JP2006507815A - Biological assays using gradients formed in microfluidic systems - Google Patents

Abstract

The present invention discloses an apparatus for monitoring chemotaxis or chemical invasiveness. The present invention further provides flexible assay systems and multiple assays that can be used to test biological interactions and biological systems. A laminar flow gradient that mimics the gradient situation that exists in vivo is employed.

Description

This application claims the priority of US Provisional Applications Nos. 60 / 419,980 and 60 / 419,976, filed Oct. 22, 2002. These contents are cited in this specification for reference.

The present invention relates generally to devices for monitoring chemotaxis and / or chemical invasiveness. The present invention also generally relates to biological assays performed in gradients formed within a microfluidic system.

  Test devices such as those used for cell migration are well known. Such devices are disclosed, for example, in US Pat. Nos. 6,329,164, 6,238,874, and 5,302,515.

  The three processes involved in cell migration are chemotaxis, chemotaxis and chemoinvasion. Chemotaxis is defined as the movement of cells induced by a concentration gradient of soluble chemotaxis stimuli. Tactility is defined as the movement of cells in response to a concentration gradient of stimuli bound to a substrate. Chemical invasiveness is defined as the movement of cells into the barrier or gel matrix and / or the movement of cells through the barrier or gel matrix. Investigations on cell migration and the effects of external stimuli on such behavior are widely conducted in modern biological research. In general, this study involves exposing cells to external stimuli and investigating the cellular response. By placing living cells in different environments and exposing them to different external stimuli, both the internal workings of the cells and the effects of external stimuli on the cells are measured, recorded and better understood can do.

  Cell migration in response to chemical stimuli is a particularly important issue to consider in order to understand the processes of various diseases and thus develop and evaluate therapeutic candidates for these diseases. By recording the migration of a cell or group of cells in response to a chemical stimulus, eg, a therapeutic agent, the effect of the chemical stimulus can be better understood. Typically, studies of disease processes in various medical fields, such as cancer research, immunology, angiogenesis, wound treatment, and neurobiology, include analysis of chemotaxis and chemoinvasiveness of living cells. For example, in the field of cancer research, cell migration is an important consideration in understanding the process of metastasis. During metastasis, typical solid tumor cancer cells loosen their attachment with neighboring cells, escape these tissues, and break down these tissues by degrading the extracellular matrix of other tissues until they reach blood vessels or lymph vessels. By invading and traversing the basal layer and endothelium of the duct, it must enter the circulation, exit the circulation elsewhere in the body, and survive and proliferate in the new environment where these cells eventually exist Don't be. Thus, cancer cell migration studies can help understand the metastatic process and develop therapeutics that potentially inhibit this process. In the field of inflammatory diseases, cell migration is also an important consideration in understanding the inflammatory response. During the inflammatory response, leukocytes migrate to the damaged tissue site and help fight infection or heal wounds. Leukocytes migrate through the capillaries and adhere to the endothelial cells that line the capillaries. The leukocytes then push between the endothelial cells and crawl across the basal layer by using digestive enzymes. Therefore, studying leukocytes that migrate across endothelial cells and invade the basal layer understands the inflammatory process, and inflammatory diseases such as adult respiratory distress syndrome (ARDS), rheumatoid arthritis, and inflammatory skin It can help in developing therapeutics that inhibit this process in disease. Cell migration is also an important consideration in the field of angiogenesis. When capillaries sprouting from existing small blood vessels, the endothelial cells initially extend from the walls of the existing small blood vessels, generating new capillary branches, and pseudopods guide the growth of the capillary buds into the surrounding connective tissue . The new growth of these capillaries allows cancer growth to expand and spread, contributing to blindness that may be associated with, for example, diabetes. Conversely, a deficiency in capillary generation may contribute to tissue death in the myocardium, for example after a heart attack. Therefore, studying endothelial cell migration as new capillaries are formed from existing capillaries understands angiogenesis and optimizes drugs that block blood vessel growth or improve vascular function Can be helpful in above. In addition, cell migration studies can also provide insight into the processes of tissue regeneration, organ transplantation, autoimmune diseases, and many other degenerative diseases and conditions.

  Cell migration assays are often used when conducting these types of studies. Commercially available devices for forming such assays are based on transwell systems (containers separated by a thin porous membrane to form upper and lower compartments), or This may be adopted. To study cell chemotaxis, cells are placed in the upper compartment and migratory stimulants are placed in the lower compartment. After sufficient incubation time, the effect of stimulation on cytochemotactic properties across the membrane is studied by fixing, staining and counting the cells.

  To study chemical invasiveness, the membrane pores are blocked by covering the membrane with a uniform layer of MATRIGEL® matrix. Cells are seeded on the MATRIGEL® matrix in the upper compartment and the chemoattractant is placed in the lower compartment. Invasive cells attach to the matrix, invade and pass through the porous membrane. Non-invasive cells do not migrate through the blocked holes. After sufficient incubation time, the effect of stimulation on cell invasion across the membrane is studied by fixing, staining and counting the cells.

  The use of transwells has several drawbacks. Assays employing transwells require laborious protocols that are not easily adapted for high throughput screening and processing. Due to the structure of the transwell system, it is difficult to integrate existing robotic liquid handling systems and automatic image acquisition systems. Thus, most screening and processing, eg cell counting, is done by hand, and this is a time consuming, tedious and expensive process. Cell counts are also subjective and often involve statistical estimates. Specifically, only randomly selected representative regions may be counted due to the time and expense associated with examining the entire filter. Furthermore, even if these regions are counted, the engineer himself must make a decision when taking into account cells that have only partially migrated through the filter.

  Transwell-based assays have inherent limitations imposed by the thin films utilized in the transwell system. The thickness of the membrane is only 50-30 microns (μm), and the chemical concentration gradient formed across the membrane is temporary and lasts only for a short time. Currently available transwell assays are satisfactory when the cytochemotactic assay requires that the chemotaxis gradient is generated over a long distance (> 100-200 μm) and is stable for over 2 hours. Cannot be implemented.

  Notwithstanding the above, perhaps the most notable drawback of transwells is that chemotaxis and chemical invasion are not observed in real time. In particular, changes in cell morphology during chemotaxis development cannot be observed in real time when using a transwell. In the case of transwells, these cells are killed when they are fixed on a slide that is needed for observation. As a result, once a cell is observed, it is no longer reintroduced into the assay or studied at the time of subsequent exposure to the test agent. Therefore, in order to study cell progression and changes in cell morphology in response to a test drug, it is necessary to carry out simultaneously on samples that are to be observed at various times before and after the introduction of the test drug. . A single chemotaxis assay requires dozens of filters in addition to the positive and negative controls required to obtain reliable data in light of the need for multiple samples for each test It may be. Each of the filters needs to be individually examined and counted, which is a cumbersome and time consuming task.

  Recently, devices for measuring chemotaxis and chemical invasiveness have become available. These apparatuses employ a configuration in which two wells are shifted in the horizontal direction. Such a configuration of the device was introduced by Sally Zigmond in 1977 (hereinafter referred to as the “Zigmond device”), a 25 mm (mm) x 75 mm slide with two grooves 4 mm wide and 1 mm deep Made of glass, these grooves are separated by 1 mm bridges. One of the grooves is filled with an attractant and the other groove is filled with a control solution, thus creating a concentration gradient across the bridge. Cells are then added to the other groove. Two holes are provided at each end of the slide to receive the pink lamp. The clamp holds the cover glass in place during cell incubation and observation. Due to the size and configuration of the Zigmond chamber, this chamber does not allow integration with existing robotic liquid handling systems and automatic image acquisition systems. Furthermore, similar to transwell-based systems, changes in cell morphology during chemotaxis development cannot be observed in real time using the Zigmond chamber. This is because the cells are fixed to the slide for observation. In addition, the pin clamp must be assembled with an Allen wrench, so the device requires special handling, positioning and alignment prior to performing the assay. Such handling and positioning of the cover glass on the slide glass and the rigidity of the cover glass can potentially damage or interfere with the surface treatment on the bridge.

  A chemotaxis device that attempts to solve the problem of not performing real-time observation is the “Dunn chamber”. The Dunn chamber consists of a specially constructed microslide with a central circular sink and a coaxial annular groove. In assays using the Dunn chamber, cells migrate towards chemotaxis stimuli on coverslips placed upside down on the Dunn chamber. The cells are monitored overnight using a phase contrast microscope equipped with a video camera connected to a computer equipped with an image capture board.

  In addition to the problem of the cover glass being rigid and not integrated into existing robotic liquid handling systems, the main problem with the Dunn chamber assay is a very small number of cells (typically 10). It is only monitored. The average behavior of this very small number of samples is not typical for the whole population. The second major problem is that replication is very limited. Each control chamber and each processing chamber must be viewed with a separate microscope, each equipped with a camera and computer as well.

  Another chemotaxis device known to those skilled in the art is disclosed in US Pat. No. 6,238,874 (Jarnigan et al.) (The '874 patent). The '874 patent discloses various embodiments of test devices that can be used to monitor chemotaxis. However, the Jarnigan et al. Device is disadvantageous because it cannot be easily sealed, assembled, peeled and disassembled. Therefore, it is difficult to maintain a chemically or biologically prepared surface during assembly. Thus, the test apparatus of the '874 patent is more suitable for single use. It is also difficult to break down and collect cells without damaging the cells or without disturbing the cell location.

  The prior art has failed to provide a test device that is easy to assemble and disassemble, such as a device for monitoring chemotaxis and / or chemical invasiveness. In addition, the prior art failed to provide a test device for monitoring cell migration, not limited to measuring the effects of chemoattractants, chemical repellents and chemical stimulants on chemotaxis / chemical invasiveness. is doing.

SUMMARY OF THE INVENTION The present invention is a method for monitoring chemotaxis or chemical invasiveness, an apparatus including a housing, wherein each of a plurality of chambers in the housing includes one or more first wells. A first well region, a second well region including one or more second wells, and a channel region including one or more channels connecting the first well region and the second well region to each other, A method is provided that includes providing the apparatus, wherein the housing defines the plurality of chambers. The method further introduces one or more soluble test substances into the one or more first wells or the one or more channels and creates a dynamic solution concentration gradient along the longitudinal axis of the chamber Including doing. The method further includes introducing a first sample containing cells into the one or more second wells or the one or more channels and monitoring the chemotaxis or chemoinvasiveness of the cells. .

  The present invention makes it possible to optionally include a gel matrix within the channel of the above-described embodiments.

Detailed Description of Embodiments As shown in FIG. 1A, according to one embodiment of the present invention, a device 10 for monitoring chemotaxis / chemical invasiveness includes a housing 10a. The housing 10a includes a support member 16 and an upper member 11. The upper member 11 is attached to the support member 16 by being disposed so as to be in substantially fluid-tight conformal contact with the support member 16. In the context of the present invention, “conformal contact” means substantially fluid-tight contact that is substantially conformal in shape. Support member 16 and upper member 11 are configured such that they together define a discontinuous chamber 12 as shown. As shown by way of example in the embodiment of FIG. 1B, preferably the chemotaxis / chemoinvasive device 10 includes a plurality of discontinuous chambers. The discontinuous chamber 12 includes a first well region 13 a including one or more first wells 13 and a second well region 14 a including one or more second wells 14. The second well region is further offset in a horizontal direction relative to the first well region in the test orientation of the device. “Test orientation” of a device means the spatial orientation of the device under test. As shown in FIG. 1C, the device 10 further includes a channel region 15a including one or more channels 15 that connect the first well region 13a and the second well region 14a to each other. In the embodiment of FIGS. 1A-2C, each well region includes a single well and the channel region includes a single channel. As shown in FIG. 1C, each well is defined by a through-hole provided in the upper member 11 and the upper surface U of the support member 16 corresponding to the well 13 and the well 14, respectively. Specifically, the side surface of each well 13 and 14 is defined by the wall of the through hole of the upper member 11, and the bottom of the wells 13 and 14 is defined by the upper surface U of the support member 16. Note that “top” (top), “bottom”, “top” and “side” are defined with respect to the test orientation of the device for the present invention. As collectively shown in FIGS. 1A and 1C, the length L of the channel region 15a is defined in the longitudinal axis direction of the channel region 15a, and the depth D of the channel region 15a is defined with respect to the upper surface U of the support member 16. The width W is defined in the normal direction with respect to the length L and the depth D of the channel region 15a. According to one embodiment of the present invention, the first well 13 of the chamber is a test agent that is a soluble test substance and / or an immobilized test biomolecule that potentially affects chemotaxis or chemical invasiveness. Adapted to accept. Examples of biomolecules include DNA, RNA, proteins, peptides, carbohydrates, cells, chemicals, biochemicals, and small molecules. The second well 14 of the chamber is adapted to receive a cellular biological sample. The immobilized biomolecule is a biomolecule that is attracted to the support member 16 by an attractive force that exists in the environment surrounding the support member 16, such as a solvating force and a turbulent force. Examples of test agents include chemical repellents, chemotaxis inhibitors, and chemoattractants such as growth factors, cytokines, chemokines, nutrients, small molecules and peptides. Alternatively, the first well 13 of the chamber is adapted to receive a biological sample of cells and the second well 14 of the chamber is adapted to receive a test agent.

