Apparatus and method for isolating extracellular matrix material

Abstract

To provide a device, a method and a system for isolating an extracellular matrix body.SOLUTION: The present invention discloses devices, methods and systems for isolating extracellular matrix material from a biological sample for use in diagnosis and prognosis of a subject. The device can have a restricted channel for isolating extracellular matrix bodies and a uniform flow channel for precise pressure measurement.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to devices, methods and systems for isolating extracellular matrix bodies. More particularly, the present invention discloses devices, methods and systems for isolating extracellular matrix bodies from biological samples for use in the diagnosis and prognosis of a subject. [Background technology]

[0002] Traditional methods for disease diagnosis and prognosis require analyzing or isolating a small portion of a biological sample. Analysis of components from this microfraction can be used for disease diagnosis and prognosis. For example, individual cells and even individual nucleic acid molecules can be detected and analyzed. However, a drawback of these traditional methods is that they rely on very small amounts of biological material and correspondingly small signals for decision-making. Useful signals may be lost if other biological material is not measured.

[0003] Conventional methods for diagnosing and prognosing disease often require the collection of biological samples, the isolation of known structures from them, and further analysis. For example, intact organ tissues, whole cells, exosomes, and other known structures can be isolated and characterized. A drawback of such methods is that they cannot detect or characterize disease when the known structures do not readily reflect the disease state.

[0004] Further drawbacks of conventional methods for disease diagnosis and prognosis include the use of biomarkers, such as exosomes, that are associated only with specific, well-known structural elements of a subject's biological sample. These methods are often inherently limited because the biomarkers are not directly related to the pathology of interest. Conventional methods often attempt to consider multiple biomarkers that may be remotely or partially associated with a disease in the hope of statistically determining a diagnostic answer. A combination of biomarkers is routinely required, making results highly unpredictable.

[0005] A further drawback of conventional devices is their inability to accurately measure flow rate and pressure in complex fluids containing biological components, where interactions between the biological components and the measuring device can cause fluctuations in flow rate and pressure.

[0006] What is needed are devices and systems for isolating particles from biological samples for diagnostic purposes that can provide increased sample isolation related to a particular biology. More particularly, what is needed are devices, methods and systems for isolating significant components from biological samples for use in disease diagnosis and prognosis.

[0007] There is an urgent need for methods and devices for isolating particles from biological samples that isolate the appropriate fraction of more pathological biological material and that allow for appropriate fluid differential pressure and flow measurement. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides devices and systems for isolating particles from biological samples that are applicable to a variety of diseases and conditions. Methods and systems for isolating particles from biological samples are provided that can increase biologically relevant sample isolation. The devices and methods of the present invention can be used to isolate key components from biological samples for use in disease diagnosis and prognosis. [Means for solving the problem]

[0009] In some embodiments, the methods and devices of the present invention for isolating particles from biological samples can be used with various types of biological samples, including bodily fluids, tissues, and cells. In certain embodiments, the disclosed methods can isolate relevant fractions of biological material that closely correspond to disease states.

[0010] The disclosed methods and devices can be used to isolate significant fractions of biological samples, followed by analysis of their components relevant to disease diagnosis and prognosis. In some embodiments, significant amounts of biological material and correspondingly improved signal levels are obtained for decision-making. Aspects of the invention include preserving the composition and properties of extracellular matrix bodies (EMBs) as indicators of disease.

[0011] In some aspects, the present invention improves the ability to detect or characterize disease using extracellular matrix bodies, which can more readily reflect the disease state.

[0012] In additional aspects, the disclosed devices and methods can provide enhanced signals for appropriate biomarkers that correlate specific biological fractions with disease states. The disclosed biomarkers can be correlated with disease and provide diagnostic tools.

[0013] The devices of the present disclosure can provide accurate measurements of flow and pressure in fluids containing biological components by maintaining continuous and substantial flow within the device so that sensors can accurately measure differential pressure and flow rate. The devices and systems disclosed herein can provide improved measurements of flow and pressure in fluids containing biological components through the arrangement of channels that maintain continuous and substantial flow. In some embodiments, the devices of the present disclosure can have one or more channels that do not substantially restrict fluid flow so that continuous and substantial flow is maintained within the system.

[0014] The disclosed devices and systems can isolate unique subpopulations or subset fractions of a biological sample. In some embodiments, the unique subset fraction of a biological sample can be associated with a disease. In certain embodiments, the unique subset fraction of a biological sample can be substantially composed of extracellular matrix bodies.

[0015] In a further aspect, the present disclosure provides devices and methods for isolating, detecting, and / or analyzing ultrastructural components of fluids containing biological materials or molecules. In certain embodiments, the ultrastructural components may be associated with disease.

[0016] Embodiments of the present invention can be used to isolate, extract, and utilize extracellular matrix bodies (EMBs), which are a source of multiple and specific disease biomarkers.

[0017] The present invention can be used in a variety of applications, including devices for isolating, detecting, and analyzing compositions of extracellular matrix bodies, bioparticles, and complexes.

[0018] In certain aspects, the extracellular matrix body can act as a biomarker through its morphological characteristics. In further aspects, the extracellular matrix body can act by containing isolated biochemical markers that can be present in disease pathways.

[0019] Aspects of the present invention can further provide diagnostic systems that include devices for detecting and measuring biomarkers via disease-associated extracellular matrix bodies (EMBs) and / or biological particles or complexes.

[0020] In further embodiments, the present disclosure describes methods and apparatus for preparing and analyzing samples of biological material.

[0021] Biological samples may include bodily fluids, tissues, cells, and the like.

[0022] Embodiments of the present invention include:

[0023] 1. An apparatus for isolating a fraction of a biological sample, comprising: one or more restricted channels having an inlet end and an outlet end, the inlet end and the outlet end being in fluid communication via the channel; a plurality of spaced apart obstacles disposed in the restriction channel to provide resistance to flow, the spacing between obstacles decreasing in a direction from the inlet end to the outlet end; and an inlet reservoir for holding a fluid, the inlet fluid reservoir being in fluid communication with the inlet end of the restriction channel; one or more uniform flow channels having an inlet end and an outlet end, the inlet end and the outlet end being in fluid communication through a channel, the inlet end being in fluid communication with the inlet reservoir; An apparatus comprising:

[0024] The above apparatus, further comprising: a pressure source for applying pressure to the fluid in the inlet reservoir; and / or a flow sensor in fluid communication with the inlet reservoir for measuring the flow rate and pressure of the fluid in the inlet reservoir.

[0025] The device may further comprise an outlet reservoir in fluid communication with the outlet ends of the restriction channel and the uniform flow channel.

[0026] The above device, wherein the restriction channel comprises a barrier band having perforations of at least about 1 micrometer, or at least about 2 micrometers, or at least about 4 micrometers, or at least about 10 micrometers, or at least about 25 micrometers, or at least about 50 micrometers, or at least about 100 micrometers, or at least about 200 micrometers, or at least about 500 micrometers.

[0027] The above device, wherein the restriction channel comprises perforations of about 1 to 4 micrometers, or about 1 to 15 micrometers, or about 4 to 35 micrometers, or about 4 to 100 micrometers, or about 4 to 200 micrometers.

[0028] The above device, wherein 1-90% of the flow in the device is in the uniform flow channel, or 1-75% of the flow in the device is in the uniform flow channel, or 1-50% of the flow in the device is in the uniform flow channel, or 1-25% of the flow in the device is in the uniform flow channel.

[0029] The restriction channel and the uniform flow channel may be integrated into the same chip or substrate. The restriction channel and the uniform flow channel may be on different chips or substrates. The restriction channel may be a microfluidic channel.

[0030] The device further comprising means for analyzing the biological sample in the channel.

[0031] The above device, further comprising means for analyzing the proteomic, lipidomic, transcriptomic, or carbohydrate composition of the biological sample in the channel.

[0032] The above apparatus further comprising means for measuring the level of the isolated fraction of the sample in the channel.

[0033] The above device further comprising a means for measuring the level of a biomarker in the isolated fraction of the sample in the channel.

[0034] The apparatus, wherein the plurality of obstacles comprise pillars integral with the channel.

[0035] The above device, wherein the plurality of obstacles include one or more of a portion of a human or animal uvea, a portion of a human or animal cornea-retina, or a portion of a human or animal chorionic retina.

[0036] The plurality of obstacles may be composed of glass beads, magnetic beads, gel particles, dextran particles, or polymer particles.

[0037] The biological sample may consist of human or animal body fluids, blood, tissues, or cells. The biological sample comprises a carrier fluid.

[0038] The device, wherein the biological sample comprises one or more reagents.

[0039] The device, wherein the restricted channel further comprises binding sites for binding biomarkers or biomolecules of the sample.

[0040] The biological sample may be from a subject undergoing diagnosis or prognosis.

