture of compounds or may have a different mixture of compounds. Further, when the mixtures of the compounds are the same, each channel 24 may receive a different concentration of that mixture. Yet, even further, each channel 24 may receive the mixture of the compounds, with each channel 24 having a different ratio of compounds to each other.
  • Such assay systems can be used to test among many numerous biological interactions, the effects of chemical or drugs on cells or other biomolecules.
  • a master of the device according to the present invention is made using photolithography.
  • a silicon substrate is patterned based on a negative pattern of the top member using a suitable photoresist.
  • PDMS polydimethyl siloxane
  • the thus poured PDMS layer is allowed to cure in an oven at about 30°C for about 17 hours.
  • the device is washed thoroughly with 2% Micro-90 (a product of international Products Co ⁇ .), rinsed for 10 minutes at 70° C in "Sonic Bath,” and rinsed with de-ionized water, followed by a rinsing with 100% ethanol.
  • the PDMS layer is then dried under nitrogen.
  • a pre-cleansed glass slide such as a rectangular one having dimensions of about 4.913 +/- 0.004 inches (in.) by about 3.247 +/-0.004 in. and a thickness of about 1.75 millimeters (mm), mm, is washed three times with ethanol and twice with methanol.
  • the surfaces of the PDMS layer and the glass slide to be bound together are both plasma oxidized for about 84 seconds.
  • the PDMS layer and the glass slide are then pressed together using forceps to squeeze out air pockets therebetween. In this manner, a fluid-tight, conformal contact is established between the PDMS layer as top member and the glass slide and support member.
  • the conformal contact between the PDMS layer and the glass slide is reversible.
  • Neutrophils are isolated from a volume of 5 milliliters (ml) of human blood from a healthy volunteer.
  • the 5 ml of blood is diluted with Hanks Balance Salt Solution (HBSS) in a 1 :2 ratio thereby increasing the total volume of blood to equal 15 ml.
  • HBSS Hanks Balance Salt Solution
  • the whole blood dilution is layered over 10ml of Ficoll-Paque Plus (obtained from Amersham Pharmacia Biotech AB, catalog # 17-1440-02).
  • the blood is then centrifuged for 30 minutes at 400g at room temperature.
  • the supernatant is aspirated off without disturbing the pellet.
  • the pellet is resuspended on 10 ml of HBSS and 150 ⁇ l of 6% dextran to make up a 1% solution.
  • the red blood cells are allowed to settle for at least one hour at room temperature.
  • the neutrophils remain inside the supernatant while the red blood cells mostly settle down forming a pellet.
  • the supernatant is pipetted out and diluted in a 1 :2 ratio using HBSS. This suspension is centrifuged for 10 minutes at a velocity of 600g. The supernatant is aspirated and the pellet is dissolved in 19 ml of deionized water. After one minute, the pellet is resuspended in 1 ml of 10X PBS. This suspension is centrifuged at 400g for 10 minutes.
  • the red blood cells are lysed in this process and the remaining cells are mostly neutrophils.
  • the resulting pellet may be dissolved in media containing BSA in order to avoid the clumping of cells after a prolonged period of time at room temperature.
  • the cell density is determined by counting the number of cells using a hemocytometer.
  • the channel is washed four times using 0.1% Bovine Serum Albumin (BSA) in Phosphate Buffer Saline (PBS). After this last wash, all the liquid inside the wells is pipetted out leaving only liquid in the channel. 20 ⁇ l of 0.1% BSA in PBS is added to the first well and 10 ⁇ l of BSA in PBS is added to the second well.
  • BSA Bovine Serum Albumin
  • neutrophils obtained from the method described in part A in 60 ⁇ l of media are added to the first well of each chamber (about 10 to about 10 cells per well of a 24 well plate, in volume of 60 ⁇ l of media per well) (non-labeled and fluorescently labeled monocytic cell lines-U937 (obtained from ATCC, catalog # TIB-202 and THP-1 (obtained from ATCC, catalog # CRL-1593.2) as well as other primary leukocytes may also be used.
  • Digital images are taken on a Zeiss inverted microscope using AXIOCAMTM beginning 15 seconds after the sample comprising leukocytes is added to the first well. Data is analyzed on AXIOVISIONTM software. Time-lapsed images are taken every 30 seconds for 5 minutes and 15 seconds. 10X objective lens is used to view and record the number of cells rolling along the channel.