  In one embodiment of the invention, a soluble test substance is used as the test agent and the channel region 15a preferably contains a gel matrix. The gel matrix moves from the first well region 13a to the second well as the solute diffuses from the higher concentration region (well region 13a) through the semi-permeable matrix (gel matrix) to the lower concentration (well region 14a). It enables the formation of a solution concentration gradient towards region 14a. When the soluble test substance comprises a chemoattractant, the cell releases the enzyme, e.g. matrix metalloprotease, so that the cell migrates through the matrix in the direction of the solution concentration gradient towards the well region 13a, This matrix must be decomposed. Subsequently, the chemotaxis and chemical invasiveness of the cells can be observed, measured and recorded.

  In one embodiment of the invention that utilizes an immobilized biomolecule as a test agent, the biomolecule is preferably of the support member 16 located below the channel region 15a and below the through hole of the well region 13a. Immobilized or bonded on the part. The biomolecule concentration decreases along the longitudinal axis of the device from the well region 13a toward the well region 14a to form an immobilized biomolecule surface concentration gradient, and the cell biological sample is against this surface gradient. Respond potentially. The cell chemotaxis can then be observed, measured and recorded.

  With respect to the specific specifications of the device 10, the upper member 11 is formed from a material adapted to provide conformal contact with the support member 16, preferably reversible conformal contact. According to embodiments of the present invention, in particular, the flexibility of such materials is substantially fluid tight with the support member 16 by attaching the top member to the top surface U of the support member 16 in a conformal manner. Allows to form a seal. The conformal contact should preferably be strong enough to prevent slippage of the upper member on the support member surface. If the conformal contact is reversible, the top member can be formed from a material that has structural integrity to allow it to be removed by a simple stripping process. Thereby, the upper member 11 is removed, and the cells at a predetermined position can be collected. The stripping process preferably does not interfere with any surface treatment or cell location of the support member 16. Physical striations, pockets, SAMs, gels, peptides, antibodies, or carbohydrates can be placed on the support member 16 and subsequently the top member 11 is supported without damaging these structures. 16 can be covered. In addition, the substantially fluid tight seal provided between the top member 11 and the support member 16 by conformal contact between the top member 11 and the support member 16 allows fluid to leak from one chamber to an adjacent chamber. And prevent contaminants from entering the well. The sealing action preferably occurs virtually instantaneously without the need to maintain external pressure. Conformal contact eliminates the need to use a sealant to seal between the upper member 11 and the support member 16. While embodiments of the present invention include the use of sealants, the fact that the use of sealants is prevented according to preferred embodiments provides another embodiment that reduces costs and saves time, and further Eliminates the risk of contamination of each chamber 12 by the sealant. Preferably, the upper member 11 is made of a material that does not deteriorate and is not easily damaged by being used in multiple tests and that provides considerable variability in the structure of the upper member during manufacture. It is formed. More preferably, the material can be selected to allow the top member to be formed using photolithography. In preferred embodiments, the material comprises an elastomer, such as silicone, natural or synthetic rubber, or polyurethane. In a more preferred embodiment, the material is polydimethylsiloxane (“PDMS”).

  In another embodiment of the invention, the apparatus 10 includes a housing defining a chamber, the chamber including a first well region including one or more first wells; a first well including one or more second wells. A two-well region; and a channel region including a plurality of channels connecting the first well region and the second well region to each other. Preferably, the second well region is offset in a horizontal direction relative to the first well region in the test orientation of the device.

  According to a preferred embodiment of the method of the present invention, a silicon master that is a negative image of any desired configuration of the top member 11 can be formed using standard photolithography procedures. For example, any advantageous specification can be met by changing the dimensions of the chamber 12, such as the size of the well regions 13a and 14a, or the length of the channel region 15a. Once a suitable configuration of the master is selected and the master is made according to this configuration, the material is spin cast, injected, or injected onto the master and cured. Once the mold is formed, this process can be repeated as often as necessary. This process not only provides high flexibility in the construction of the top member, but also allows the top member to be replicated in large quantities. The present invention also contemplates different methods of manufacturing apparatus 10, including, for example, e-beam lithography, laser assisted etching, and transfer printing.

  The apparatus 10 preferably conforms to the industry standard microtiter plate footprint. As such, device 10 preferably has the same outer dimensions and overall size as an industry standard microtiter plate. In addition, if the chamber 12 includes multiple chambers, the chamber 12 itself, or the wells of each chamber 12, may have the same pitch as that of an industry standard microtiter plate. The term “pitch” as used herein refers to the distance between each vertical centerline between adjacent chambers or adjacent wells in the test orientation of the device. The embodiment of the apparatus 10 shown in FIG. 1B includes 48 chambers configured in a standard 96 well plate format, each well adapted to the space of each macrowell of the plate. The size and number of multiple chambers 12 can correspond to standard 24-, 96-, 384-, 768- and 1536-well microtiter plate footprints. For example, for a 96-well plate, the device 10 can include 48 chambers 12, and thus can perform 48 tests, and for a 384-well plate, the device 10 includes 192 chambers 12 192 tests can therefore be performed. The present invention also contemplates any other number of chambers and / or wells arranged in any suitable configuration. For example, if the pitch or footprint standards change, or if new applications require new dimensions, the device 10 can be easily changed to accommodate these new dimensions. By matching the exact dimensions and specifications of the standard microtiter plate, the embodiment of the device 10 will be advantageously adapted to the existing infrastructure of fluid handling, storage, registration and detection. Thus, embodiments of the device 10 can lead to high-throughput screening because it allows robotic fluid handling and automatic detection and data analysis. In addition, the upper member 11 can employ several different variations and embodiments. Depending on the test parameters, for example if chemotaxis or chemoinvasiveness is to be monitored, depending on the cell type, cell number, or the distance at which chemotaxis or chemoinvasiveness is required The chamber 12 of the upper member 11 can have various embodiments. Some of these are discussed herein. For discontinuous chamber 12, the shape, size, position, surface treatment and number of channels in channel region 15a, and the shape and number of wells 13 and 14 may vary.

  Regarding the shape of the channel region 15a, each channel 15 in the channel region 15a is not limited to a particular cross-sectional shape when viewed in a plane perpendicular to its longitudinal axis. For example, the cross section of any given channel 15 may be hexagonal, circular, semicircular, oval, rectangular, square, or any other polygonal or curved shape.

  With respect to the dimensions of the channel 15, the length L of a given channel 15 can vary depending on various test parameters. For example, the length L of a given channel 15 can vary in relation to the distance at which chemotaxis occurs. For example, the range of length L of a given channel 15 can be from about 3 μm to about 18 mm, allowing the cell to travel a sufficient distance, thus reducing cell chemotaxis and chemoinvasiveness. It can provide enough opportunities for observation. The width W and depth D of a given channel 15 can also vary as a function of various test parameters. For example, the width W and depth D of a given channel 15 depends on the size of the test cell and whether a gel matrix is added to the given channel 15 in a chemotaxis / chemoinvasive device. May change. In general, the width W and depth D of a given channel 15 may be approximately within the diameter of the test cell. To reduce random cell movement, at least one of the width W and depth D of a given channel 15 is less than the cell diameter if no gel matrix is placed in the given channel 15 Desirably, this allows the cell to “push” a given channel toward the test agent once it is activated. If a given channel 15 contains a gel matrix, the depth D and width W of a given channel 15 may be greater than the diameter of the test cell. For example, referring to the embodiment of FIGS. 1A-2C, if suspension cells of about 3-12 μm in diameter, such as leukocytes, are present in the well 14 and the channel 15 does not contain a gel, the range of width W of the channel 15 is about Should be from 3 to about 20 μm, and the depth D range of channel 15 should be from about 3 to about 20 μm, but at least one of channel 15 depth D or width W is Should be smaller than the diameter. If leukocytes are present in the well 14 and the channel 15 contains a gel matrix, the width W of the channel 15 should be about 20 to about 100 μm and the depth D of the channel 15 The range should be about 20 to about 100 μm, and both width W and depth D of channel 15 can be larger than the diameter of the test cell. Similarly, if the adherent cells of 3-10 microns in diameter prior to adherence, such as endothelial cells, are present in the well 14 and the channel 15 does not contain a gel, the width W and depth D range of the channel 15 is about 3 ˜about 20 μm, but at least one of the width W or depth D of the channel 15 should be less than the diameter of the test cell. If adherent cells are present in well 14 and channel 15 contains a gel matrix, the width W and depth D range of channel 15 should be about 20 to about 200 μm and the channel Both the width W and the depth D of 15 may be larger than the diameter of the test cell.

  As seen in FIGS. 2A-2C, the channel 15 connects the first well 13 to the second well 14 on each side of the well (FIGS. 2A and 2C) or in the central region of the well (FIG. 2B). Can do.

  The number of channels in the channel region 15a between the well regions 13a and 14a can also vary. The channel region 15a can include a plurality of channels, for example, as shown in FIGS. As seen in FIG. 3A, in a preferred configuration, the length L of each channel 15i-n between well 13 and well 14 is the same. In another embodiment, as seen in FIG. 3B, the length L of each channel 15i-15n of the channel region 15a starts from the channel 15i provided in the side portion 12a of the chamber 12, It increases in the direction of the well 14 when viewed toward the channel 15n provided in the side portion 12b. In one embodiment, as seen in FIG.3B, the length L of each successive channel in the plurality of channels 15 of the chamber 12 is the channel width W relative to the preceding channel of the plurality of channels. The channel inlets provided in one of the first well region and the second well region, for example, the well region 13a shown in the drawing, are aligned along the width W direction of the channel. In this configuration, cells that move in any particular channel exit the channel and enter the well 14 at points that are longitudinally offset from each other, but the section of the channel region 15a closest to the well region 13a can vary Cells that eventually enter different channels are aligned in the width W direction of the channels and positioned so that there is no longitudinal shift between these channels. Thus, when comparing two adjacent channels, the first cell group entering channel 15i has an entry position that is not longitudinally shifted relative to the different second cell group entering channel 15j, but The first cell group exiting channel 15i has an exit point that is longitudinally offset from the second cell group exiting channel 15j. For the different embodiments of the present invention shown in FIG. 3C, the width W of each channel 15i-15n is determined from the channel 15i provided in the side portion 12a of the chamber 12, It increases toward the channel 15n provided in 12b. Preferably, the width W or the depth D of each successive channel of the plurality of channels increases in the direction of the channel width W with respect to the preceding channel of the plurality of channels. Alternatively, the depth D of each successive channel can be increased along the channel width W direction (not shown). As will be apparent to those skilled in the art, various embodiments for changing the dimensions of the channel in the channel region 15a are within the scope of the invention. For example, the length of channels 15i-15n need not increase continuously from channels 15i to 15n, as shown in FIG. 3B. Instead, the channels 15i-15n can have a length that varies without following a specific order or pattern.