[0041] The device as described above, further comprising a serpentine fluid mixing region in the restricted channel.The device as described above, wherein the restricted channel or continuous flow channel has a fluorinated coating.

[0042] The present invention further comprises: flowing a biological sample from the inlet end to the outlet end of the device of claim 1; and reversing the direction of fluid flow toward the inlet end of the device; The present invention contemplates a method for extracting extracellular matrix bodies from a biological sample.

[0043] 1. A microfluidic system for isolating a fraction of a biological sample, comprising: 1. A microfluidic device comprising: one or more restricted channels having an inlet end and an outlet end, the inlet end and the outlet end being in fluid communication via the channel; a plurality of spaced apart obstacles disposed in the restriction channel to provide resistance to flow, the spacing between the obstacles decreasing in a direction from the inlet end to the outlet end; and an inlet reservoir for holding a fluid, the fluid reservoir in fluid communication with the inlet end of the restriction channel; and one or more uniform flow channels having an inlet end and an outlet end, the inlet end and the outlet end being in fluid communication through the flow channel, the inlet end being in fluid communication with an inlet reservoir; a microfluidic device comprising: a drive unit including a pressure source; a source unit including a fluid source, the pressure source being in fluid communication with the fluid source and the inlet reservoir of the microfluidic device; a sensor unit including a sensor in fluid communication with the inlet reservoir, the sensor measuring the flow rate and pressure of the fluid in the inlet reservoir and transmitting the flow rate and pressure data to the processor; and an on-chip analyzer unit, comprising one or more means, for analyzing the isolated fraction in the microfluidic device and sending the analysis data to a processor; and Processor for receiving and displaying flow, pressure and analysis A system including:

[0044] A composition comprising a fraction of a biological sample extracted from a device of the present disclosure, said composition being usable in human or animal therapy, said composition being usable in the diagnosis or prognosis of a subject.

[0045] A method for preparing a biological sample, comprising isolating extracellular matrix bodies from the biological sample. The extracellular matrix bodies may have a size of 0.5 to 5,000 micrometers, or 1 to 1,000 micrometers, or 1 to 200 micrometers, or 4 to 100 micrometers. The isolation of the extracellular matrix bodies may be performed by ultrafiltration or centrifugation. The isolation of the extracellular matrix bodies may be performed using the device of the present disclosure.

[0046] The above method further comprising anchoring the extracellular matrix body to a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

[0047] A method for preparing biological samples in which extracellular matrix bodies are fixed to glass surfaces using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking. [Brief explanation of the drawings]

[0048] [Figure 1] FIG. 1 is a plan view of one embodiment of a microfluidic chip of the present invention. In this embodiment, a silicon wafer master 101 is printed with a pattern of three microfluidic channel chips 103. The silicon wafer 101 can be used as a substrate. Photoresist can be poured onto the substrate and exposed to UV light to form the pattern of the microfluidic channel chips 103. The wafer and photoresist together form a mold into which PDMS can be poured. Once solidified, the PDMS can be peeled off the mold, yielding three microfluidic chip templates per wafer. The templates can then be attached to glass slides to form the final microfluidic chips.

[0049] [Figure 2] Figure 2 shows a top view of a microfluidic chip insert for one embodiment of a device of the present invention. The chip has two restriction channels 203, each 2500 μm wide and 25,000 μm long in this example. Restriction channels 203 contain pillars of various diameters and spacing, indicated by circles. The chip has a third uniform flow channel 205 with pillars of uniform size and spacing that do not significantly restrict flow. The chip has an inlet reservoir 201 and an outlet reservoir 207, which also contain larger pillars. The dashed arrows indicate the direction of flow from the inlet reservoir to the outlet reservoir.

[0050] [Figure 3] Figure 3 is a plan view corresponding to Figure 2. Figure 3 shows PDMS polymer pillars 301 of various sizes represented by circles. The flow of biofluids through the three channels is indicated by dashed arrows.

[0051] [Figure 4] Figure 4 is a plan view corresponding to the absorption tank of Figure 2. Figure 4 shows pillars 401, represented by circles. The flow of biofluid through the three channels is indicated by dashed arrows.

[0052] [Figure 5] Figure 5 is a plan view corresponding to the inlet reservoir region of Figure 2. Pillars 501, represented by circles, are shown in Figure 5. The flow of biofluid through the three channels is indicated by dashed arrows.

[0053] [Figure 6] Figure 6 is a plan view corresponding to the channel region of Figure 2. Figure 6 shows pillars 601, represented by circles. Biofluid flow through the channel is indicated by dashed arrows. Microfluidic channel devices of the invention may have regions with different pillar or obstacle size and / or spacing to create turbulent or restricted flow.

[0054] [Figure 7] Figure 7 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 7 shows pillars 701, represented by circles. Biofluid flow through the channel is indicated by dashed arrows. The transition from the 50 μm gap between pillars to the 25 μm gap within the restrictive channel is shown.

[0055] [Figure 8] Figure 8 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 8 shows pillars 801, represented by circles. Biofluid flow through the channel is indicated by dashed arrows. This shows the transition from large to small gaps between pillars in the restriction channel.

[0056] [Figure 9]Figure 9 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 9 shows pillars 901, represented by circles. The flow of biofluid through the channel is indicated by dashed arrows.

[0057] [Figure 10] Figure 10 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 10 shows pillars 1001, represented by circles. The flow of biofluid through the channel is indicated by dashed arrows. This view shows a channel with regions of blunt pillar obstructions 1001 that can create turbulence.

[0058] [Figure 11] Figure 11 is an enlarged plan view corresponding to outlet reservoir 1107 in Figure 2. Figure 11 shows pillars 1101, 1103, and 1105 of various sizes. Biofluid flow through the channels is indicated by dashed arrows. In this embodiment, the outer restricting channels each contain a barrier 1102 formed by very small, closely spaced pillars.

[0059] [Figure 12] Figure 12 is an enlarged plan view corresponding to inlet reservoir 1201 of Figure 2. Figure 12 shows pillars 1203 of various sizes. Outer restriction channel 1207 contains pillars of various sizes and spacing. Uniform flow channel 1205 contains pillars of uniform size and spacing. The direction of biofluid flow through the outer channels is indicated by dashed arrows.

[0060] [Figure 13] 13 is a plan view of a microfluidic chip according to one embodiment of the device of the present invention. Three microfluidic inserts are shown. The flow direction of the biofluids is indicated by the dashed arrows.

[0061] [Figure 14]Figure 14 is a perspective view of one embodiment of a microfluidic channel device of the present invention having blunt pillar-like obstacles to flow 1401. Figure 14 is an enlarged version of Figure 15. The flow direction of the biofluid is indicated by the dashed arrows.

[0062] [Figure 15] Figure 15 is a perspective view of one embodiment of a microfluidic channel device of the present invention. Figure 15 corresponds to the channel region of Figure 2. Figure 15 shows blunt pillar-like obstacles 1501 with varying spacing within the restriction channel. In this embodiment, the restriction channel can have pillar obstacles 1501 organized into bands with varying spacing between pillars. The direction of biofluid flow is indicated by the dashed arrows.

[0063] [Figure 16] 16 is an elevated side view of an embodiment of a microfluidic chip of the present invention. An inlet reservoir 1605 is in fluid communication with a fluid line 1601 for introducing biofluids and / or other fluids into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and a hole 1604 defined in a glass cover slide. The biofluid passes through the inlet reservoir 1605 and reaches a microfluidic channel 1606. The direction of biofluid flow is indicated by the dashed arrow.

[0064] [Figure 17] Figure 17 is an enlarged plan view corresponding to the inlet region of Figure 2, showing the location of probe 1602 of Figure 16. The direction of biofluid flow is indicated by the dashed arrows.

[0065] [Figure 18]Figure 18 is an elevated side view of an embodiment of a microfluidic chip 1614 of the present invention. An inlet reservoir is in fluid communication with a fluid line 1601 for introducing biofluid into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and a hole 1604 defined in a glass cover slide 1613. The biofluid passes through the inlet reservoir to a microfluidic channel 1606 and flows to an outlet reservoir 1607. A probe adjuster 1612 may be provided to adjust the height of the probe 1602 to create a good seal with the probe adapter 1603 and hole 1604. The direction of biofluid flow is indicated by the dashed arrow.

[0066] [Figure 19] Figure 19 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 19 shows pillars 1701 represented by circles. For this embodiment, some representative lengths in micrometers of the pillar band regions in the outer channel are shown.

[0067] [Figure 20] Figure 20 shows a magnified plan view micrograph corresponding to the channel region of Figure 2. Figure 20 shows the pillars as dots. In this example, some representative lengths in micrometers of the pillar band region of the outer channel are shown. The direction of flow of the biological fluid is indicated by the dashed arrows.