  • N c(lt,me /texposure)
  • c conversation factor for determining the actual distance the cells traveled. This factor may vary from image to image.
  • l t , me the length of the leukocytes migration in the captured image.
  • texposure the exposure time of the image.
  • t eXposure is 100 milliseconds (ms) when the flow rate is about 0.1 dynes/cm to about 20 dynes/cm .
  • Neutrophils are isolated according to the method disclosed in section II, part
  • the device is incubated for two hours at room temperature in a dish with a cover in order to keep the wells from drying out. After the incubation, the channel is washed four times using 0.1 % Bovine Serum Albumin (BSA) in Phosphate Buffer Saline (PBS). After this last wash, all the liquid inside the wells is pipetted leaving only liquid in the channel. 20 ⁇ l of 0.1%) BSA in PBS is added to the first well and 10 ⁇ l of BSA in PBS is added to the second well.
  • BSA Bovine Serum Albumin
  • PBS Phosphate Buffer Saline
  • neutrophils isolated from part A in 60 ⁇ l of media 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 volume of 60 ⁇ l of media per well) (non-labeled and fluorescently labeled monocytic cell lines-U937 and THP-1 as well as primary leukocytes may also be used).
  • Digital images are taken on a Zeiss inverted microscope using AXIOCAMTM beginning 15 seconds after the sample comprising leukocytes is added to the first well. Data is analyzed on AXIOVISIONTM software. Time-lapsed images are taken every 30 seconds for 5 minutes and 15 seconds. 10X objective lens is used to view and record the number of cells rolling along the channel and adhering to the channel.
  • Placement of Leukocytes and Endothelial Cells in Chamber 10 ⁇ L of a 10X dilution of MATRIGELTM (obtained from BD Bioscience, catalog # 356231) is added to the first well of the device fabricated according to the method disclosed in Section I. lO ⁇ L are added to the first well and the microcapillary action draws the solution into the channel . The MATRIGELTM is then allowed to gel for about 15 minutes at room temperature. Another option is to coat the channel with 1 mg/mL concentration of fibronectin (obtained from GibcoBRL, catalog # 33016-015) that is obtained by diluting the stock concentration of fibronectin using a 0.1 % BSA solution. 5 ⁇ L of fibronection at a concentration of 1 mg/mL are pipetted into the first well and microcapillary action draws the solution in to the channel.
  • MATRIGELTM obtained from BD Bioscience, catalog # 356231
  • the endothelial cells are prepared for seeding.
  • Cells are obtained from Clonetics at Bio- Whittaker in cryogenic vials. They are grown in T75 flasks until ready to be split using 0.025%) Trypsin/EDTA.
  • the cells are seeded on the channel at a density of lxlO 5 cells per 5 ⁇ l of media per assay for approximately two days to form a confluent monolayer of endothelial cells. During these two days, the endothelial cells are replenished with 40 ⁇ L of fresh media added into each well.
  • the endothelial cells are exposed to a concentration of lng/ml of TNF- ⁇ (other chemokines may alternatively be used) for a period of four hours at 37°C.
  • TNF- ⁇ other chemokines may alternatively be used
  • the TNF- ⁇ is washed using 60 ⁇ L of fresh media twice. The volume of media inside each well is replaced with 15 ⁇ L of fresh media.
  • Neutrophils isolated from Section II, part A in 60 ⁇ l of media are added to the first well of chamber the (about 10 to about 10 cells per well of a 24 well plate, in volume of 60 ⁇ l of media per well) (non-labeled and fluorescently labeled monocytic cell lines-U937 and THP-1 as well as primary leukocytes may also be used.) If a monocytic cell line is used, the cells are fluorescence labeled using cell tracker probes (obtained from Molecular Probes, catalog #s C-2925 and C-2927). The cells are incubated with a l ⁇ M concentration of probes for 30 minutes at 37°C. The media is then changed and the cells are placed inside an incubator for an additional 30 minutes.
  • V cOtime /texposure)
  • c conversation factor for determining the actual distance the cells traveled. This factor may vary from image to image, l t i me : the length of the leukocytes migration in the captured image. exposure : the exposure time of the image.