  For the surface treatment of a given channel 15, cells on the sidewall of a given channel 15, eg endothelial cells as seen in Figure 4B, to simulate in vivo conditions where the cell is surrounded by other cells 40 can be coated. Examples of endothelial cells include human umbilical vein endothelial cells or highly endothelial venule cells. In another embodiment, a given channel 15 has a gel matrix, such as gelatin, agarose, collagen, fibrin, any natural or synthetic extracellular proteinaceous matrix, or, as an example, MATRIGEL® ( Becton Dickenson Labware) or ECM GEL (Sigma, St. Louis, Mo.) The hydrogel is filled. Preferably, the gel has a substantially high water content and is sufficiently porous to allow cellular chemotaxis or chemical invasiveness. As described above, the gel facilitates the formation of a solution concentration gradient along the longitudinal axis of the chamber 12 when a test agent containing a soluble test substance is placed in the well 13. In addition, by adding a gel matrix to a given channel 15, the natural environment within the body is simulated. This is because enzymatic degradation through the extracellular matrix is an important step in the invasive process.

According to the present invention, the individual wells of each well region 13a or 14a can have any shape and size. For example, the top profile of a given well may be circular as shown in FIGS. 1A-2C or trapezoidal as shown in FIGS. Alternatively, the top profile of a given chamber may be generally “8” shaped, as shown in FIG. When using a soluble test substance, preferably as a test agent, the shape of the well 13 is such that the soluble test substance is easily accessible to the channel 15, thereby the required solution concentration gradient along the length L of the channel 15. Is supposed to form. Preferably, the shape of the well 14 is such that cells accumulate, for example, in the corners, sides or other dead spaces of the well 14, so that migration from the first well 14 is delayed, trapped, or prevented. There is no such thing. The accumulation of cells that can occur in the dead space of the well 14 is not limited to any particular number of cells, but if a large number of cells are used, there is a greater risk of cells accumulating in the corners of the well 14. Therefore, to maximize accessibility to concentration gradients and to minimize cell “wasting” when utilizing large cell samples, it is most important to have a sufficiently high percentage of cells, specifically from channel 15. Forming the shape of the well 14 so that cells in the region of the separated well 14 can migrate from the well 14. In a different embodiment that also addresses the problem of cell waste, the well 14 can be shaped to have a higher probability that all cells have access to the concentration gradient. For example, as seen in FIG. 8, the length Lw of the well 14 in the top view is minimized to reduce the surface area of the well. As such, the cells are closer to the concentration gradient formed in the channel 15. In a preferred embodiment, the L _w of the well 14 in the top view is about 1 mm to about 2 mm.

  In addition to discontinuous changes in chamber 12 components, the present invention also considers changes in the entire chamber 12 as well as changes between chambers. With respect to the entire chamber 12, in one embodiment, the size of the chamber 12 is formed so that the intact chamber 12 fits the area normally required for a single well of a 96-well plate. . In this configuration, 96 different assays can also be performed in 96 well plates. In another embodiment, modifying the chamber 12 changes the ratio of 1: 1 first well to second well as present in the previous embodiment. For example, as seen in FIG. 9, the apparatus 10 has a first well region 13a having a plurality of first wells 102, 103, 104 and 105 connected to each other, and a plurality of wells 106, 107, 108 and 109. The chamber 12 includes a second well region 14a and a channel region 15a having a plurality of channels 15 connecting each of the first wells to each of the second wells. Each well in the first well region 13a can receive the same test agent, and each well in the second well region 14a can receive a different cell type. Alternatively, each well in the first well region 13a can receive a different test agent, and each well in the second well region 14a can receive the same cell type. This configuration allows several different cell types or different test agents to be tested simultaneously. In another embodiment seen in FIG. 10, each channel 15 of the channel region 15a includes the illustrated subchannel. This arrangement not only allows several different cell types or different test agents to be tested simultaneously, but also performs several tests of each test agent or cell type.

  FIG. 11 illustrates another chamber configuration of a chemotaxis / chemoinvasive device according to another embodiment of the present invention. In this embodiment, the chamber 12 includes a first well region 13a connected to a second well region 14a including a single well 14 by a channel region 15a including a single channel 15. The first well region contains a plurality of first wells 17a, 18a and 19a, and a plurality of capillaries including a first peripheral capillary 17, a central capillary 18 and a second peripheral capillary 19, and the capillaries include a plurality of capillaries. Connected to each of the first wells. All three capillaries merge at the junction to become channel 15. The channel 15 is connected to the second well region 14a. The well region 13a is not limited to one containing only three capillaries, and can contain any number of additional capillaries (not shown). The first wells 17a-19a can be adapted to receive, for example, a solution of a test biomolecule. Biomolecules flow into channel 15 and are able to non-specifically adsorb to the area of the surface through which the solution containing the test biomolecule flows. The first wells 17a-19a are also adapted to subsequently receive cells.

  With regard to changes between chambers, in one embodiment, the length L of each channel 15 increases along one or more dimensions of the top member 11 when viewed from one chamber to an adjacent chamber. In another embodiment, all chambers 12 have channels 15 of the same length L. The width W of each channel 15 can vary and increase along one or more dimensions of the top member 11 when viewed from one chamber to an adjacent chamber. In another embodiment, all chambers 12 have channels 15 of the same width W. FIG. 4A is a top view of one embodiment of the present invention in which different chambers within the upper member 11 have various channel sizes and shapes, such sizes and shapes being in a particular order No pattern or arrangement. By employing such a modified configuration, the best channel region configuration for a given test can be obtained. In other words, if an optimal channel region configuration is determined, a new assay plate configured to meet only those specifications can be employed.

  The support member 16 of the device 10 provides a support on which the upper member 11 rests and can be formed from any material suitable for this function. Suitable materials are known to those skilled in the art and are glass, polystyrene, polycarbonate, PMMA, polyacrylate and other plastics. Where the device 10 is a chemotaxis, tactile and / or chemoinvasive device, the support member 16 preferably comprises a material that is compatible with cells that can be placed on the surface of the support member 16. Suitable materials can include standard materials used in cell biology, such as glass, ceramic, metal, polystyrene, polycarbonate, polypropylene, and other plastics including polymeric thin films. A preferred material is glass having a thickness of about 0.1 mm to about 2 mm. This is because glass makes it possible to see cells with optical microscopy techniques.

  Similar to the top member 11, the support member 16 can have several different embodiments. Specifically, the configuration and surface treatment of the support member 16 are variable.

  As can be seen in the side view of the support member 16 shown in FIG. Is inclined. In the illustrated embodiment, the predetermined regions correspond to the bottom surface of each well, that is, the surface 16 a corresponding to the bottom surface of the well 13 and the surface 16 b corresponding to the bottom surface of the well 14. The front surface 16c corresponds to the bottom surface of the channel 15. In this embodiment, the given configuration facilitates the suspension cells to flow in the direction of the downward slope of the upper surface 16b of the support member 16, whereby the suspension cells are easily exposed by the concentration gradient. It becomes like this. When a soluble test substance is used as a test agent in the well 13 of the device 10, the upper surface 16a of the support member 16 is also channeled by tilting downward at an angle of less than 90 ° to the horizontal plane. Test substance exposure to 15 can be facilitated to facilitate the formation of a solution concentration gradient.

  The support member 16 can also have processing means on its surface or embedded in the surface. This processing means can serve a number of functions including, for example, facilitating placement, attachment or movement of the test cells, and simulating in vivo conditions. Numerous surface features and chemicals can be used alone or in conjunction with this treatment means.

  For example, in one embodiment, the support member 16 comprises a patterned self-assembled monolayer (SAM) on a gold surface or other suitable material. SAMs are typically monolayers formed from molecules with functional groups that each selectively attach to a specific surface, with the remainder of each molecule interacting with neighboring molecules in the monolayer. To form a relatively ordered array. By using SAM, various controls of biological interactions can be employed. For example, SAMs can be arranged or modified to have various “heads” to generate biospecific surface “islands” surrounded by bioinert head regions. Furthermore, the SAM can be modified to have a “switchable surface”. A “switchable surface” can be configured to capture cells and then modified to release the captured cells. Furthermore, it may be desirable to utilize a bioinert support member material to resist non-specific adsorption of cells, proteins or any other biological material. Therefore, it may be advantageous to use a SAM on the support member 16.

  The present invention also contemplates detecting and analyzing cell chemotaxis and chemical invasiveness by using any system known to those skilled in the art, as seen in FIG. Specifically, the present invention uses any system known to those skilled in the art to visualize changes in cell morphology as cells move across channel 15, cells within channel 15 Consider measuring the distance traveled and quantifying the number of cells that move to a particular point in the channel 15. As such, the present invention considers chemotaxis and chemoinvasive “real time” and “endpoint” analysis. In one embodiment, the device 122 includes an observation system 120 and a controller 121. The controller 121 is connected to the observation system 120 via a line 122. By positioning and programming the controller 121 and the observation system 120, the chemotaxis and chemical invasiveness of the cells in the device can be observed, recorded and analyzed. The observation system 120 can be any of a number of systems including a microscope, a high speed video camera, and a series of individual sensors. Examples of microscopes include phase contrast, fluorescence, luminescence, differential interference, dark field, confocal laser scanning, digital deconvolution, and video microscopes. Each of these embodiments can observe or sense cell movement and behavior before, during and after introduction of the test agent. At the same time, the observation system 120 can generate signals for the controller 121 to interpret and analyze. This analysis can include measuring the cell's physical movement over time, as well as changes in the cell in shape, activity level, or any other observable characteristic. In each case, the behavior of the test cell can be observed in real time, later, or both. The observation system 120 and the controller 121 enable real-time observation via a monitor. These allow subsequent playback via some type of recording system integrated with or connected to these components. For example, in one embodiment, cell behavior during a desired observation period is recorded on a VHS format video tape by a standard video camera. The video camera is positioned in the vertical eyepiece of the three-eyepiece compound microscope or in the main body of the inverted microscope and is attached to a high quality video recorder. The video recorder is then captured in digitizing means, such as a PCI frame grabber, and the analog data is converted to digital form. The electronically readable (digitized) data is then accessed and processed by a suitable video analysis system, such as the system disclosed in US Pat. No. 5,655,028. The above specification is incorporated herein by reference in its entirety. Such a system is commercially available from Solltech Inc. (Auckland, Iowa) under the trademark DIAS®. Software that can assist in distinguishing cells from debris and other detection artifacts that may be present in the sample should be particularly advantageous. In either case, these components can also analyze these cells as they progress through a response to the test agent.

  In one embodiment, the present invention uses an automated analysis system as shown in FIG. 15 to analyze data measuring the cell migration distance in channel 15, and to identify a specific point in channel 15. Consider quantifying the number of cells that move up to. FIG. 15 is a block diagram of an automatic analysis system 100 including, for example, an image preprocessing stage 110, an object identification stage 120, and a migration analysis stage 130. Image pre-processing stage 110 can receive digital image data of chamber 12 from a digital camera or other image forming device as described above. The data typically includes a plurality of image samples at various spatial locations (called “pixels” for short) and can be provided as color data or grayscale data. Image preprocessing stage 110 may allow other stage algorithms to operate by changing the captured image data. The object identification stage 120 can identify an object from inside the image data. Various subjects can be identified based on the test to be performed. For example, the object identifier can identify the channel 15, cell or cell group from within the image data. The migration analysis stage 130 can perform the migration analysis specified during the test.

  FIG. 15 shows a number of blocks that can be included within the image preprocessing stage 110. In effect, the image pre-processing stage 110 cancels image artifacts that may be present in the captured image data as a result of imperfections in the image former or apparatus. In one embodiment, the image preprocessing stage 110 may include an image equalization block 140. Equalization 140 can be utilized in embodiments where sample values of captured image data do not occupy the entire quantization range available for the data. For example, an 8-bit grayscale system allows 256 different quantization levels (0-255) corresponding to the input data. Due to imperfections in the imaging process, pixel values can be limited to a narrow range, ie the first 20 quantization levels (0-20). Equalization 140 can rescale the sample values to ensure that these values occupy the full range available in the 8-bit system.

  In another embodiment, the equalization block 140 can rescale the sample values based on color or wavelength. Conventional cell analysis techniques often cause cells to appear in a predetermined color or a predetermined wavelength. This makes it possible to distinguish these cells from other materials captured by the imager. For example, when fluorescence is applied, cells emit light at a predetermined wavelength. When nuclear staining is applied, the cell nucleus is stained with a material that causes the cell nucleus to appear with image data having a predetermined color. The equalization block 140 can rescale sample values having components that match these expected colors or wavelengths. By doing this, the equalization block 140 effectively removes other colors or wavelengths with results that may be advantageous in subsequent image processing.