[0068] [Figure 21] Figure 21 shows a top view of one embodiment of a microfluidic device corresponding to Figure 2. Biofluid can be introduced into the inlet region reservoir 2202 using a delivery probe 2201. The direction of biofluid flow to the outlet reservoir region 2203 is indicated by the dashed arrows. The expanded view of this example shows some representative lengths in micrometers of the pillar band regions in the outer channel. In this embodiment, the dotted lines in the exploded view indicate the potential tortuous path of the biofluid between obstacles.

[0069] [Figure 22] FIG. 22 shows one embodiment of a microfluidic system of the present invention having a processor, a fluid drive unit, a fluid source unit, a sensor unit, an on-chip unit, and an off-chip unit.

[0070] [Figure 23] Figure 23 shows that aqueous humor from a patient with primary open-angle glaucoma increased the pressure within the microfluidic device. Figure 23 shows the relative change in pressure (mmHg) within the artificial pillar network formed by pillars within the microfluidic channel upon injection of human aqueous humor obtained from a patient with severe primary open-angle glaucoma. The fluid flow rate was kept constant at 2 μl / min, and the baseline system pressure was measured using an external pressure sensor. A sample of human aqueous humor was injected at the time indicated by the arrow and the letter "a." The pressure gradually increased, reaching a maximum of approximately 41 mmHg after 27 minutes. Figure 23 shows that aqueous humor from a patient diagnosed with POAG glaucoma increased the pressure within the device.

[0071] [Figure 24] Figure 24 (top) shows a confocal micrograph of the microfluidic chip after capturing EMBs from human aqueous humor of a patient with primary open-angle glaucoma. The EMB proteins were labeled with the fluorescent marker carboxyfluorescein succinimidyl ester (CFSE, marked by an arrow). The circles represent pillars in the restriction channel. Figure 24 (bottom) shows EMBs isolated in the microfluidic channel around the pillars.

[0072] [Figure 25]Figure 25 shows the isolation of extracellular matrix bodies from a biological fluid using size-exclusion filters. Bovine vitreous humor was filtered through a 5 μm cellulose acetate syringe filter, followed by a 1 μm syringe-tip filter, followed by a 0.45 μm syringe-tip filter, and finally a 0.22 μm filter. Each fraction was characterized using wide-field microscopy. Figure 25 shows unconcentrated bovine vitreous humor 4301 aspirated into a 1 mL syringe using a 22 g needle 4305 and extruded through a 5 μm syringe-tip filter 4309. The filtrate was collected and filtered through a 1 μm syringe-tip filter 4311. The filtrate was collected and filtered through a 0.45 μm syringe-tip filter 4313. The filtrate was collected and extruded through a 0.22 μm syringe-tip filter 4315. Biofluid fractions were collected after each filtration step for light microscopy. Microscopic images showed that large bodies were reduced when the filtrate was passed successively through smaller filter sizes.

[0073] [Figure 26] Figure 26 shows representative transmission electron microscopy (TEM) images for the isolation of EMBs using size-exclusion filters. Figures 26a and 26b demonstrate the presence of extracellular matrix bodies in the natural biological fluid of bovine vitreous humor. To isolate and recover extracellular matrix bodies from this complex biological fluid, sequential syringe-based filtration was performed using cellulose filters with pore sizes ranging from 5 μm to 0.22 μm. The extracellular matrix bodies were stained with Alcian blue. Figure 26c shows a fraction of bovine vitreous isolated by sequential filtration through a 5 μm syringe-tip filter. Figure 26d shows a fraction of bovine vitreous isolated by sequential filtration through a 1 μm syringe-tip filter. Figure 26e shows a fraction of bovine vitreous isolated by sequential filtration through a 0.45 μm syringe-tip filter. Figure 26f shows a fraction of bovine vitreous isolated by sequential filtration through a 0.22 μm syringe-tip filter. The images show a relative decrease in larger ECM bodies as the filtrate is passed through successively smaller filter sizes.

[0074] [Figure 27] Figure 27 shows the isolation of extracellular matrix bodies from a biological fluid using centrifugation. Sequential centrifugation yielded four pellets: 9705, 9713, 9721, and 9729. Bovine vitreous was resuspended in 9701, placed in a 1 ml tube, and centrifuged (Sorvall Legend RT) at 350 g for 10 minutes at 4°C to form Pellet 1 (9705). Fifty microliters of the supernatant was saved for analysis and labeled Supernatant 1 (9709). The remaining supernatant was then transferred to a new tube and centrifuged at 2000 g for 10 minutes at 4°C (Eppendorf, 5417R series, F45-30-11 Eppendorf rotor) to form Pellet 2 (9713). Fifty microliters of the supernatant was saved for analysis and labeled Supernatant 2 (9717), and the remaining supernatant was transferred to a new tube. The supernatant was then centrifuged at 10,000g for 10 minutes at 4°C to form Pellet 3 (9721). 50 μl of the supernatant was saved for analysis and designated Supernatant 3 (9725), and the remaining supernatant was transferred to a new tube. The supernatant was then centrifuged at 20,000g for 10 minutes at 4°C to form Pellet 4 (9729). 50 μl of the supernatant was saved for analysis and designated Supernatant 4, and the remaining supernatant was transferred to a new tube.

[0075] [Figure 28]Figure 28 shows representative transmission electron microscopy (TEM) images for the isolation of EMBs by sequential centrifugation. Figure 28a shows extracellular matrix bodies present in bovine vitreous. In Figure 28b, a representative TEM micrograph of a sample taken from the pellet after centrifugation at 450 g showed extracellular matrix bodies present in the pellet fraction. Similarly, in Figure 28c, a representative TEM micrograph of a sample taken from the pellet after centrifugation at 2,000 g showed extracellular matrix bodies present in the pellet fraction. In Figure 28d, a representative TEM micrograph of a sample taken from the pellet after centrifugation at 10,000 g showed extracellular matrix bodies present in the pellet fraction. In Figure 28e, a representative TEM micrograph of a sample taken from the pellet after centrifugation at 20,000 g showed extracellular matrix bodies present in the pellet fraction.

[0076] [Figure 29] 29 shows the dose-response behavior of the compound bivalirudin TFA on intraocular pressure (IOP) in bovine vitreous. Differential pressure is measured using a microfluidic device of the present invention.

[0077] [Figure 30] 30 shows the dose-response behavior of the compound colistin sulfate on intraocular pressure (IOP) in bovine vitreous humor, where the differential pressure is measured using a microfluidic device of the present invention.

[0078] [Figure 31] 31 shows the dose-response behavior of the compound polymyxin B sulfate on intraocular pressure (IOP) in a bovine vitreous humor glaucoma model, where differential pressure is measured using a microfluidic device of the present invention.

[0079] [Figure 32]Figure 32 illustrates the isolation and extraction of extracellular matrix bodies in a restricted channel of a microfluidic device of the present disclosure. Figures 32a and 32b show representative micrographs of a microfluidic chip perfused with bovine vitreous humor biofluid. The letter "p" indicates pillars within the channel. After perfusion, extracellular matrix bodies were isolated between and around the pillars. Figures 32c and 32d show representative micrographs of the channel after extraction of the extracellular matrix bodies. The extracellular matrix bodies were removed from the chip, resulting in substantially fewer bodies after extraction.

[0080] [Figure 33] FIG. 33 shows that biomarkers of extracellular matrix bodies were detected by off-chip proteome analysis of isolated and extracted bovine extracellular matrix bodies by LC / MS.

[0081] [Figure 34]Figure 34 shows the isolation and subsequent extraction of extracellular matrix bodies in the restricted channel of a microfluidic device of the present disclosure. The image scale bar is 50 μm. Figure 34a shows a representative wide-field micrograph of a microfluidic device perfused with bovine vitreous humor suspended in phosphate-buffered saline (pH 7.0) and counterstained for hyaluronic acid with Alcian blue (gray signal, bright field). Figure 34a shows the signal from extracellular matrix bodies (arrows) trapped between the pillars (p) of the device. The chip was perfused with the biological fluid for at least 60 minutes. After perfusion, aggregates were isolated between the pillars and observed in a clump-like form. Substances smaller than the extracellular matrix body material were expelled through the outlet port. Figure 34b shows the extraction of extracellular matrix bodies from the restricted channel of the microfluidic device. The device was perfused with mild detergent, 0.1% sodium dodecyl sulfate, and SDS, and the flow direction was reversed from the outlet to the inlet. Figure 34b shows that substantially fewer objects were present in the channel after elution, indicating that the objects had been extracted. Figure 34c shows a high-power image of extracellular matrix bodies (arrows) trapped between pillars (p) after perfusion. Figure 34d shows extraction and backflow with detergent, again demonstrating a significant reduction in bodies within the channel after extraction.