  • t exposure is 100 ms when the flow rate is about 0.1 dynes/cm 2 to about 20 dynes/cm 2 .
  • Neutrophils are isolated according to the method disclosed in section II, part A. Placement of Leukocytes, P-selectin, and P-selectin Antibodies in the Chamber
  • the device is incubated for two hours at room temperature in a dish with a cover in order to keep the wells from drying out. After the incubation, the channels of each well are washed four times using 0.1 % Bovine Serum Albumin (BSA) in Phosphate Buffer Saline (PBS).
  • BSA Bovine Serum Albumin
  • PBS Phosphate Buffer Saline
  • 100 ng/mL of P- selectin antibody is pipetted into the first well of chamber #1; 10 ng/mL of P- selectin antibody is pipetted into first well of chamber #2; and Ing/mL of P-selectin antibody is pipetted into the first well of chamber #3; 100 ⁇ g/mL of P-selectin antibody is pipetted into the first well of chamber #4; and 0.1 %> BSA in PBS is pipetted into the first well of chamber #5.
  • the device is incubated for thirty minutes at room temperature in a dish with a cover in order to keep the wells from drying out.
  • the channels are washed first with 20 ⁇ l of BSA, then with lO ⁇ l of BSA and then 0.1% BSA in PBS.
  • Neutrophils in 20 ⁇ l of media are added to the first well of each chamber (about 10 3 to about 10 6 per well of a 24 well plate, in volume of 20 ⁇ l of media per well) (non-labeled and fluorescently labeled monocytic cell lines-U937 and THP-1 as well as primary leukocytes may be used).
  • Digital images are taken on a Zeiss inverted microscope using AXIOCAMTM beginning 15 seconds after the sample comprising leukocytes is added to the first well. Data is analyzed on AXIOVISIONTM software.
  • Time-lapsed images are taken every 30 seconds for 5 minutes and 15 seconds.
  • 10X objective lens is used to view and record the number of cells rolling after the treatment with P-selectin antibody.
  • a lOOng/mL dilution of the antibody is a prefened concentration to inhibit the rolling of the cells.
  • the number of leukocytes that roll and adhere to the endothelium are reduced in the presence of anti-P selectin.
  • the device is incubated for two hours at room temperature in a dish with a cover in order to keep the wells from drying out. After the incubation, the channels of each well are washed four times using 0.1% Bovine Serum Albumin (BSA) in Phosphate Buffer Saline (PBS).
  • BSA Bovine Serum Albumin
  • PBS Phosphate Buffer Saline
  • E- selectin antibody is pipetted into the first well of chamber #1; 10 ng/mL of E- selectin antibody is pipetted into first well of chamber #2; and Ing/mL of E-selectin antibody is pipetted into the first well of chamber #3; 100 ⁇ g/mL of E-selectin antibody is pipetted into the first well of chamber #4; and 0.1 % BSA in PBS is pipetted into the first well of chamber #5.
  • the device is incubated for thirty minutes at room temperature in a dish with a cover in order to keep the wells from drying out. After incubation, the channels are washed four times with 0.1 %> BSA in PBS.
  • Neutrophils in 20 ⁇ l of media 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 volume of 20 ⁇ l of media per well) (non-labeled and fluorescently labeled monocytic cell lines-U937 and THP-1 as well as primary leukocytes may be used).
  • Digital images are taken on a Zeiss inverted microscope using AXIOCAMTM beginning 15 seconds after the sample comprising leukocytes is added to the first well. Data is analyzed on AXIOVISIONTM software. Time-lapsed images are taken every 30 seconds for 5 minutes and 15 seconds. 10X objective lens is used to view and record the number of cells rolling after the treatment with E-selectin antibody.
  • a lOOng/mL dilution of the antibody is a prefened concentration to inhibit the rolling of the cells.
  • the number of leukocytes that roll and adhere to the endothelium are reduced in the presence of anti-E selectin.
  • Endothelial cells are placed and activated in four different channels of four chambers (#l-#4) according to the method disclosed in section IV, part B.
  • a fifth (#5) chamber endothelial cells are placed in the channel, but are not activated.