  Image rotation is another image artifact that can arise from an incomplete image forming device. Channel 15 is generally more likely to be aligned with the columns and rows of pixels in the image data, but if this alignment is improved, further analysis can be facilitated. Thus, in one embodiment, the image preprocessing stage 110 includes an image alignment block 150 that counteracts this artifact by rotating the captured image data. Once rotational artifacts are removed from the captured image data, the images from the individual channels 15 are then likely to match a regular row or column array of pixel data.

  FIG. 16 illustrates a method of operation for the image alignment block 150 according to one embodiment of the present invention. This operation method will be described in relation to the image data example shown in FIG. In the example of FIG. 17, channel 15 is generally aligned with a row of image data if there are no rotation artifacts. In order to cancel the rotation artifacts, the image preprocessor can identify an image data band that coincides with the boundary between the second well 14 and the channel 15 itself (block 1010). In the case of FIG. 17, this band can constitute a column 310. In general, the region of the second well 14 is brighter than the region of the channel 15 because there are more cells present here. Thus, a histogram of image data values along the estimated direction of channel 15 can appear as shown in FIG. Band 310 can be distinguished from sudden changes in image data values along this direction.

  Once the column of image data to be considered is identified, the column 310 can be divided into two bounding boxes 320, 330 (block 1020). By summing the image data intensities of each of the two bounding boxes and comparing the sums with each other, the orientation of the rotation artifacts can be determined (blocks 1030, 1040). In the case of the example of FIG. 17, rotation artifacts cause additional second wells 14 to enter the region of the bounding box 320 instead of the bounding box 330 (clockwise artifact). The image data can be rotated counterclockwise until the total value of each bounding box 320, 330 is balanced.

  If the image intensity of the first bounding box is higher than the image intensity of the second bounding box 330, the image data can be rotated in the first direction (block 1050). If the image intensity of the second bounding box 330 is higher than the image intensity of the first bounding box 320, the image data can be rotated in the second direction (block 1060). Then, once the image intensity is balanced, the method 1000 can end and the rotation artifacts have been corrected.

  Returning to FIG. 15, the image pre-processing stage 110 may also process the captured image data by cropping the image to the area occupied by the channel 15 itself (block 160). As described above, each test bed can include a pair of wells interconnected by a plurality of channels. In most migration analyses, it is sufficient to measure the movement or activity of cells only within channel 15. It is not necessary to consider the activity in the second well 14 or the first well 13. In such an embodiment, the image preprocessing stage 110 can remove pixels located outside the channel 15 by trimming the image data.

  The image preprocessing stage 110 may include a threshold processing block 170 that performs threshold detection on the image data. The threshold processing block 170 can truncate any sample with a rescaled value that does not exceed a predetermined threshold to zero. Such threshold processing is useful for removing noise from captured image data. In one embodiment, the threshold processing block 170 can be integrated with the equalization block 140 described above. The threshold processing block 170 need not exist as a separate element. In some implementations, particularly in implementations where the equalization block 140 scales pixel values according to wavelength components, the threshold processing block 170 can be omitted entirely. The output of the image preprocessing stage 110 may be an input to the object identification stage 120.

  The object identification stage 120 identifies the channel itself and possibly objects including individual cells from within the image data. According to one embodiment, channel 15 can be identified in the fluorescence system by creating a histogram of fluorescence along the major axis in the fluorescence system (block 180). FIG. 19 shows image data that can be measured from the example of FIG. The main axis can coincide with the boundary between the well adapted to receive cells and the channel region. The light intensity from inside the channel region 15a can be summed along this axis to obtain the data set shown in FIG. In the second stage, the data set is “expanded” (block 190). Expansion can be achieved by applying a high pass filter to the data set or any other similar technique. FIG. 20 shows the expanded data set of FIG.

  Channels can be identified from the data set of FIG. The position of the candidate channel 15 can be identified as matching the relative maximum value of the data set. Alternatively, the candidate position of the boundary between the channels 15 can be determined from the relative minimum value in the data set of FIG. From the parameter set known for the channel region 15a itself, the final set of channel 15 positions can be determined. For example, if channel 15 is known to have a regular spacing between channels 15, any candidate channel 15 position that would violate this spacing can be excluded from consideration.

  Returning to FIG. 15, in addition to identifying channel 15, individual cells can be identified in the image data (block 200). In applications where cells are marked with nuclear staining, all that is required to identify individual cells is that the image processing device identify and count the number of marked nuclei. The appearance of the nucleus is a number of points of a given color. In applications using fluorescent cells, the identification of individual cells is more complicated. Individual cells can be identified relatively easily, and these cells appear as objects of relatively uniform areas in the image data. It is more difficult to identify a large number of cells clustered together. In this case, the number of cells can be determined from the area or radius of the cluster in the image data. Clusters are likely to appear in images with some area or cluster radius. By comparing the area or radius of the cluster with the area or radius of individual cells, the number of cells can be manipulated. Of course, identification of individual cells can be omitted depending on the requirements of migration analysis.

  The final stage in the image processing system is the migration analysis 130 itself. In one embodiment, coordinate data for each cell in channel 15 can be collected and recorded. However, some tests do not require this complexity. In the first embodiment, it is sufficient to identify the number of cells present in channel 15. In this case, identification of individual cells can be avoided by simply summing the amount of fluorescence detected in each channel 15. From this measurement, the cell number can be derived without investing the processing costs of identifying individual cells. The above description provides image analysis associated with the test single channel 15. Of course, depending on the requirements of the migration analysis 130, it may be desirable to generate a number of different channel 15 image samples. Further, it may be desirable to generate a single channel 15 image sample at different times. Such a test scenario can be accommodated by repeating the above image processing for different channels 15 and different time points.

  According to one embodiment, the image processing can take into account manufacturing defects of the individual channels 15. During image processing, manufacturing defects can prevent cells from migrating into channel 15. In one embodiment, when system 100 counts a large number of cells in channel 15 (or derives the number from the identified cell location), system 100 may compare the number to an expected threshold. it can. When that number falls below the expected threshold, the system 100 can exclude channel 15 from the migration analysis. In fact, this expectation threshold can be established as the lowest number of cells that are likely to enter a properly configured cell, given that the test conditions are analyzed under migration analysis. If the actual number of cells falls below that threshold, it can be concluded that conditions exist that block channel 15.

  The above-described operations and processes of the analysis system 100 can be performed by a multipurpose processor that executes software, such as a computer, workstation, or server. Alternatively, some of the operations and processes may be provided in a digital signal processor or application specific integrated circuit (spoken “ASIC”). In addition, these tasks and processes, specifically related to image preprocessing, can be assigned within the processor of the digital microscope system. Such variations are fully within the scope of the present invention.

  The present invention also contemplates the use of the above-described embodiments of apparatus 10 to assay various components of chemotaxis / chemoinvasiveness. Overall, the present invention provides a first assay that includes high-throughput screening of a test drug to determine whether the test drug affects chemotaxis / chemoinvasiveness. The test agent generally comprises a soluble test substance or an immobilized test biomolecule and is generally disposed within the first well region 13a of the chamber 12 of the device 10. Act as a chemoattractant and promote or initiate chemotaxis / chemical invasiveness, or act as a chemical repellent and repel chemotactic / chemical invasiveness, or inhibitor To determine which test drugs affect chemotaxis / chemical invasiveness by acting as, and stopping or inhibiting chemotaxis / chemical invasiveness, then performing a second assay to test Compounds can be screened. The test compound generally comprises a therapeutic agent or chemotaxis / chemoinvasive inhibitor and is generally introduced into the second well region 14a containing the cellular biological sample. Screening test compounds will determine whether and how they affect cell migration in response to test drugs.

  Specifically, a chemotaxis / chemoinvasive assay according to one embodiment of the present invention includes a device 10 that includes a housing that includes a top member 11 attached to a support member 16. The top member and support member are configured such that they together define a discrete assay chamber 12. The discontinuous assay chamber 12 includes a first well region 13a connected to a second well region 14a by a channel 15. The first well region 13a includes one or more first wells 13, each of the one or more first wells 13 being adapted to receive a test agent. The second well region 14a includes one or more second wells 14 that are offset in a horizontal direction with respect to the first well region 13a in the test orientation of the device. Each of the one or more second wells 14 is adapted to receive a cell sample. The channel 15 includes one or more channels that connect the first well region 13a and the second well region 14a to each other. The test agent received in the first well 13 is a soluble test substance and / or an immobilized test biomolecule. When the test drug contains an immobilized test biomolecule, the biomolecule is immobilized on the upper surface U of the support member 16 constituting the bottom surface of the well region 13a and the upper surface U of the support member 16 constituting the bottom surface of the channel region 15a. It becomes.

Examples of cell biological samples include lymphocytes, monocytes, leukocytes, macrophages, mast cells, T cells, B cells, neutrophils, basophils, eosinophils, fibroblasts, endothelial cells, epithelial cells, neurons Tumor cells, motile gametes, bacterial and fungal motility forms, cells involved in metastasis, and any other cell type involved in response to inflammation, injury and infection. Well region 14a can receive only one cell type or any combination of the cell type examples described above. For example, as described above, it is often desirable to more effectively create an environment similar to in vivo conditions by providing a mixed cell population. The well region 14a can also receive cells in a specific cell cycle phase. For example, the well region 14a is capable of receiving the leukocytes in G ₁ phase or G ₀ phase.

Examples of soluble test substances include chemoattractants, chemical repellents, or chemotaxis inhibitors. As described above, the chemoattractant is a chemotactic substance that attracts cells, and when placed in the well region 14a, causes the cells to migrate toward the well region 14a. The chemical repellent substance is a chemotactic substance that repels cells, and when arranged in the well region 14a, causes the cells to migrate away from the well region 14a. A chemotaxis inhibitor is a chemotaxis substance that inhibits or stops chemotaxis, and when placed in the well region 14a, inhibits or does not allow cells to migrate from the well region 14a. . Examples of chemoattractants include hormones such as T ₃ and T ₄ , epinephrine and vasopressin; immunological substances such as interleukin-2, epidermal growth factor, and monoclonal antibodies; growth factors; peptides; small molecules; Is mentioned. Cells can act as chemoattractants by releasing chemotactic factors. For example, in one embodiment, a sample containing cancer cells can be added to well 13. Samples containing different cell types can be added to well 14. As cancer cells grow, they can release factors that act as chemoattractants that attract the cells in well 14 to migrate towards well 13. In another embodiment, endothelial cells are added to well 13, and the endothelial cells are activated by adding a chemoattractant such as TNF-α or IL-1 to well 13. Leukocytes are added to the well 14 and the leukocytes can be attracted to the endothelial cells in the well 13.

  Examples of chemical repellents include stimulants such as benzalkonium chloride, propylene glycol, methanol, acetone, sodium dodecyl sulfate, hydrogen peroxide, 1-butanol, ethanol and dimethyl sulfoxide; and toxins such as cyanide, carbonyl cyanide Chlorophenylhydrazones, endotoxins and bacterial lipopolysaccharides; viruses; pathogens; and pyrogens.

Examples of immobilized biomolecules include chemoattractants, chemical repellents, and chemotaxis inhibitors as described above. Further examples of immobilized chemoattractants include chemokines, cytokines and small molecules. Further examples of chemoattractants include IL-8, GCP-2, GRO-α, GRO-β, MGSA-β, MGSA-γ, PF ₄ , ENA-78, GCP-2, NAP-2, IL -8, IP10, I-309, I-TAC, SDF-1, BLC, BRAK, Bolecaine, ELC, LKTN-1, SCM-1β, MIG, MCAF, LD7α, Eotaxin, IP-110, HCC-1, HCC -2, Lkn-1, HCC-4, LARC, LEC, DC-CK1, PARC, AMAC-1, MIP-2β, ELC, Exodus-3, ARC, Exodus-1, 6Ckine, Exodus-2, STCP-1 , MPIF-1, MPIF-2, eotaxin-2, TECK, eotaxin-3, ILC, ITAC, BCA-1, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MCP-1, MCP-2 , MCP-3, MCP-4, MCP-5, RANTES, eotaxin-1, eotaxin-2, TARC, MDC, TECK, CTACK, SLC, lymphotactin and fractalkine; and other cells. Further examples of chemical repellents include receptor agonists and other cells.