[0082] [Figure 35]Figure 35 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. A microfluidic device was injected with a fluid containing homogenized bovine vitreous suspended in a biological fluid. After perfusion of the fluid through the device, the fluid passed through the inlet and exited through the outlet. Large extracellular matrix bodies (arrows) were trapped between the pillars (denoted Lp). The chip was perfused with a blocking solution to prevent nonspecific antibody binding prior to antibody staining. Next, an anti-fibronectin primary antibody was injected, the sample was incubated for 2 hours, and washed to label the protein fibronectin, a known extracellular matrix component and integrin-binding protein. This was followed by incubation with a goat anti-rabbit FITC secondary antibody for 1 hour and washing. The microfluidic chip was then imaged with wide-field fluorescence and bright-field microscopes. Figure 35 shows a representative wide-field fluorescence micrograph. This image shows the extracellular matrix bodies within the microfluidic channel. The spacing between the large pillars (Lp) was approximately 100 μm. Punctate signals within the body indicate fibronectin staining (anti-fibronectin Ab, Alexa488-labeled goat anti-rabbit secondary antibody, FITC, white signal).

[0083] [Figure 36] Figure 36 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. Figure 36a shows a representative micrograph brightfield image of stained extracellular matrix bodies in the channel (arrow). The image scale bar is 20 μm. The control image had no fluorescent signal, indicating that the signal in Figure 36a is specific to fibronectin. Figure 36b again shows stained extracellular matrix bodies in the channel (arrow). The image scale bar is 50 μm. Again, the control image had no fluorescent signal, indicating that the signal in Figure 36b is specific to fibronectin.

[0084] [Figure 37]Figure 37 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. Figure 37 shows representative micrographs of on-chip immunohistochemical staining of Perlecan protein, a component of the extracellular matrix of cartilage and a known cancer biomarker, in a biological fluid containing extracellular matrix bodies. A fluid containing homogenized bovine vitreous suspended in the biological fluid was injected into the microfluidic chip. After perfusion of the biological fluid through the device, the sample flowed through the inlet and out through the outlet. Large extracellular matrix bodies (arrows) were trapped between pillars (marked "p"). The chip was perfused with a blocking solution to prevent nonspecific antibody binding before antibody staining. Perlecan was labeled by injecting anti-Perlecan primary antibody, incubating the sample for 2 hours, and washing. This was followed by incubation with goat anti-rabbit TRITC secondary antibody for 1 hour and washing. The microfluidic chip was then imaged using wide-field fluorescence and bright-field microscopes. Figure 37 shows extracellular matrix bodies within the microfluidic channels between pillars (p). The punctate signals represent perlecan staining (white signal). There was no fluorescent signal in the control image, indicating that the signal in Figure 37 is specific to perlecan.

[0085] [Figure 38] Figure 38 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. Figure 38a shows a representative micrograph bright-field image of on-chip immunohistochemical staining of perlecan. There was no fluorescent signal in the control image, indicating that the signal in Figure 38a is specific to perlecan. The image scale bar is 10 μm. Figure 38b shows a representative micrograph bright-field image of on-chip immunohistochemical staining of perlecan. There was no fluorescent signal in the control image, indicating that the signal in Figure 38b is specific to perlecan. The image scale bar is 10 μm. Figure 38 also demonstrates that Alcian blue, a marker for hyaluronic acid, can be used as a stain for EMB.

[0086] [Figure 39]Figure 39 shows visualization of extracellular matrix bodies on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and staining of collagen with picrosirius red dye. Figure 39 shows the signal of the extracellular matrix bodies (dark staining), indicating that EDC crosslinking held the extracellular matrix bodies to the surface.

[0087] [Figure 40] Figure 40 shows visualization of extracellular matrix bodies on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and staining of collagen with picrosirius red dye. Figure 40 shows the signal of collagen chains within the extracellular matrix bodies (dark staining), indicating that EDC crosslinking retains the extracellular matrix bodies on the surface. Figure 40 also demonstrates that picrosirius red staining can be used for EMB staining.

[0088] [Figure 41] Figure 41 shows off-chip analysis of extracellular matrix bodies visualized on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and DNA staining with Hoechst dye. Figure 41 shows the signal for DNA in the extracellular matrix bodies (Hoechst dye, DAPI filter, white signal, DNA).

[0089] [Figure 42] Figure 42 shows on-chip isolation and detection of extracellular matrix bodies. Figure 42 shows representative photomicrographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline at pH 7.0, counterstained for hyaluronic acid with Alcian blue, dark stain, and bright field. Figure 42 shows signal from extracellular matrix bodies trapped near the large pillars (circles) of the device. The chip was perfused with the biological fluid for at least 60 minutes. Extracellular matrix bodies were observed in clusters. Image scale bar is 50 μm.

[0090] [Figure 43] Figure 43 shows on-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 43 shows a representative low-power fluorescence micrograph of a channel after perfusion of bovine vitreous humor extracellular matrix bodies, counterstained for proteins with carboxyfluorescein succinimidyl ester (CFSE). Fluid containing homogenized bovine vitreous was injected into the microfluidic chip. After perfusion of the fluid through the device, the fluid passed through the inlet and out through the outlet. Large extracellular matrix bodies were trapped near pillars (denoted "p"). The image scale bar is 25 μm.

[0091] [Figure 44] Figure 44 shows on-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 44 shows representative micrographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline at pH 7.0, counterstaining for collagen with picrosirius red, dark staining, and bright field. Figure 44a shows signal from extracellular matrix bodies between the pillars (marked "p") of the device. The chip was perfused with biological fluid for at least 60 minutes. Figure 44b shows the same image with a fluorescent filter, demonstrating collagen detection with picrosirius red (light gray signal). The image scale bar is 50 μm. Figures 44c and 44f show similar images at higher power. The image scale bar is 10 μm.

[0092] [Figure 45]Figure 45 shows the frequency and size distribution of human extracellular matrix bodies present in human aqueous humor from healthy and pre-disease states, glaucoma suspects, and pre-glaucoma patients. Aqueous humor was collected from eight patients with intraocular pressures ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other methods. Extracellular matrix body size was determined by crosslinking the samples to glass slides using carbodiimide-EDC fixative, staining with uranyl acetate, and imaging with a wide-field microscope. Size was quantified using an automated program (ImageJ) for all eight samples. The size (area) of extracellular matrix bodies ranged from approximately 1.67 μm² to approximately 67 x 10³ μm². Figure 45 shows the number of extracellular matrix bodies in the range of 0 to 200 μm².

[0093] [Figure 46] Figure 46 shows the frequency and size distribution of human extracellular matrix bodies present in human aqueous humor from healthy and pre-disease states, glaucoma suspects, and pre-glaucoma patients. Aqueous humor was collected from eight patients with intraocular pressures ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other methods. Extracellular matrix body size was determined by crosslinking the samples to glass slides using carbodiimide-EDC fixative, staining with uranyl acetate, and imaging with a wide-field microscope. Size was quantified using an automated program (ImageJ) for all eight samples. The size (area) of extracellular matrix bodies ranged from approximately 1.67 μm² to approximately 67 x 103 μm². Figure 46 shows the number of extracellular matrix bodies ranging from 201 to 1000 μm².

[0094] [Figure 47]Figure 47 shows the frequency and size distribution of human extracellular matrix bodies present in human aqueous humor from healthy and pre-disease states, glaucoma suspects, and pre-glaucoma patients. Aqueous humor was collected from eight patients with intraocular pressures ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other methods. Extracellular matrix body size was determined by crosslinking samples to glass slides using carbodiimide-EDC fixative, staining with uranyl acetate, and imaging with a wide-field microscope. Size was quantified in all eight samples using an automated program (ImageJ). The size (area) of extracellular matrix bodies ranged from approximately 1.67 μm² to approximately 67 x 103 μm². Figure 47 shows the number of extracellular matrix bodies ranging from 1,001 to 67,000 μm².

[0095] [Figure 48] Figure 48 shows the size distribution of bovine vitreous extracellular matrix bodies isolated and extracted using the microfluidic device of the present invention. Extracellular matrix bodies in bovine vitreous humor biofluid after isolation and extraction from the microfluidic device of the present invention. The chip was perfused with the biofluid for at least 60 minutes. After 60 minutes of perfusion, ECM bodies were isolated near the restrictor channel pillars. The chip was then treated with detergent (0.1% sodium dodecyl sulfate, SDS), and the sample was extracted from the chip using reverse flow, allowing the bodies to flow out the inlet. Fractions of the eluate were collected at 10-minute intervals for a total of 80 minutes. Samples were mounted on glass slides, stained with Alcian blue, and imaged using a wide-field microscope. Size was quantified using an automated program (ImageJ). The maximum size (area) of extracellular matrix bodies was approximately 16 x 103 μm2. Figure 48 shows the number of extracellular matrix bodies in each elution fraction, which increased over time. This experiment demonstrated that the microfluidic device of the present invention can be used to isolate and extract extracellular matrix bodies of various sizes.