  • 100 ⁇ g/ml of P-selectin antibody is pipetted into the first well of chamber #1 ;
  • 100 ⁇ g/ml of E-selectin antibody is pipetted into the first well of chamber #2;
  • 100 ⁇ g/ml of VCAM-1 antibody is pipetted into the first well of chamber #3; and 100 ⁇ g/ml of BSA in PBS is pipetted into the first well of chamber #4.
  • the device is incubated for thirty minutes at room temperature in a dish with a cover in order to keep the wells from drying out. After incubation, the channels are washed four times with 0.1% BSA in PBS. Neutrophils in 20 ⁇ l of media 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 volume of 20 ⁇ l of media per well) (non-labeled and fluorescently labeled monocytic cell lines-U937 and THP-1 as well as primary leukocytes may be used). Digital images are taken on a Zeiss inverted microscope using AXIOCAMTM beginning 15 seconds after the sample comprising leukocytes is added to the first wells. Data is analyzed on AXIOVISIONTM software. Time-lapsed images are taken every 30 seconds for 5 minutes and 15 seconds. 10X objective lens is used to view and record the number of cells rolling after the treatment with the antibodies as seen in Figure 10.
  • EXAMPLE VII SELECTIVE ACTIVATION OF ENDOTHELIAL CELLS BY DELIVERY OF TNF- ⁇ IN A GRADIENT CREATED BY LAMINAR FLOW
  • TNF- ⁇ was delivered to the lawn of endothelial cells via laminar flow to "activate" the endothelial cells.
  • Each stream of solutions containing TNF- ⁇ were at different concentrations, thus creating a gradient pe ⁇ endicular to the channel. This gradient effectively delivered TNF- ⁇ to the lawn of endothelial cells at different concentrations at different positions on the lawn of cells.
  • Leukocytes were then flowed over the lawn of activated endothelial cells. Referring to Figure 12, only those endothelial cells that were activated by TNF- ⁇ provide suitable "attachment" sites for the leukocytes.
  • the leukocytes did not attach equally to the entire lawn, but attached to the areas of the endothelial cell lawn that had been exposed to high concentrations of TNF- ⁇ and did not attach to those areas of the lawn that had been exposed to low concentrations of TNF- ⁇ , or those areas not exposed to TNF- ⁇ at all. These results indicate that there was indeed a creation of a concentration gradient of TNF- ⁇ by the laminar flow.

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Abstract

L'invention concerne un dispositif destiné à contrôler la migration des leucocytes. Elle concerne également un procédé mettant en oeuvre le dispositif de l'invention pour contrôler la migration des leucocytes en présence d'un écoulement de cisaillement physiologique, et ainsi simuler des états physiologiques d'un vaisseau sanguin in vivo. L'invention concerne aussi un procédé d'utilisation du dispositif pour cribler à haute capacité plusieurs substances d'essai. Elle concerne en outre un système de dosage souple et de nombreux dosages pouvant être mis en oeuvre pour tester des interactions et des systèmes biologiques. Des gradients d'écoulement laminaire sont utilisés pour simuler des situations de gradient présentes in vivo.
EP03719923A 2002-04-24 2003-04-24 Dispositif et procede destines a controler la migration des leucocytes Withdrawn EP1502108A4 (fr)

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US241445 1994-05-11
US37478102P 2002-04-24 2002-04-24
US37478302P 2002-04-24 2002-04-24
US37477902P 2002-04-24 2002-04-24
US374779P 2002-04-24
US374781P 2002-04-24
US374783P 2002-04-24
US206112 2002-07-29
US10/206,112 US6893851B2 (en) 2000-11-08 2002-07-29 Method for arraying biomolecules and for monitoring cell motility in real-time
US10/241,445 US7326563B2 (en) 2000-11-08 2002-09-12 Device and method for monitoring leukocyte migration
US41998002P 2002-10-22 2002-10-22
US419980P 2002-10-22
PCT/US2003/012764 WO2003091730A1 (fr) 2002-04-24 2003-04-24 Dispositif et procede destines a controler la migration des leucocytes

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WO2003091730A1 (fr) 2003-11-06
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EP1502108A4 (fr) 2007-03-21
AU2003223722A1 (en) 2003-11-10

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