  In order to perform a test, for example a chemotaxis and / or chemical invasive assay utilizing soluble test substances, the test device 10 is first fabricated. A preferred embodiment of a method for manufacturing a device according to the present invention is described below. A master that is the negative of the top plate 11 can be fabricated by standard photolithography procedures. A predetermined material is spin-coated or injection-molded on the master. The predetermined material is then cured and peeled off from the master to form the upper member 11 which is placed on the support material 16.

  A rigid frame having a standard microtiter plate footprint is preferably disposed around the outer peripheral surface of the upper member 11. In one embodiment, a gel matrix is injected into the well region 13a, thereby allowing the gel matrix to flow into the channel region 15a. After installing the gel matrix, excess gel is removed from the well regions 13a, 14a. In another embodiment, no gel matrix is added to the channel region 15a. Subsequently, the cellular biological sample is placed in the well region 14a and the test substance is placed in the well region 13a. In one embodiment, a low concentration of test substance is placed in the well region 14a to activate the cells and facilitate the initiation of the assay. Alternatively, depending on the test cell and the soluble test substance used, the soluble test substance can be introduced during or after placing the cell in the well region 14a. When the soluble test substance is introduced, the solution concentration gradient of the test substance is caused by the diffusion method from the well region 13a containing the test drug to the well region 14a containing the cell biological sample, along the longitudinal axis of the channel region 15a. Formed along. The secondary effect of this solution gradient is the formation of a physisorbed (immobilized) gradient. Once this solution gradient is established, some portion of the solute of the test substance can be adsorbed on the support member 16. This adsorbed layer of test solute can also contribute to chemotaxis and chemical invasiveness. The cellular biological sample responds to this concentration gradient and either migrates towards a higher concentration of the test substance, or migrates away from the higher concentration of the test substance, or a higher concentration of the test substance Inhibition of movement can be shown in response to. Through such chemotaxis in response to a gradient, the chemotactic effect of a chemotactic substance can be measured. Measure the cell migration distance and the time it takes for the cell to reach a given point in the channel region 15a or the amount of time it takes for the cell to reach a given point in the well region 14a (from the chemotactic substance) Chemotaxis is assayed by (in the case of chemical repellents that cause cells to move away).

  Another embodiment of the apparatus 10 that includes another configuration of the chamber 12 is utilized to create a solution concentration gradient using a network of microfluidic channel regions. For this embodiment seen in FIG. 14, the first well region 13 a of the chamber 12 has first wells 20, 21 and 22 connected to the channel 15 by the network of microfluidic capillaries 23. Specifically, the first well region 13a includes a plurality of first wells, and the first wells include a plurality of capillaries 24 connected to each of the plurality of first wells and a plurality of sub capillaries 25. And connected by. The sub-capillaries 25 are branched such that each of them is connected to each of a plurality of capillaries at one end and to the channel 15 at the other end. Each first well 20, 21, 22 receives a different concentration of soluble test substance. After three different concentrations of soluble test substance are injected into the three first wells 20, 21, 22 simultaneously, the solution stream moves down the channel region network, continuously splits and mixes, Then re-merge. After generation of several branched subcapillaries, each subcapillary containing a different population of soluble test substance is merged into a single channel 15, creating a concentration gradient across channel 15, perpendicular to the flow direction. Form.

  According to another embodiment of the invention for monitoring tactility, in any one of the embodiments of the test device of the invention, for example the embodiment shown in FIGS. 1A-14, the support member 16, preferably A biomolecule is immobilized on the upper surface U constituting the bottom surface of the channel 15 and the well region 13a. The concentration of the biomolecule increases or decreases along the longitudinal axis of the device from the upper surface of the support member 16 constituting the bottom surface of the well region 13a toward the upper surface U of the support member 16 constituting the bottom surface of the well region 14a. As a result, a surface gradient is formed. After the test biomolecule is immobilized on the support member 16, the upper member is placed on the support member 16, and a rigid frame having a standard microtiter footprint is placed around the outer peripheral surface of the upper member 11. , And add cells to the well region 14a. In another embodiment, after the test biomolecule is immobilized on the support member 16 and the top member is over the support member 16, the gel matrix is added to the channel region 15a. Subsequently, cells are added to the well region 14a. The cellular biological sample potentially responds to the concentration gradient of immobilized biomolecules and migrates towards a higher concentration of test biomolecules or away from the higher concentration of test biomolecules Or exhibit inhibition of movement in response to higher concentrations of the test biomolecule. The surface gradient can be increased linearly or as a quadratic, cubic, or logarithmic function, or can be increased in any surface profile that can be approximated by increasing or decreasing in steps.

K. Efimenko and J. Genzer, “How to Prepare Tunable Planar Molecular Chemical Gradient”, 13 Applied Material , 2001, 20, Oct. 16; US Pat. No. 5,514 (cited herein for reference). The test biomolecule can be bound on the upper surface U of the support member 16 by various specific or non-specific approaches known to those skilled in the art, as described in A surface gradient can be formed on the upper surface U. For example, a microcontact printing technique, or any other method known to those skilled in the art, can be used to immobilize the SAM layer that provides hexadecanethiol on the upper surface U of the support member 16. The support member 16 is then exposed to high energy light through a desired gradient micropattern photolithography mask or a grayscale mask with a continuous gradation from white to black. Removal of the mask leaves a surface gradient of SAM that provides hexadecanethiol. The support member 16 is then immersed in a solution of alkanethiol terminated with ethylene glycol. The region of the support member 16 with SAM providing hexadecanethiol will rapidly adsorb biomolecules, and the region of the support member with SAM providing oligomers of ethylene glycol groups will resist protein adsorption. . The support member 16 is then immersed in a solution of the desired test biomolecule, and this biomolecule is rapidly adsorbed only in the region of the support member 16 containing SAM providing hexadecanethiol, and the immobilized biomolecule Form a surface gradient.

  In another embodiment, in any one of the embodiments of the test device of the present invention, eg, the embodiment shown in FIGS. Immobilized on member 16 and a surface concentration gradient is formed. In this embodiment, a discrete concentration solution containing the test biomolecule is continuously placed in the well region 14a to allow the test biomolecule to non-specifically adsorb to the support member 16. . For example, first, a 1 milligram / milliliter (mg / ml) solution can first be placed in the well region 14a; second, a 1 microgram / milliliter (μg / ml) solution can be placed in the well region 14a. Finally, a 1 nanogram / milliliter (ng / ml) solution can be placed in the well region 14a. As a result of the different concentrations of test biomolecules in the solution, the amount of adsorption on the support member 16 will also differ.

Utilizing another embodiment of the apparatus 10 containing another configuration of the chamber 12 as seen in FIG. 11, the concept of laminar flow consisting of a plurality of parallel liquid streams (a method known to those skilled in the art) Based on this, an immobilized biomolecule surface gradient is formed. Based on this concept, when two or more flows with a low Reynolds number merge to form a single flow with a low Reynolds number, the combined flows flow parallel to each other without turbulent mixing. According to one embodiment, the chemotactic biomolecule solution is placed in 17a and 19a and the protein solution is placed in 18a. These solutions can flow into the channel region 15a while being affected by gentle suction in the well region 14a. Biomolecules adsorb nonspecifically to the surface region over which the biomolecule-containing solution flows, forming a surface gradient. The wells are then filled with a suspension of cells and the motility of the cells towards an increasing concentration gradient of the biomolecule is observed and monitored. S. Takayama et al., “Patterning Cell and their Environment Using Multiple Laminar Fluid Flows in Capillary Networks” Pro. Natl. Acad. Sci. USA , Vol . 96, 5545-5548, May 1999 I want to be.

  The present invention also contemplates assays that use both soluble and surface concentration gradients to determine whether soluble test substances or immobilized test biomolecules have a greater effect on cell migration. In this embodiment, the assay is performed by forming the surface gradient described above, the assay is performed by forming the solution gradient described above, the assay is performed by forming both types of gradients, and these Compare the results of the three assays. With respect to the combined gradient assay, in any one of the embodiments of the test apparatus of the present invention, such as the embodiment shown in FIGS. The test biomolecule is immobilized on the upper surface of the support member 16 located on the side, and the concentration of the biomolecule decreases from the well region 13a toward the well region 14a along the longitudinal axis of the chamber 12. In addition, a soluble test substance is added to the well region 13a. Such an embodiment forms a surface concentration gradient and a soluble chemotaxis concentration gradient that decrease in the same direction. If the combined concentration gradient has a synergistic effect on cell migration, both gradients are cell receptors that bind to the chemotactic ligand of the soluble chemotactic substance and cell receptors that bind to the immobilized biomolecule. Should be used to screen both the body. Both types of receptors are identified as important and therapeutic agents that target both of these receptors, or one that targets one receptor and the other targets another receptor Can be screened for. When the combined concentration gradient does not have a synergistic effect, individual gradients that more strongly promote cell migration can be identified and cell receptors that bind to the chemotactic ligand of the test agent that forms that gradient Can be targeted.

  Identifying optimal pairs of chemotactic ligands and receptors is important in understanding the biological pathways associated with cell migration and developing therapeutic agents that target these pathways. Thus, the present invention as a whole uses chemotaxis test agents to determine which chemotaxis receptors expressed on the cell surface have the greatest impact on chemotaxis and / or chemoinvasiveness. Make it possible to identify. In one embodiment, the present invention allows high throughput screening of a given class of chemoattractant. This chemoattractant is known to attract specific cell types that have receptors on the cell surface for each chemoattractant in this class. Such screening identifies which receptors are more strongly involved in chemotaxis and / or chemical invasive processes. After identifying this receptor, the present invention identifies the receptor as potential depending on whether chemotaxis and / or chemical invasiveness is desired to be promoted or prevented. Consider high-throughput screening for therapeutic agents that block or bind to this receptor. In another embodiment, the present invention allows for high-throughput screening of different chemoattractants known to bind to the same receptor on the surface of a particular cell type and thereby which chemoattractant ligands It can be determined whether the / receptor pair has a greater effect on chemotaxis and / or chemical invasiveness. After identification of this ligand / receptor pair, does the present invention target this receptor and whether chemotaxis and / or chemical invasiveness is desired to be promoted or prevented? Accordingly, high-throughput screening of therapeutic agents that block or activate this receptor will be considered.

  The present invention also provides for the chemistry of specific cell types with various chemotactic receptors on the cell surface to identify which receptors are more strongly involved in chemotaxis and / or chemoinvasive processes. Also contemplated are high-throughput screening of certain classes of chemotaxis inhibitors known to inhibit chemotaxis. After identifying this receptor, the present invention allows high-throughput screening of therapeutic agents that also potentially block this receptor (if such an effect is desired).

  In one embodiment of the invention, an assay is performed to determine if the test compound inhibits cancer cell invasiveness. In this embodiment, in any one of the embodiments of the test apparatus of the present invention, eg, the embodiment shown in FIGS. 1A-14, untreated cancer cells are placed in the well region 14a of the chamber 12 and tested. Place the drug in the well region 13a. Measure and record cell chemotaxis and invasiveness. After identifying a suitable test agent (a test agent that chemically attracts cancer cells), another assay is performed in chamber 12. In this subsequent assay, cancer cells are placed in the well region 14a and a test compound, eg, a therapeutic agent, is also placed in the well region 14a. In another embodiment, the test compound is also placed in the channel region 15a. If the gel matrix is to be added to the channel region 15a, the test compound can be mixed with the gel matrix before the gel contacts the channel region 15a during fabrication of the device 10. The next sample of the test drug identified in the first assay is placed in the well region 13a to determine the chemotaxis and invasiveness of cells treated with the test compound, and the chemotaxis of cells not treated with the test compound. And compared with invasiveness. The anti-cancer potential of a test compound is measured by the chemotaxis and invasive rate of the treated cancer cells compared to the untreated cancer cells.