[0096] [Figure 49]Figure 49 demonstrates off-chip analysis of extracellular matrix bodies retained by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinking. Figure 49a shows a representative TEM image of extracellular matrix bodies from native bovine vitreous humor obtained by EDC crosslinking. EDC fixation allowed for the observation of extracellular matrix bodies. Figure 49b shows a similar image taken without EDC crosslinking. No extracellular matrix bodies were observed without EDC fixation.

[0097] [Figure 50] Figure 50 shows the isolation of extracellular matrix bodies from a human plasma sample from a patient with early-stage pancreatic ductal adenocarcinoma (PDAC) compared with a healthy control. Figure 50a shows a representative wide-field fluorescence microscopy image of a PDAC sample. Figure 50a shows extracellular matrix bodies trapped in a microfluidic channel. Figure 50a further shows that large extracellular matrix bodies (arrows) are trapped between pillars (marked "p"). The width between pillars was approximately 100 μm. Punctate signals from the packed extracellular matrix bodies indicate fibronectin staining (anti-fibronectin Ab, Alexa488-labeled goat anti-rabbit secondary Ab, FITC, white signal). Staining revealed abundant signal and punctate staining within the EMB (arrows in Figure 50a). Figure 50b shows a similarly obtained fluorescence micrograph of an age-matched healthy control human plasma sample. Figure 50b shows significantly reduced fibronectin signal (gray signal, arrows in Figure 50b). When treated under identical conditions, the healthy control signal was much smaller than the disease signal. Image scale bar is 20 μm. DETAILED DESCRIPTION OF THE INVENTION

[0098] The present invention discloses devices and systems for isolating particles from biological samples, which can be used to isolate and treat a variety of diseases and conditions by isolating and using biological materials, substances, and / or molecules. The devices and systems for isolating particles from biological samples can be used to enhance the level of sample isolation relevant to the biology of interest. The devices and methods of the present invention can be used to isolate key components from biological samples for use in disease diagnosis and prognosis.

[0099] The present device for isolating particles from biological samples can be used with various types of biological material and samples, including bodily fluids, blood, tissues, cells, and tumors. In certain embodiments, the disclosed method can isolate relevant fractions of biological material corresponding to a pathological condition.

[0100] This disclosure provides devices that can be used to isolate a substantial fraction of a biological sample and provide for analysis of its components relevant to the diagnosis and prognosis of disease. In some embodiments, substantial amounts of biological material and correspondingly improved signal levels can be obtained.

[0101] Aspects of the present invention include isolating and preserving the composition and properties of extracellular matrix bodies (EMBs) from biological fluids or materials. By preserving the composition and properties of extracellular matrix bodies (EMBs) isolated or extracted from biological samples, fluids, or materials, the EMBs can be used to diagnose or signal disease, or to monitor chemical or biological processes or changes in the sample material.

[0102] In some aspects, the present invention enhances the ability to detect or characterize extracellular matrix bodies that can more readily reflect a disease state.

[0103] In a further aspect, the disclosed devices and methods can provide increased signals for relevant biomarkers that link specific biological fractions to disease states. The disclosed biomarkers can be associated with disease and provide diagnostic tools.

[0104] The devices of the present disclosure can provide improved measurements of flow and / or pressure within a fluid containing a biological component by maintaining continuous and substantial flow within the device. In some embodiments, when substantial flow is maintained, the sensor can accurately measure differential pressure and flow. The devices and systems disclosed herein can provide improved measurements of flow and pressure within a fluid containing a biological component by arranging channels that maintain continuous and substantial flow. In some embodiments, the devices of the present disclosure can have one or more channels that do not substantially restrict fluid flow, such that continuous and substantial flow is maintained within the system.

[0105] The devices disclosed herein can provide improved measurements of flow and pressure within fluids containing biological components by using one or more uniform flow channels that maintain continuous and substantial flow in addition to a restricted channel.

[0106] In certain embodiments, the device of the present invention will have a flow rate that is 1-90% of the flow rate from a uniform flow channel, or 1-75% of the flow rate from a uniform flow channel, or 1-50% of the flow rate from a uniform flow channel, or 1-25% of the flow rate from a uniform flow channel.

[0107] In certain embodiments, the device of the present invention has a flow rate of at least 25% of the flow rate from a uniform flow channel, or at least 50% of the flow rate from a uniform flow channel, or at least 75% of the flow rate from a uniform flow channel, or at least 90% of the flow rate from a uniform flow channel.

[0108] In some embodiments, extracellular matrix bodies having diameters of about 0.5 to about 5,000 micrometers or more can be confined within a microfluidic channel or cause blockage of a flow channel, resulting in increased pressure.

[0109] In additional embodiments, extracellular matrix bodies having diameters of about 1 to about 200 micrometers or greater can be confined within a microfluidic channel or cause blockage of the channel, increasing the pressure.

[0110] In a further aspect, the present disclosure provides devices and methods for isolating, detecting, and / or analyzing ultrastructural components of fluids containing biological materials or molecules. In certain embodiments, the ultrastructural components may be associated with disease.

[0111] Embodiments of the present invention can be used to isolate, extract, and utilize extracellular matrix bodies (EMBs), which are a source of multiple and specific biomarkers.

[0112] The present invention includes devices for isolating, detecting, and analyzing the composition of extracellular matrix bodies, biological particles, and complexes for a variety of applications.

[0113] In certain aspects, extracellular matrix bodies can function as biomarkers through their morphological characteristics. In further aspects, extracellular matrix bodies can function by containing isolated biochemical markers that may be involved in disease pathways.

[0114] Aspects of the present invention can further provide diagnostic systems that include devices for detecting and measuring biomarkers via disease-associated extracellular matrix bodies (EMBs) and / or biological particles or complexes.

[0115] In further embodiments, the present disclosure describes methods and apparatus for preparing and analyzing samples of biological material.

[0116] Extracellular matrix bodies (EMBs) can be complex and consist of proteins, lipids, carbohydrates, nucleic acid molecules, bioparticles, vesicles such as extracellular vesicles and exosomes, and combinations thereof.

[0117] Samples of biological material include body fluids, tissues, and cells.

[0118] Examples of bodily fluid samples include any bodily fluid, including whole blood, plasma, blood components, SCF, urine, semen, synovial fluid, pleural fluid, vaginal fluid, gastric fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, ear fluid, breast milk, other bodily fluids, and combinations thereof.

[0119] In a further aspect, the microfluidic devices and systems of the present invention can be used to isolate and extract biological particles from a sample.

[0120] The microfluidic devices and systems of the present invention can be used to purify or separate extracellular matrix bodies or complexes greater than about 0.5 micrometers in diameter, down to particles of about 5,000 micrometers in diameter, or particles greater than about 2 micrometers in diameter, down to particles of about 700 micrometers in diameter.

[0121] In a further aspect, the microfluidic devices and systems of the present invention can be used to measure the relative viscosity and flow properties of biological and clinical fluids.

[0122] In certain aspects, the microfluidic devices and systems of the present invention can be used to isolate and extract disease-associated bioparticles from a sample.

[0123] In some aspects, the microfluidic devices and systems of the present invention can be used to measure intraocular pressure in ocular fluids.

[0124] Equipment and Systems The present invention provides microfluidic devices and systems for measuring pressure and / or flow in fluids.

[0125] The present invention improves the measurement of flow and pressure in fluids by providing continuous and substantial flow and volumetric flow rates, allowing for accurate measurement of differential pressure and / or flow rate.

[0126] In a further aspect, the microfluidic devices and systems of the present invention can be used to detect, isolate and extract biological particles or other components of biological materials or fluids.

[0127] The microfluidic devices and systems of the present invention can be used to measure the relative viscosity and flow properties of bio- and clinical fluids.

[0128] The microfluidic devices and systems of the present invention may consist of a microfluidic chip that can be held on a substrate.

[0129] In some aspects, microfluidic devices of the present invention can have flow channels with obstacles that can affect and / or restrict the flow of fluid within the channel.

[0130] In some aspects, microfluidic devices of the invention can have channels with bands of obstacles. The bands of obstacles can traverse the width of the channel such that the bands of obstacles affect the flow and flux of fluid along the channel.

[0131] In some embodiments, the spacing between obstacles may be constant across the strip. The spacing between obstacles within a channel may be used to control the size of the barriers within the channel for fluid flow.

[0132] In additional embodiments, microfluidic devices of the present invention can have channels with multiple, continuous band-like obstacles. In certain embodiments, the spacing between the band-like obstacles can decrease in multiple bands along one length of the channel. Consequently, the spacing between obstacles within a band can increase in multiple bands along the length of the channel in the opposite direction.