  For another example of the use of the chemotaxis and chemoinvasive devices of the present invention, the device can be used to assay a cellular response to an inflammatory response. Local infection or injury in any tissue of the body attracts leukocytes into the damaged tissue as part of the inflammatory response. The inflammatory response is mediated by a variety of signal molecules generated within the damaged tissue site by mast cells, platelets, nerve endings and leukocytes. These mediators act on capillary endothelial cells and cause these cells to loosen their association with adjacent endothelial cells, making the capillaries more permeable. Endothelial cells also express cell surface molecules that are stimulated to recognize specific carbohydrates. These carbohydrates are present on the surface of leukocytes in the blood and attach these leukocytes to the endothelial cells. Other mediators released from the damaged tissue act as chemoattractants, allowing the bound leukocytes to migrate between the capillary endothelial cells and into the damaged tissue. To investigate leukocyte chemotaxis, in one embodiment, channel region 15a is treated to simulate conditions in human capillaries during an inflammatory response. For example, the side wall of the channel region 15a is coated with, for example, an endothelial cell that expresses a cell surface molecule such as selectin as shown in FIG. 4B. Then, in any one of the embodiments of the test apparatus of the present invention, eg, the embodiment shown in FIGS. 1A-14, white blood cells are added to the well region 14a and a known chemoattractant is added to the well region 13a. . Other suitable cell types that can be added to the well region 14a are neutrophils, monocytes, T and B lymphocytes, macrophages, or other cell types involved in response to injury or inflammation. The chemotaxis of leukocytes crossing the channel region 15a toward the well region 13a is observed. Depending on the type of infection to be investigated, different categories of leukocytes can be used. For example, in one embodiment for examining cytochemotactic response to bacterial infection, the well region 14a receives neutrophils. In another embodiment for examining cytochemotactic response to viral infection, the well region 14a receives T cells.

  In another embodiment that simulates the process of angiogenesis, as is known to those skilled in the art, the growth factor applied to the cornea is reduced from the periphery of the dense angiogenic tissue surrounding the cornea to a low center of the cornea. Induce new blood vessel growth towards density angiogenic tissue. Thus, in the case of another example assay that utilizes chemotaxis and chemoinvasive devices, in any one of the embodiments of the test device of the present invention, such as the embodiments shown in FIGS. The cells are placed in the well region 13a, and the endothelial cells are placed in the well region 14a. Growth factors are added to the well region 13a and endothelial cell chemotaxis is observed, measured and recorded. Alternatively, because angiogenesis is important in tumor growth (to supply oxygen and nutrients to the tumor mass), instead of adding growth factors to well region 13a, angiogenic factors such as vascular endothelial growth factor (VEGF) Can be added to the well region 13a, and normal endothelial cells can be added to the well region 14a. In another embodiment, also related to the examination of angiogenesis, mast cells, macrophages, and adipocytes that release fibroblast growth factor during tissue repair, inflammation and tissue growth are placed in the well region 13a, Endothelial cells are then placed in the well region 14a. During angiogenesis, the capillary buds grow into the surrounding connective tissue so that the channel region 15a can be filled with a gel matrix to further simulate in vivo conditions.

  There are several variations and embodiments of the assay described above. One embodiment involves the number of channels connecting the well region 13a and the well region 14a of the chamber 12 of the device 10. In one embodiment as shown in FIG. 3A to 3C, there are a plurality of channels connecting the well region 13a and the well region 14a. Multiple channels allow multiple assays to be performed simultaneously using a single cellular biological sample. In such embodiments, all assays are performed under uniform and consistent conditions, thus providing statistically more accurate results. For example, each assay begins with exactly the same number of potentially migrating cells and exactly the same concentration of test agent. Once a concentration gradient is formed, each assay is exposed to the gradient over the same time. These multiple channels also provide a surplus if the assay fails.

  Another embodiment of the cell invasiveness and chemotaxis assay of the present invention is the well region of the chamber 12 in any one of the embodiments of the test apparatus of the present invention, such as the embodiment shown in FIGS. Including placing cells within 14a. The cells can be patterned to form a specific array on the upper surface U of the support member 16 that forms the bottom surface of the well region 14a. Alternatively, the cells can simply be deposited in the well region 14a without a specific pattern or arrangement. If the cells are patterned to form a particular array on the top surface of the support member 16 that forms the bottom surface of the well region 14a, preferably the support member that forms the bottom surface of the well region 14a during fabrication of the device 10 The upper surface of 16 is first patterned with cells, and then the upper member 11 is placed on the support member 16. It is desirable to accurately measure the distance and time that a cell travels by monitoring the movement of the cell from a predetermined “start” location. As such, in one embodiment, the cells are immobilized on a support member located below the first well, such that the viability of the cells is maintained and the location of the cells can be defined or Since it is patterned, chemotaxis and invasiveness can be observed. Several techniques are known to those skilled in the art for forming discontinuous arrays on a support member by immobilizing and patterning cells. A preferred technique is described in co-pending application 60 / 330,456. In the case of one embodiment, by using the cell position patterning member, the cells are patterned in a definable region on the upper surface U of the support member 16 constituting the bottom surface of the well region 14a of the upper member 11. .

  For example, the top member 11 is fabricated with a standard 96-well microtiter plate footprint so that the wells 13 and 14 correspond to the size and shape of the macrowell of the microtiter plate (not shown). , The cell location patterning member has a contoured area corresponding to the size and shape of the wells 13 and 14, and thus corresponding to the size and shape of the macrowell of the microtiter plate. Each contoured region has a micro through hole through which cells are patterned. In order to pattern the cells, the cell location patterning member is brought into contact with the support member 16, and the contoured region of the cell location patterning member is a portion of the upper surface U of the support member 16 constituting the bottom surface of the well region 14a. When aligned and the upper member 11 is brought into contact with the support member 16, the contoured region will eventually correspond to the well region 14a. Cells can then be deposited on the cell location patterning member, these cells passing through the through holes of the cell location patterning member and corresponding to the through holes corresponding to the second well region 14a of the chamber 12. It is possible to gradually exit onto a support member located below the area. Next, the upper member 11 is placed on the support member 16 so that the through hole 14a is positioned on the region of the support member 16 where cells are patterned. As a result of these patterning steps, a discontinuous array of cells is formed in the well region 14a.

  Preferably, the cell location patterning member comprises an elastomeric material such as PDMS. The use of PDMS for the patterned member provides a substantially fluid tight seal between the patterned member and the support member. This substantially fluid tight seal is preferred between these two components. This is because if such a seal exists between the patterned member and the support member, the cells placed in the well are less likely to enter the adjacent well. The arrangement of micro-through holes in the patterned member may be rectangular, hexagonal, or another array in which cells will be patterned in their respective shapes. The width of each microthrough hole may vary depending on the cell type and the number of cells to be patterned. For example, if both the cell and the through-hole width are 10 microns, only one cell will deposit through each micro-through hole. Thus, in this example, when the width of the micro through hole is 100 microns, a maximum of about 100 cells can be deposited.

  The present invention also provides a test apparatus according to the present invention in any one of the embodiments shown in FIGS. 1A to 14, for example, two or more kinds on the upper surface of the support member 16 constituting the bottom surface of the well region 14a. Consider patterning cell types. One type of cell rarely exists in isolation in vivo, but instead has contact with and communicates with other cell types, thus having a system that can assay cells in an environment more similar to the body environment It is desirable. For example, because cancer cells are never found in isolation and are surrounded by normal cells, an assay configured to test the effect of a drug on a cancer cell will have the cancer cell in that assay surrounded by normal cells. It becomes more accurate. In the case of an anticancer drug test, in any one of the embodiments of the test apparatus of the present invention, for example, the embodiment shown in FIGS. 1A to 14, cancer cells are placed on the upper surface of the support member 16 constituting the bottom surface of the well region 14a. The cancer cells can then be surrounded by stromal cells by patterning and then by a separate patterning procedure. In order to pattern two different cell types on the upper surface of the support member 16 that constitutes the bottom surface of the well region 14a, a micro cell position patterning member as described above is brought into contact with the support member 16 to form a cell position pattern. When the contouring region of the shaping member is aligned with the upper surface U portion of the support member 16 that constitutes the bottom surface of the well region 14a, and the upper member 11 is brought into contact with the support member 16, the contouring region finally enters the well region 14a. Will respond. Next, cells of the first type can be deposited on the cell position patterning member, and these cells pass through the micro-through holes of the patterning member, and the upper surface of the support member 16 constituting the bottom surface of the well region 14a. Can gradually get out on the U part. The microcell location patterning member can then be removed from the support member 16. A macrocell location patterning member having a contoured area corresponding to the size and shape of the wells 13 and 14 can thus correspond to the size and shape of the macrowell of a 96 well microtiter plate. The macro cell location patterning member has macro through holes. The macro through hole of the macro cell location patterning member includes an area larger than the surface area defined by the micro through hole of the micro cell location patterning member, but more than the surface area defined by the well region 14a of the chamber 12. Is also small. The macrocell location patterning member can then be brought into contact with the support member 16. The second type of cells can then be deposited on the macrocell location patterning member, and when the top member 11 is brought into contact with the support member 16, these cells will have macrothrough holes in the macropatterning member. Through this, it is possible to gradually come out on the portion of the upper surface U of the support member 16 constituting the bottom surface of the well region 14a. As a result of such a patterned arrangement, the second type cells can surround and “stack” the first type cells. If it is only desired to stack the first type cells on the second type cells, the micro cell location patterning member used to deposit the first cell type, or the first type cell to deposit A second type of cell can be deposited using a different microcell location patterning member having exactly the same configuration as the patterning member used. After the cells are patterned on the support member 16, the through-hole of the upper member 11 corresponding to the well region 14a includes the region patterned with the cells by bringing the upper member 11 into contact with the support member 16. It will be. As a result, the cells are effectively immobilized in a specific array within the well region 14a.

  No matter how many different cell types are patterned on the top surface of the support member 16 that constitutes the bottom surface of the well region 14a, the cells are patterned on the support member by several methods known to those skilled in the art. Can. For example, cells can be patterned on the support member 16 by using SAM. There are several techniques known to those skilled in the art for patterning cells by using SAM. Some of these techniques are described in U.S. Pat. And 5,976,826 (Singhvi et al.), Which are incorporated herein by reference.

  Several methods for labeling cells to observe and measure the aforementioned parameters are known to those skilled in the art. In one embodiment, an unpurified sample containing the cell type is incubated with a stain that is absorbed differently by the various cell types. The cell is then placed in the well region 14a of the chamber 12 in any one of the embodiments of the test apparatus of the present invention, such as the embodiment shown in FIGS. Individual stained cells are detected based on color or intensity contrast using any suitable microscopic technique, and location coordinates are assigned to such cells. In another embodiment, the crude cell sample is incubated with one or more detectable reporters. Each reporter can selectively bind to the specific cell type and impart a characteristic fluorescence to all labeled cells. The cell is then placed in the well region 14a of the chamber 12 in any one of the embodiments of the test apparatus of the present invention, such as the embodiment shown in FIGS. An appropriate wavelength of light is then applied to the sample to detect fluorescent cells and assign position coordinates to these cells. As will be apparent to those skilled in the art, various methods can be utilized to distinguish selected cells from other components in the unpurified sample. For example, these methods can include dyes, radioisotopes, fluorescent agents, chemiluminescent agents, beads, enzymes and antibodies. Specific labeling of cell types can be accomplished using, for example, fluorescently labeled antibodies. Cell labeling processes are known to those skilled in the art, as are various fluorescent dyes that can be used for labeling specific cell types.

  The cells of the selected type are in a mixed population of cells, eg, a detectable reporter that selectively and / or preferentially binds to such cells, or a selected combination of detectable reporters. It can also be differentiated. For example, labeling can be accomplished using monoclonal antibodies that selectively bind to expressed CDs, antigens and receptors, and the like. Examples of tumor cell antigens include CD13 and CD33 present on myeloid cells; CD10 and CD19 present on B-cells; and CD2, CD5 and CD7 present on T-cells. As will be apparent to those skilled in the art, a number of markers are available that distinguish a variety of known cell markers. In addition, additional markers are subsequently discovered. Such markers useful for labeling cells, whether currently known or discovered in the future, can be utilized to practice the present invention.

  There are few, if any, absolutely specific markers for a single cell type, so label two or more markers, and each marker will have a different label for each selected cell type. It is desirable to have it. Detecting multiple labels corresponding to each selected cell type should help ensure that chemotaxis and chemical invasive analysis is limited to that cell only.

  The invention further provides support means; support means and means for defining a discontinuous chamber attached to the support means by being arranged in fluid-tight conformal contact with the support means. . The discontinuous chamber includes a first well region that includes one or more first wells; and one or more second wells that are horizontally displaced relative to the first well region in the test orientation of the apparatus. A second well region including: a channel region including one or more channels connecting the first well region and the second well region to each other. An example of a support means includes the support member 16 shown in FIGS. 1A, 1B, 12 and 13, whereas an example of the means attached to the support means is shown in FIGS. 1A-11, 13 and 14. An upper member 11 is included. Other such means will be apparent to those skilled in the art.

  In another embodiment, the present invention provides a method for assaying and studying biological phenomena that depend on or are responsive to gradient formation and / or flow conditions. Such biological phenomena include many of the processes in the body, such as cell surface interactions that occur during leukocyte attachment and rolling. In addition, for studies involving chemotaxis, chemotaxis and cell migration, assays that study such cell motility in the presence of gradient and / or flow conditions are of greater use.

  Various types of gradients are useful in biological system studies. Such useful gradients include static gradients. The static gradient has a concentration that is fixed or set, or substantially fixed or set. An example of a static gradient is the gradient of immobilized molecules on the surface. An example of a static gradient is the use of different concentrations of immobilized biomolecules (proteins, antibodies and nucleic acids, etc.) or immobilized chemical moieties (drugs and small molecules). Other useful gradients include dynamic gradients. The dynamic gradient has a concentration that can be varied. An example of a dynamic gradient is a fluid flow gradient with molecules of varying concentration. An example of a fluid gradient includes the use of fluid streams containing biomolecules with varying concentrations, such as growth factors, toxins, enzymes, proteins, antibodies, carbohydrates, drugs, or other chemicals and small molecules.

In one embodiment of the invention, a dynamic / solution based gradient is formed by laminar flow techniques. Laminar flow techniques typically involve two or more fluid streams from two or more different sources. These fluid streams are made into a single stream together and are formed to flow parallel to each other without turbulent mixing. Fluids with different properties, such as varying low Reynolds, flow in parallel and these fluids do not mix unless turbulent flow is present. Since the fluid does not mix, it forms a pseudo channel (due to the fact that there is no physical separation between the fluids). The generation of solutions and surface gradients is discussed in US Patent Application No. 2002/0113095 and paper (Jeon, Noo Li et al., Langmuir , 16, 8311-8316 (2000)). Both of these references are hereby incorporated by reference.

  In these contexts, a PDMS microfluidic device was utilized to generate a gradient through the capillary microfluidic network. Multiple solutions containing different chemicals were introduced into three separate inlets and these solutions were able to flow through the capillary network. Repeated merging, mixing and splitting of the fluid streams resulted in a distinguishable mixture having a distinguishable composition in each of the branch channels. Upon remerging all of the branches, a concentration gradient was established across the outlet channel, perpendicular to the flow direction. See FIG.

  By combining the device of the present invention with the formation of a dynamic gradient, a vast number of assay parameters can be generated by modifying any part of the device. For example, the devices disclosed herein can be combined with cell patterning techniques in conjunction with the introduction of dynamic gradients to create a variety of conditions for testing a large number of biological interactions. . In addition, these devices and assays are useful for drug discovery and drug testing. This is because when a cell or biological material is present in vivo, and thus when the cell or biological material is not exposed to a gradient, the number of cells or biological materials is increased. This is because substances behave differently ex vivo.

  Thus, in one embodiment of the invention, cells can be patterned across the channel. Cell patterning can be achieved by methods known to those skilled in the art, as well as methods disclosed in the present invention (eg, using microcontact printing or using elastomeric stencils). A solution containing any desired biomolecule or chemical / drug can then be flowed across the patterned cells. In addition, by first treating with a biomolecule, such as an activator, the cell more closely reproduces the biological system. These cells can then be exposed to chemicals or drugs. For example, by creating a gradient with laminar flow, different amounts of biomolecules or chemicals / drugs can be supplied to the patterned cells, and thus the concentration of each biomolecule or chemical / drug. The effects can be tested against each other at the same time. Thus, such comparisons performed simultaneously in parallel reduce the variability of conditions between assays.

  By creating a dynamic gradient in laminar flow in combination with the device of the present invention, a number of assay configurations are provided. For example, a vast number of assays can be formed by varying the combination of cells on the surface, biomolecules in the channel and compounds in the channel.

  Different assay configurations are possible for immobilized cells or other immobilized biomolecules such as proteins, antibodies, nucleic acids and the like. In one embodiment, a single cell type is immobilized throughout the channel region. In another embodiment, the cell type mixture is immobilized such that one cell type is located per region. In another embodiment, the cell type mixture is immobilized throughout the channel region. This may be advantageous for monitoring the interaction between cells. In yet another embodiment, different cell types are immobilized in each different region.

  In addition to various immobilization schemes, the center of further flexibility in assay design lies in the biomolecules present in the channel. For example, in one embodiment, one type of biomolecule is present in each channel at the same concentration. In another embodiment, one type of biomolecule is present in each channel at different concentrations. In another embodiment, different types of biomolecules are present in each channel. In another embodiment, there is a mixture of biomolecules in each channel. Each channel may have the same mixture or a different mixture. For the same mixture, the ratio or concentration of different biomolecules may be different in each channel.

  Similarly, for compounds such as drugs or test substances, the present invention provides flexibility in assay design. For example, in one embodiment, a single compound is present in all channels consistently at the same concentration. In another embodiment, the same compound is present in all channels, but each channel has a different concentration of that compound. In another embodiment, each channel has a different compound. In another embodiment, there are two or more compounds. If there are two or more compounds, each channel can have the same compound mixture or different compound mixtures. Furthermore, if the compound mixture is the same, each channel can receive a different concentration of the mixture. Still further, each channel can receive a mixture of compounds with each channel having a different compound ratio.

  By using such an assay system, the effects of chemicals or drugs on cells or other biomolecules can be tested from among a number of biological interactions. For example, the IC50 of a compound can be measured by using a laminar flow gradient of the compound present at low to high concentrations that is flowed across the immobilized biomolecule by the apparatus and assay of the present invention.

  As will become apparent from the foregoing, numerous modifications and variations can be provided without departing from the true spirit and scope of the novel concepts of the present invention. For example, different embodiments of the device of the invention can be combined. Embodiments of the present invention contemplate different types of assays, such as those in which the test agent contains a buffer instead of a chemotactic agent. For such assays, cell migration through channel region 15a is observed without the presence of a chemotaxis gradient.

  It will be appreciated that the disclosure of the present invention is intended to be illustrative of the invention, which is not intended to limit the invention to the specific embodiments. It is intended that the disclosure be supplemented by the appended claims and all modifications that come within the spirit thereof.

Example 1: Chemical Invasive Device Fabrication Procedure A silicon wafer (6 inches) is spin coated with photoresist (SU8-50) at 2000 rpm for 45 seconds. After the wafer is baked on a hot plate at 115 ° C. for 10 minutes, the wafer is allowed to cool to room temperature. A mask aligner (EVG620) is used to expose the photoresist film through the photomask. Following a 45 second exposure, a further hard bake is performed at 115 ° C. for 10 minutes. Allow the silicon wafer to cool to room temperature for more than 30 minutes. Propylene glycol methyl ether acetate (PGMEA) is used to remove this uncrosslinked photoresist. The wafer is dried under a stream of nitrogen and the patterned photoresist is ready for subsequent processing.

  In one embodiment, the patterned photoresist is spin coated with another SU8-100 layer at 1500 rpm for 45 seconds. A mask aligner is used to selectively expose the macro features (ie, wells) of the top member, but not the channel region connecting the wells and other regions of the top member. After post-exposure processing and photoresist removal, the master contains multiple layered features. By repeating this process, a macro component of about 3mm in height is introduced on the master.

  When a PDMS prepolymer is cast to a master, the PDMS prepolymer faithfully replicates the configuration requirements within the master. When casting, add PDMS in an amount slightly less than the height of the macro component. After curing PDMS at 65 ° C for 4 hours, peel the PDMS from the silicon master, thoroughly clean with soap and water, and rinse with 100% ethanol. The glass support member is also cleaned and rinsed with ethanol. The PDMS membrane and glass support member are oxidized with plasma for 1 minute. In this case, the side to be coupled to each other is directed upward. The PDMS membrane is then placed on the glass support member and pressure is applied to remove any air bubbles that may have formed between the PDMS membrane and the glass support member. The assembled device is then cooled to 4 ° C. Within 15 minutes of plasma oxidizing the PDMS membrane and glass support member, 20 microliters (μl) of Matrigel (other hydrogels can be used) are poured into the first well and allowed to flow into the capillary. Matrigel is cured by placing the apparatus at room temperature for 15 minutes. The excess gel is then removed from the top member well using a vacuum and Pasteur pipette.

Example 2: Cytochemical Invasion Assay Placement of Cells and Test Agents in Chamber The first and second wells of the upper member chamber are filled with phosphate buffered saline, PBS. The bottom of the second well can be treated with fibronectin (1 mg / ml) or other extracellular matrix protein for 30 minutes followed by 2 washes with PBS. After aspirating PBS, place astrocytoma cells (U87-MG) in 50 μl of freshly heated medium in the second well (25,000 cells per well in a 24-well plate, volume per well 50 μl medium). Cells are deposited through the second well of the chamber and adhere to the bottom of the second well.

  Overnight in a 37 ° C. incubator, leave cells to attach and spread in second well. At the start of the test, the cell medium is replaced with fresh medium without serum. 10 μg bFGF (basic fibroblast growth factor) per ml of medium is added to the first well of each chamber.

Image acquisition and data analysis
Use AXIOCAM® to take digital images with a Zeiss inverted microscope. Analyze data with AXIOVISION® software. Take time-lapse images every day for 4 days at the same time.

Example 3: Cytochemical Invasion Inhibition Assay Using Solution Gradient Placement of cells and test agents in chamber
For the three chambers, fill the wells of each chamber of the top member with PBS. The bottom of the second well can be treated with fibronectin (1 mg / ml) or other extracellular matrix protein for 30 minutes followed by 2 washes with PBS. After aspirating PBS, U87-MG is placed in 50 μl of freshly heated medium in the second well (10,000 cells per well in a 24-well plate, 50 μl medium per well). Cells are deposited through the second well of each chamber and adhere to the bottom of the second well.

  Overnight in a 37 ° C. incubator, leave cells to attach and spread in second well. At the start of the test, the cell medium is replaced with fresh medium without serum or with 1% serum. 1 μg of bFGF (basic fibroblast growth factor) per ml of medium is added to the first well of the chamber. A solution gradient is allowed to form over 1 hour.

  For three different chambers, place 100 μM LY294002 in the second well of chamber # 1, 10 μM LY294002 in the second well of chamber # 2, and 1.0 μM in the second well of chamber # 3 Put LY294002.

Image acquisition and data analysis
Use AXIOCAM® to take digital images with a Zeiss inverted microscope. Analyze data with AXIOVISION® software. Take time-lapse images every day for 4 days at the same time.

Example 4: Immobilization of biomolecules on a support member After assembling the device as described above, the channel region is filled with an ethanol solution containing (CH ₃ CH ₂ O) ₃ Si (CH ₂ ) ₃ NH ₂ . After 20 minutes, wash the channel region with ethanol at room temperature. The siloxane monolayer formed on the support member is crosslinked by incubating the device at 105 ° C. for 1 hour. Residues are removed by washing the apparatus with ethanol. The channel region is filled with a solution of diisocyanate, ie hexamethylene diisocyanate or tolyl diisocyanate (1% in acetonitrile or N-methylpyrrolidinone). The diisocyanate is allowed to react with the terminal amino group of the siloxane monolayer formed on the support member for 2 hours. Wash off diisocyanate. The channel region is filled with heparan sulfate or other sulfated carbohydrates (eg, diacetylated forms such as heparin, heparin fragments, lectins containing sulfated sugars). By allowing heparan sulfate to react with the support member, an immobilized species is formed. Wash away heparan sulfate solution and other reagents. A chemokine solution (any chemokine selected from CC, CXC, CX3C or XC group can be used) is introduced into the channel region. Due to electrostatic interactions, chemokines with higher pI (˜9-10) are adsorbed onto the negatively charged sulfated support members.