[0133] For example, a microfluidic device of the present invention may have a restriction channel in which multiple bands that are continuous in one direction have obstacle-to-obstacle spacing of 1000 micrometers, which is adjacent to a band that is spaced 500 micrometers apart, which is adjacent to a band that is spaced 200 micrometers apart, which is spaced 100 micrometers apart, etc. In such a case, the bands may be "adjacent to bands with 100 micrometer spacing," "adjacent to bands with 50 micrometer spacing," "adjacent to bands with 25 micrometer spacing," "adjacent to bands with 10 micrometer spacing," or "adjacent to bands with 4 micrometer spacing."

[0134] In a further embodiment, the microfluidic device of the present invention can have a channel with band-like obstacles with minimal spacing between obstacles in one band. The band with minimal spacing between obstacles can be a barrier band.

[0135] In additional embodiments, the microfluidic devices of the invention can have restriction channels in which the barrier bands have an obstacle-to-obstacle spacing of at least 1 micrometer, or at least 2, or at least 4, or at least 5, or at least 10, or at least 50, or at least 100, or at least 200 micrometers.

[0136] In certain embodiments, the microfluidic device of the present invention can be a chip that is 7 to 25 micrometers in height.

[0137] In operation, the microfluidic devices and systems of the present invention can be used to isolate, extract, and / or purify bioparticles. In certain embodiments, a restriction channel can have bands with decreasing spacing between obstacles, where one band is a barrier band. The methods of the present invention can use such a band arrangement to isolate larger particles from smaller particles and fluids by flowing a particle-containing fluid from an inlet in a direction of decreasing spacing toward the outlet of the channel. In these methods, larger particles can be sequestered and retained along the channel, while smaller particles and fluids exit the channel at the outlet. Larger particles can be extracted from the channel at the inlet by reversing the direction of flow of the extraction fluid.

[0138] In operation, the microfluidic devices and systems of the present invention can be used to isolate, extract, and / or purify bioparticles extracted from the channel at the outlet.

[0139] In some embodiments, larger particles can be extracted from the channel at the outlet by the addition of a detergent to break down the larger particles.

[0140] Figure 1 shows a top view of one embodiment of the microfluidic chip of the present invention. In this format, a silicon wafer master 101 has three microfluidic channel chip patterns 103 printed on it. The silicon wafer 101 can be used as a substrate. Photoresist can be poured onto the substrate and exposed to ultraviolet light to form the pattern of the microfluidic channel chips 103. Together, the wafer and photoresist form a mold into which PDMS can be poured. Once solidified, the PDMS can be peeled off the mold, yielding three microfluidic chip molds per wafer. These molds can be bonded to glass slides to form the final microfluidic chips.

[0141] The microfluidic chips of the present invention can have channels for restricting fluid flow, and inlets and outlets for fluid flow. A pump can be used to apply fluid head pressure at the inlet. In some embodiments, reduced pressure or vacuum can be used at the outlet to regulate the flow rate.

[0142] Figure 2 shows a top view of a microfluidic chip insert for one embodiment of the device of the present invention. The chip has two restriction channels 203, each 2500 μm wide and 25,000 μm long in this example. The restriction channels 203 contain pillars of varying diameter and spacing, represented by circles. The chip also has a third uniform flow channel 205 with pillars of uniform size and spacing that do not significantly restrict flow. The chip has an inlet reservoir 201 and an outlet reservoir 207, which also contain larger pillars. The dashed arrows indicate the direction of flow from the inlet reservoir to the outlet reservoir. The restriction channel may have a point of greatest restriction, a barrier to flow 202. The barrier 202 can restrict flow and alter the pressure within the channel and system, so that differential pressure and / or flow can be related to the composition of the fluid.

[0143] The microfluidic chip of the present invention may have one or more channels for restricted fluid flow and one or more uniform or continuous flow channels. In some embodiments, the uniform flow channel does not restrict the fluid flow within the channel. The uniform continuous flow channel may include blunt obstacles to create turbulence and / or tortuous paths for the fluid flow.

[0144] Figure 3 is a plan view corresponding to Figure 2. Figure 3 shows PDMS polymer pillars 301 of various sizes represented by circles. The flow of biofluids through the three channels is indicated by dashed arrows.

[0145] In certain embodiments, blunt or non-blunt obstacles can be provided in the restricted fluid channel to create meandering or vortex patterns in the flow in specific regions. The obstacles in the channel can be formed as pillars of circular or other shapes.

[0146] In certain embodiments, the obstruction of the restrictive channel can provide a Reynolds number of 500 or greater, or 1000 or greater, or 10,000 or greater.

[0147] In additional embodiments, a continuous uniform flow channel may be disposed between various restriction channels. In certain embodiments, uniform flow channels and restriction channels may be used in any order and in any number.

[0148] Figure 4 is a plan view corresponding to the inlet reservoir of Figure 2. Pillars 401 are shown as circles in Figure 4. The flow of biofluid through the three channels is indicated by dashed arrows.

[0149] Figure 5 is a plan view corresponding to the inlet reservoir region of Figure 2. Figure 5 shows pillars 501, represented by circles. The flow of biofluid through the three channels is indicated by dashed arrows.

[0150] Figure 6 is a plan view corresponding to the channel region of Figure 2. Figure 6 shows pillars 601, represented by circles. Biofluid flow through the channel is indicated by dashed arrows. The microfluidic channel device of the present invention has regions of varying spacing and / or size of pillars or obstacles that create turbulent or restricted flow.

[0151] In certain embodiments, the microfluidic channel devices of the present disclosure may have a region that simulates the corpus cavernosum reticulum of the eye.

[0152] The device of the present invention can include an omentum composition comprising an extracellular matrix body or complex. The extracellular matrix body or complex for use in the omentum composition can be extracted or purified from glaucomatous aqueous humor. The aqueous humor can be of animal or clinical origin.

[0153] In further embodiments, the microfluidic chips of the present invention can have one or more channels for restricting fluid flow and one or more uniform flow channels, which can include blunt obstacles to create turbulence and / or tortuous paths for the fluid flow.

[0154] In further embodiments, the microfluidic chip of the present invention may have 1 to 20 channels for restricting fluid flow and 1 to 10 uniform flow channels arranged in any order on the substrate. The uniform flow channels can be distributed in any manner relative to the restricted flow channels.

[0155] In certain embodiments, the uniform flow channels may be alternately positioned in a collinear or parallel position relative to the restricted flow channels. In additional embodiments, the uniform flow channels may be above or below the restricted flow channels. In some embodiments, the uniform flow channels may be located on a separate substrate from the chip containing the restricted channels.

[0156] In further embodiments, the uniform flow channel may provide fluid communication from the inlet reservoir to the outlet reservoir. In certain embodiments, the uniform flow channel may provide fluid communication from the outlet reservoir to a source of fluid entering the inlet reservoir.

[0157] In certain embodiments, the total cross-sectional area of ​​the uniform flow channel may be greater or less than the total cross-sectional area of ​​the restriction channels in the microfluidic devices of the present invention. In various embodiments, the uniform flow channel may be free of obstacles and may not have tortuous fluid flow. In such embodiments, the uniform flow channel may have laminar or turbulent fluid flow.

[0158] The microfluidic chip of the present invention can have one or more restriction channels to restrict fluid flow. The restricted flow can be due to various arrangements of blunt or non-blunt obstacles or pillars within the channel. In some embodiments, the pillars can present shapes to the flowing fluid, such as round, spherical, triangular, square, polygonal, diamond, fin-shaped, and combinations thereof.

[0159] Figure 7 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 7 shows pillars 701, represented by circles. Biofluid flow through the channel is indicated by dashed arrows. The transition from the 50 μm gap between pillars to the 25 μm gap within the restrictive channel is shown.

[0160] Figure 8 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 8 shows pillars 801, represented by circles. Biofluid flow through the channel is indicated by dashed arrows. This shows the transition from large to small gaps between pillars in the restriction channel.

[0161] Figure 9 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 9 shows pillars 901, represented by circles. The flow of biofluid through the channel is indicated by dashed arrows.

[0162] In further embodiments, restricted flow within a channel may result from various placements of blunt or non-blunt obstacles or pillars within the channel, with the size and spacing of the obstacles varying with distance along the channel.

[0163] In certain embodiments, the size and / or spacing of blunt or non-blunt obstacles or pillars within a restrictive channel may vary with distance along the channel. The size and / or spacing of blunt or non-blunt obstacles may decrease with distance along the channel. At some locations within the restrictive channel, the size and / or spacing of blunt or non-blunt obstacles may decrease to a level that provides maximum restriction or barrier to flow.

[0164] Figure 10 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 10 shows pillars 1001, represented by circles. The flow of biofluid through the channel is indicated by dashed arrows. This view shows a channel with regions of blunt pillar obstructions 1001 that can create turbulence.

[0165] Figure 11 is an enlarged plan view corresponding to outlet reservoir 1107 of Figure 2. Figure 11 shows pillars 1101, 1103, and 1105 of various sizes. The flow of biofluid through the channels is indicated by dashed arrows. In this embodiment, the outer restricting channels each contain a barrier 1102 formed by very small, closely spaced pillars.