Example 5: Chemotaxis inhibition assay using surface gradient
Two wells are filled with 50 μl PBS and the hydrostatic pressure is allowed to equilibrate. The hydrostatic pressure is equilibrated by adding 5 μl anti-hisx6 antibody to the first well and 5 μl buffer to the second well. By diffusion, the antibody concentration forms a gradient from the first well to the second well. After 2 hours at room temperature, the two wells are washed by adding 50 μl buffer to the second well and removing 50 μl from the first well. The solution gradient is transferred onto the surface by physisorption, thereby forming a surface gradient. A solution of IL-8 (recombinant human IL-8 with HISx6 fusion label, R + D system, catalog number 968-IL) at a concentration of 25 μg / ml is added to the channel region. Incubate the solution for 30 minutes at room temperature. Wash off excess IL-8 chemokine and decorate the surface with bound IL-8. Add neutrophils (just separated from healthy donors) to the second well. Typically, 20,000-100,000 cells are added in a volume range of 10-550 μl. Neutrophils are allowed to adhere to the support member and allowed to migrate toward higher concentrations of IL-8. Migration is inhibited by adding a polyclonal antibody against LI-8.

Example 6: Selective activation of endothelial cells by feeding TNF-α into a gradient formed by laminar flow Until the surface of the device of the present invention is coated with endothelial cells and these cells are confluent Allowed to grow (formation of “lawn” of cells). Endothelial cells were “activated” by supplying TNF-α to the endothelial cell lawn via laminar flow. Each flow of the solution containing TNF-α was at a different concentration, thus forming a gradient perpendicular to the channel. This gradient effectively supplied TNF-α to the endothelial cell lawn at different concentrations on the cell lawn at different concentrations. The leukocytes were then flushed onto the activated endothelial cell lawn. Only endothelial cells activated by TNF-α provide a suitable “attachment” site for leukocytes. White blood cells do not adhere equally to the entire lawn, adhere to the endothelial cell lawn area exposed to high concentrations of TNF-α, and the endothelial cell lawn area exposed to low concentrations of TNF-α, Or it did not adhere to the region which was not exposed to TNF-α at all. These results indicate that a concentration gradient of TNF-α due to laminar flow was indeed formed. See FIG.

The various features of the present invention will become most apparent from the following description and the accompanying drawings.
FIG. 1A is a top perspective view showing in partial cross section a portion of one embodiment of a test apparatus according to the present invention. FIG. 1B is a top perspective view showing one embodiment of the test apparatus of the present invention. FIG. 1C is a side elevation view showing one of the chambers of the test apparatus of FIG. 1B in cross-section in the longitudinal direction. FIG. 2A is a schematic top view showing another embodiment of a chamber defined in the test apparatus of the present invention, wherein the channel region defines a single channel. FIG. 2B is a schematic top view showing an embodiment of the chamber defined in the embodiment of the test apparatus according to FIG. 1B, wherein the channel region defines a single channel. FIG. 2C is a view similar to FIG. 2A showing another embodiment of a chamber defined in the test apparatus of the present invention, wherein the channel region defines a single channel. FIG. 3A is a view similar to FIG. 2A showing another embodiment of a chamber defined in the test apparatus of the present invention, wherein the channel region defines a plurality of channels having the same length. is there. FIG. 3B is a view similar to FIG. 3A showing a channel region defining a plurality of channels that increase in length from one side of the chamber toward the other side of the chamber. FIG. 3C is a view similar to FIG. 3A showing a channel region defining a plurality of channels that increase in width from one side of the chamber toward the other side of the chamber. FIG. 4A is a view similar to FIG. 1B, showing another embodiment of a test apparatus according to the present invention. FIG. 4B is an enlarged schematic top view of the channel of FIG. 4A showing the cells lateral to the channel. FIG. 5 is a view similar to FIG. 2A showing another embodiment of a chamber defined in the test apparatus of the present invention, wherein the well is trapezoidal in its top view. FIG. 6 is a view similar to FIG. 2A showing another embodiment of a chamber defined in the test apparatus of the present invention, wherein the well is trapezoidal in its top view. FIG. 7 is a view similar to FIG. 2A, showing another embodiment of a chamber defined in the test apparatus of the present invention, wherein the chamber has the shape of FIG. 8 in its top view. FIG. 8 is another embodiment of a chamber defined in the test apparatus of the present invention, wherein one well is rectangular in its top view and the other well is circular in its top view. FIG. 2B is a view similar to FIG. 2A. FIG. 9 is another embodiment of a chamber defined in the test apparatus of the present invention, in which the first well region and the second well region each define a plurality of wells, and the channel region corresponds to each of the well regions. FIG. 2B is a view similar to FIG. 2A showing an embodiment defining a plurality of channels connecting the wells. FIG. 10 shows another embodiment of a chamber defined in the test apparatus of the present invention, wherein the channel region defines a plurality of channels connecting each well of each well region, It is the same figure. FIG. 11 shows another embodiment of the chamber defined in the test apparatus of the present invention, wherein the first well region has a plurality of wells and capillaries corresponding to the respective wells, and the channel region is a single one. FIG. 2B is a view similar to FIG. 2A showing an embodiment having one channel and the second well region having a single well. FIG. 12 is a side cross-sectional view of a part of a support member according to the invention, the support member part being shown along the longitudinal axis of a chamber according to the invention. FIG. 13 is an isometric view showing a collection system according to one embodiment of the present invention. FIG. 14 is another embodiment of a chamber defined in the test apparatus of the present invention, wherein the first well region includes a plurality of wells interconnected by a capillary network, and the channel region is a single channel. FIG. 2B is a view similar to FIG. 2A, showing an embodiment in which the second well comprises a single well. FIG. 15 is a block diagram showing an automatic analysis system according to one embodiment of the present invention. FIG. 16 is a flowchart of a method according to one embodiment of the present invention. FIG. 17 is an example of image data, and shows an example in which the method of FIG. 16 can be implemented based on this. FIG. 18 is a diagram showing a histogram that can be obtained from the image data of FIG. FIG. 19 is a diagram illustrating an example of image data. FIG. 20 is a diagram showing a histogram that can be obtained from the image data example of FIG. FIG. 21 is a diagram showing the results of a test including generating a TNF-α concentration gradient via laminar flow. TNF-α was supplied to a dense “lawn” of endothelial cells. Endothelial cells that have been contacted by TNF-α are activated and can thus bind to leukocytes. The endothelial cells were then delivered leukocytes. As shown in the figure, leukocytes are bound to endothelial cell regions that receive high concentrations of TNF-α, whereas endothelial cell regions that are not or hardly exposed to TNF-α bind to leukocytes. There wasn't. FIG. 22 shows a microfluidic device for creating a laminar flow gradient.

Claims (25)

A method for monitoring chemotaxis or chemical invasiveness, comprising:
An apparatus including a housing, wherein each of a plurality of chambers in the housing
A first well region comprising one or more first wells;
A second well region comprising one or more second wells;
Providing the apparatus comprising a channel region including one or more channels connecting the first well region and the second well region to each other, the housing defining the plurality of chambers;
Introducing one or more soluble test substances into the one or more first wells or the one or more channels;
Creating a dynamic solution concentration gradient along the longitudinal axis of the chamber;
Chemotaxis or chemistry comprising introducing cells into the one or more second wells or the one or more channels; and monitoring chemotaxis or chemoinvasiveness of the cells How to monitor invasiveness.   The method of claim 1, wherein the one or more channels contain a gel matrix. Formation of dynamic solution concentration gradient:
Introducing a first fluid stream having a first concentration of a first substance into one or more chambers of the plurality of chambers; and a second fluid into one or more chambers of the plurality of chambers. The method of claim 1, comprising introducing a second fluid stream having a second concentration of material, wherein the first concentration and the second concentration are different from each other.   4. The method of claim 3, wherein the first material and the second material are the same.   4. The method of claim 3, wherein the first material and the second material are different.   The first fluid stream and the second fluid stream merge to form a single third fluid stream in fluid communication with one or more of the plurality of chambers, wherein the third fluid stream is the first and 4. The method of claim 3, comprising a second material concentration gradient, wherein the concentration gradient is substantially perpendicular to the flow direction of the third fluid stream.   The first fluid stream and the second fluid stream merge to form a single third fluid stream, and the third fluid stream then diverges into separate fourth, fifth, and sixth fluid streams. The fourth, fifth and sixth fluid streams are then recombined into a single seventh fluid stream, wherein the single seventh fluid stream is substantially laminar under the plurality of conditions. 4. The method of claim 3, wherein the method is in fluid communication with one or more of the chambers.   Introducing the cells into the one or more second wells or the one or more channels includes patterning the cells on the one or more channels along the longitudinal axis of the chamber. And introducing the soluble test substance into the one or more first wells or the one or more channels comprises introducing the soluble test substance into the one or more channels by laminar flow The method of claim 1.   The method of claim 1, wherein introducing cells into the one or more second wells or the one or more channels comprises placing a single cell type within the one or more channels. Method.   2. The introducing of cells into the one or more second wells or the one or more channels comprises placing a mixture of multiple cell types within the one or more channels. the method of.   Introducing the cell into the one or more second wells or the one or more channels, wherein each of the plurality of channels has a different cell type; The method of claim 1, comprising introducing.   12. The method of claim 11, wherein introducing different cell types into each of the plurality of channels comprises introducing different cell types into each of the plurality of channels at different concentrations.   12. The method of claim 11, wherein introducing different cell types into each of the plurality of channels comprises introducing different cell types into each of the plurality of channels at the same concentration.   Introducing the cells into the one or more second wells or the one or more channels is a single cell type for each of the plurality of channels, wherein the one or more channels are a plurality of channels; The method of claim 1 comprising introducing:   15. The method of claim 14, wherein introducing a single cell type into each of the plurality of channels comprises introducing the single cell type into each of the plurality of channels at different concentrations.   15. The method of claim 14, wherein introducing a single cell type into each of the plurality of channels comprises introducing the single cell type into each of the plurality of channels at the same concentration.   Introducing the cells into the one or more second wells or the one or more channels, wherein the one or more channels are a plurality of channels, The method of claim 1, comprising introducing.   18. The method of claim 17, wherein introducing the cell mixture into each of the plurality of channels comprises introducing the same cell mixture or a different cell mixture into each of the plurality of channels.   The one or more channels are a plurality of channels, the one or more soluble test substances are a plurality of test substances, and the one or more channels in the one or more first wells or the one or more channels 2. The method of claim 1, wherein introducing the soluble test substance includes introducing a different substance of the plurality of test substances into each of the plurality of channels.   The introduction of different substances of the plurality of test substances into each of the plurality of channels comprises introducing different substances of the plurality of test substances into each of the plurality of channels at different concentrations. 19. The method according to 19.   The introduction of different substances of the plurality of test substances into each of the plurality of channels comprises introducing different substances of the plurality of test substances into each of the plurality of channels at the same concentration. 19. The method according to 19.   The one or more channels are a plurality of channels, the one or more soluble test substances are a plurality of the same soluble test substances, and the one or more first wells or the one or more channels in the one or more channels The method of claim 1, wherein introducing one or more soluble test substances comprises introducing one of the plurality of the same test substances into each of the plurality of channels.   Introducing one of the plurality of the same soluble test substances into each of the plurality of channels comprises introducing one of the plurality of the same soluble test substances into each of the plurality of channels at different concentrations; 23. A method according to claim 22.   Introducing one of the plurality of the same soluble test substances into each of the plurality of channels comprises introducing one of the plurality of the same soluble test substances into each of the plurality of channels at the same concentration; 23. A method according to claim 22.   The housing includes a support member and an upper member, the upper member being attached to the support member by being disposed in substantially fluid-tight conformal contact with the support member. The method according to 1.

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