[0166] In further embodiments, various arrangements of blunt or non-blunt obstacles or pillars within the restriction channel can be used to restrict flow to any level. A wide range of spacing and / or patterns of blunt and / or non-blunt obstacles can be used in the restriction channel. The fluid can have a tortuous path within the restriction flow channel. The spacing of obstacles within the restriction channel and / or the tortuosity of the fluid path can increase with distance along the flow channel in the direction of flow.

[0167] Fluid output from a channel of a microfluidic chip of the invention can be collected in an outlet reservoir at the outlet end of the channel. Fluid input or output into a channel of a microfluidic chip of the invention can be achieved in a reservoir at the inlet end of the channel.

[0168] Figure 12 is an enlarged plan view corresponding to inlet reservoir 1201 of Figure 2. Figure 12 shows pillars 1203 of various sizes. Outer restriction channel 1207 contains pillars with various sizes and spacing. Uniform flow channel 1205 contains pillars of uniform size and spacing. The direction of biofluid flow through the outer channels is indicated by dashed arrows.

[0169] 13 is a plan view of a microfluidic chip in one embodiment of the device of the present invention. Three microfluidic inserts are shown. The flow direction of the biofluids is indicated by the dashed arrows.

[0170] Figure 14 is a perspective view of one embodiment of a microfluidic channel device of the present invention having blunt pillar-like obstacles to flow 1401. Figure 14 is an enlarged version of Figure 15. The flow direction of the biofluid is indicated by the dashed arrows.

[0171] Figure 15 is a perspective view of one embodiment of a microfluidic channel device of the present invention. Figure 15 corresponds to the channel region of Figure 2. Figure 15 shows pillar-like obstacles 1501 with varying spacing in a restriction channel. In this embodiment, the restriction channel can have pillar obstacles 1501 organized in bands with varying spacing between pillars. The direction of biofluid flow is indicated by dashed arrows. A continuous uniform flow channel 1517 can be located separately from the restriction channel 1515.

[0172] 16 is an elevated side view of an embodiment of a microfluidic chip of the present invention. An inlet reservoir 1605 is in fluid communication with a fluid line 1601 for introducing biofluids and / or other fluids into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and a hole 1604 defined in a glass cover slide. The biofluid passes through the inlet reservoir 1605 and reaches a microfluidic channel 1606. The flow direction of the biofluid is indicated by the dashed arrow.

[0173] Figure 17 is an enlarged plan view corresponding to the inlet region of Figure 2, showing the location of probe 1602 of Figure 16. The direction of biofluid flow is indicated by the dashed arrows.

[0174] Figure 18 is an elevated side view of an embodiment of a microfluidic chip 1614 of the present invention. An inlet reservoir is in fluid communication with a fluid line 1601 for introducing biofluid into the reservoir. The fluid line 1601 passes through a probe 1602, a probe adapter 1603, and a hole 1604 defined in a glass cover slide 1613. The biofluid passes through the inlet reservoir to a microfluidic channel 1606 and flows to an outlet reservoir 1607. A probe adjuster 1612 may be provided to adjust the height of the probe 1602 to create a good seal with the probe adapter 1603 and hole 1604. The flow direction of the biofluid is indicated by the dashed arrow.

[0175] Figure 19 is an enlarged plan view corresponding to the channel region of Figure 2. Figure 19 shows pillars 1701, represented by circles. In this embodiment, some representative lengths of the pillar band regions within the channel are shown in micrometers.

[0176] Figure 20 is a magnified plan view micrograph corresponding to the channel region of Figure 2. Figure 20 shows pillars as dots. In this embodiment, the representative spacing of pillar strips within the channel is shown in micrometers. The direction of biofluid flow is indicated by the dashed arrows.

[0177] Figure 21 is a plan view of one embodiment of a microfluidic device corresponding to Figure 2. Figure 21 shows that a biofluid can be introduced into an inlet region reservoir 2202 with a delivery probe 2201. The direction of biofluid flow to an outlet reservoir region 2203 is indicated by dashed arrows. A close-up view of this embodiment shows some representative spacings of the ribbon-like pillars in the channel in micrometers. For this embodiment, the dotted lines in the close-up view indicate possible tortuous paths of the biofluid between obstacles.

[0178] FIG. 22 illustrates one embodiment of a microfluidic system of the present invention. A processor 102 can send and / or receive control signals from a fluid drive unit 101, which supplies a drive fluid, such as compressed gas, to a fluid source unit 103. The fluid source unit 103 can contain a fluid, a biofluid, a carrier, and / or a reagent of interest. The fluid, biofluid, carrier, and / or reagent of interest can flow to a sensor unit 105, which can monitor the flow rate and / or pressure of the fluid. The fluid, biofluid, carrier, and / or reagent of interest can flow to an on-chip unit 107, which can include a microfluidic device of the present invention. The fluid, biofluid, carrier, and / or reagent of interest can enter an inlet reservoir of a microfluidic chip of the present invention in the on-chip unit 107. The fluid, biofluid, carrier, and / or reagent of interest can reach an outlet reservoir of a microfluidic chip of the present invention in the on-chip unit 107 and flow to an off-chip unit 109. The processor 102 can receive data from the sensor unit 105 and record flow rate and / or pressure. The on-chip unit 107 can include analytical tools such as illuminators and photodetectors for spectroscopy measurements. The off-chip unit 109 can include various analytical tools such as microscopy tools, imagers, and analyzers, chromatography analyzers, mass spectrometry analyzers, and / or magnetic resonance analyzers. The processor 102 can send control signals and / or receive data from the on-chip unit 107 and the off-chip unit 109.

[0179] In some embodiments, the fluid composition within a system or device of the present invention can be analyzed by various techniques. For example, the fluid composition can be analyzed by imaging techniques.

[0180] Examples of imaging techniques include electron microscopy, stereomicroscopy, wide-field microscopy, polarizing microscopy, phase contrast microscopy, multiphoton microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, light microscopy, and ultrasound microscopy.

[0181] Examples of imaging techniques include positron emission tomography, computed tomography, and magnetic resonance imaging.

[0182] Examples of assay techniques include colorimetric assays, chemiluminescent assays, spectrophotometry, immunofluorescence assays, and light scattering methods.

[0183] In some embodiments, the present invention can provide a device for measuring the pressure and flow rate of a fluid composition. In certain embodiments, the device can have a meshwork composition placed in a channel to provide resistance to flow. The meshwork composition can include any one or more of a uveal meshwork, a corneal meshwork, and a villi meshwork. Such a meshwork can be simulated, for example, using an obstruction in a restrictive channel, or can be provided by extraction of aqueous humor, body fluids, or clinical samples.

[0184] Extracellular matrix bodies or composites for use in omentum compositions may be composed of various biomolecules or composite particles and may have diameters ranging from about 0.5 to about 50,000, or 0.5 to 1,000, or 1 to 200, or 1 to 100, or 1 to 50, or 1 to 25, or 1 to 10, or 1 to 5 micrometers.

[0185] Extracellular matrix bodies or complexes that can be isolated in the devices of the present invention may have diameters ranging from about 0.5 to about 5,000, or 0.5 to 1,000, or 2 to 700, or 1 to 200, or 1 to 100, or 1 to 50, or 1 to 25, or 1 to 10, or 1 to 5 micrometers.

[0186] In some embodiments, the channel may include obstacles such as glass beads, microbeads, magnetic beads, gel particles, dextran particles, or polymer particles. The obstacles may also be composed of glass fibers, polymeric fibers, inorganic fibers, organic fibers, or metal fibers.

[0187] In additional embodiments, the devices or channels of the present invention may include binding agents, affinity detectors, or immunological agents attached to elements of the device in fluid communication with the sample fluid. The devices may also have agents for internal capture and / or detection of biomolecules from the sample. In certain embodiments, the devices can have agents for internal capture and / or detection of biomarkers in the sample fluid.

[0188] In certain embodiments, the uveal plexus or restrictive channel can have a fenestration of about 25 micrometers. The corneal retina or restrictive channel can have a fenestration of about 2-15 micrometers. The villi or restrictive channel can have a fenestration of about 1-4 micrometers or less.

[0189] The device may further include a fluid reservoir for holding the fluid composition, the fluid reservoir being in fluid communication with the inlet of the channel for introducing the fluid composition into the inlet of the channel.

[0190] The devices of the present disclosure can have a drive source or pressure source for applying pressure to the drive fluid composition. The drive fluid can enter a fluid reservoir for driving the fluid composition to the inlet of the microfluidic channel.

[0191] The device of the present invention can have a sensor unit in fluid communication with the fluid composition for measuring the flow rate and pressure of the fluid composition at the inlet of the channel and communicating the flow rate and pressure to the processor.

[0192] The signals and data from the units of the system device can be received by the processor. The processor can display the flow rate and pressure. The memory or medium can store instructions or files such as a machine-readable storage medium. The machine-readable storage medium can be non-transitory.

[0193] The processor of the present disclosure may be a general-purpose or special-purpose computer. The processor may execute instructions stored in a machine-readable storage device or medium. The processor may include an integrated circuit chip, a microprocessor, a controller, or a digital signal processor, any of which may be used to receive and / or transmit data and execute stored instructions. The processor may also perform calculations, transform data, and / or store data in a memory, medium, or file. The processor may receive and execute instructions, which may include performing one or more steps of a method of the present invention. The device of the present invention may include one or more non-transitory machine-readable storage media, one or more processors, one or more memory devices, and / or one or more user interfaces. The processor may have an integrated display for displaying data or transformed data.

[0194] In some aspects, the systems of the present disclosure may include a device having microfluidic channels. Three or more channels may be disposed within a microfluidic chip.

[0195] The disclosed system can include an on-chip unit having one or more detectors for analyzing the fluid composition within the channel or exiting the inlet or outlet of the channel. The detector can also be positioned to detect the fluid composition within the channel.

[0196] The systems of the present disclosure can include an off-chip unit having one or more detectors for analyzing the fluid composition extracted from the microfluidic channel.

[0197] In certain embodiments, the extracellular matrix bodies or composites for use in the omentum compositions in the systems or devices of the present disclosure can include fixatives, stabilizing components, or cross-linking components capable of transforming the structure into a stable, homogenous composition.

[0198] Examples of stabilizing components include fixatives described herein, cross-linking compounds described herein, organic solvents, polypeptides, and pharmaceutically acceptable organic salts.

[0199] The crosslinked extracellular matrix body or composite may be reversibly crosslinked or irreversibly crosslinked.

[0200] In some embodiments, the devices of the present invention can include an extracellular matrix body or composite as an omentum composition that can be used to identify or screen for active agents. The omentum composition may also include a drug delivery vehicle.

[0201] In additional embodiments, the device of the present invention can be used to measure the amount or level of extracellular matrix bodies or complexes in a test sample. Measuring the amount or level of extracellular matrix bodies or complexes in a test sample can provide a diagnostic marker level for the test sample. The device of the present invention can be used to identify glaucoma or pre-glaucoma in a subject.

[0202] In a further embodiment, the device of the present invention can be used to measure pressure that can be related to the amount or level of extracellular matrix bodies or complexes in a test sample. The pressure value in the channel can be directly related to the amount or level of extracellular matrix bodies or complexes in the test sample.

[0203] In certain embodiments, the devices of the present invention can be used to measure an assay value that can be related to the amount or level of an extracellular matrix body or complex in a test sample. The assay value of the composition in the channel can be directly related to the amount or level of the extracellular matrix body or complex in the test sample.

[0204] Examples of assays include colorimetric assays, chemiluminescent assays, spectrophotometric assays, immunoassays, or light scattering assays.

[0205] Means for analyzing samples within microfluidic devices include analytical tools such as spectroscopy and spectroscopic analysis, and illumination sources and photodetectors for immunolabeling and detection, as further illustrated in the examples herein.

[0206] Means for analyzing samples within a microfluidic device include illumination sources and imaging tools such as microscopes, as further illustrated in the examples herein.

[0207] Extracted Compositions and Methods In some embodiments, the composition may comprise a fraction of a biological sample extracted from a microfluidic device.

[0208] In certain embodiments, compositions extracted from the microfluidic device may be used in the treatment of the human or animal body.

[0209] In additional embodiments, compositions extracted from the microfluidic device may be used for the diagnosis or prognosis of a subject.

[0210] The isolated and / or extracted extracellular matrix body composition can be combined with a pharmaceutical carrier and one or more pharmaceutical excipients.

[0211] The morphology of the isolated and / or extracted extracellular matrix bodies may be altered by the isolation and / or extraction process.

[0212] The isolated and / or extracted form of the extracellular matrix body may be chemically modified.

[0213] In some embodiments, the composition of the extracellular matrix body can be isolated and / or extracted for use in treating the human or animal body.

[0214] In further embodiments, the composition may comprise a sample from which the extracellular matrix bodies have been removed by an isolation and / or extraction process for use in treating the human or animal body. In certain embodiments, at least 25%, or at least 50%, or at least 75%, or at least 90% of the extracellular matrix bodies of the sample have been removed by an isolation and / or extraction process for use in treating the human or animal body.

[0215] In some embodiments, extracting a composition from a microfluidic device can be a method of preparing a biological sample for use in the diagnosis or prognosis of a subject.

[0216] In certain embodiments, the method for isolating extracellular matrix bodies can be performed by ultrafiltration or centrifugation, or by a microfluidic device of the present disclosure.

[0217] Embodiments of the present invention further include immobilizing the extracellular matrix body on the glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

[0218] All publications, including patents, published patent applications, and non-patent publications, referred to herein are each expressly incorporated herein by reference in their entirety for all purposes.

[0219] Although the foregoing disclosure has been described in detail by way of example for clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be understood by this disclosure and made without undue experimentation within the scope of the appended claims, which are set forth by way of example and not limitation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of the features, materials, elements, or limitations of the various exemplary components, examples, and claimed embodiments.

[0220] The terms "a," "an," "the" and similar terms in the claims describing the invention are intended to be interpreted as including both the singular and the plural. [Example]

[0221] Example 1. Pressure and flow measurement in a microfluidic device using disease-relevant biofluids. Isolation of extracellular matrix bodies in a microfluidic device Figure 23 shows that aqueous humor from a patient with primary open-angle glaucoma increased the pressure within the microfluidic device. Figure 23 shows the relative change in pressure (mmHg) within a microfluidic model spongiform retina upon injection of human fluid from a patient with severe primary open-angle glaucoma. The flow rate through the microfluidic channel was constant at 2 μl / min, and the baseline system pressure was measured with an external pressure sensor. The human aqueous humor sample was injected at the time point indicated by the arrow and letter "a." The pressure steadily increased, reaching a maximum of approximately 41 mmHg at 27 minutes. Figure 23 shows that aqueous humor from a patient diagnosed with POAG glaucoma increased the pressure within the device.

[0222] Example 2. Isolation of disease-associated extracellular matrix bodies in a microfluidic device Figure 24 (top) is a confocal micrograph of the microfluidic chip after isolation of EMBs from human aqueous humor from a patient with primary open-angle glaucoma at the end of the experiment shown in Figure 23. Figure 24 (top) shows the protein content of the aqueous humor labeled with the fluorescent marker carboxyfluorescein succinimidyl ester (arrows). Circles indicate pillars in the restriction channel. Figure 24 (bottom) shows EMBs isolated in the microfluidic channel sandwiched between pillars (arrows).

[0223] Example 3. Isolation of extracellular matrix bodies from biofluids using size exclusion filters This example demonstrates that EMBs can be isolated by size exclusion and distinguished from smaller particles, such as free extracellular vesicles or other vesicles.

[0224] Figure 25 shows the isolation of extracellular matrix bodies from a biofluid using size exclusion filters. Bovine vitreous humor was filtered through a 5 μm cellulose acetate syringe filter, followed by a 1 μm syringe tip filter, then a 0.45 μm syringe tip filter, and finally a 0.22 μm filter. Each fraction was characterized using wide-field microscopy.

[0225] Figure 26 shows representative wide-field microscopy images for the isolation of EMBs using size-exclusion filters. Figures 26a and 26b demonstrate the presence of extracellular matrix bodies in the native biofluid of bovine vitreous humor. To isolate and recover extracellular matrix bodies from the complex biofluid, serial syringe filtration was performed using cellulose filters with pore sizes ranging from 5 μm to 0.22 μm. The extracellular matrix bodies were fixed to glass slides with EDC and stained with Alcian blue stain. Figure 26c shows a fraction of bovine vitreous isolated by serial filtration through a 5 μm syringe-tip filter. Figure 26d shows a fraction of bovine vitreous isolated by serial filtration through a 1 μm syringe-tip filter. Figure 26e shows a fraction of bovine vitreous isolated by serial filtration through a 0.45 μm syringe-tip filter. Figure 26f shows a fraction of bovine vitreous isolated by serial filtration through a 0.22 μm syringe-tip filter. The image shows the relative decrease in larger ECM bodies as the filtrate is passed through successively smaller filter sizes.

[0226] This example demonstrates that EMBs can be isolated by size exclusion. Analysis of the isolated EMBs revealed that they consist of DNA, RNA, proteins, and hyaluronic acid or collagen, which are components of the extracellular matrix.

[0227] The vitreous humor is a highly hydrated tissue with a water content of 98-99.7%, and is essentially composed of an extracellular matrix. The main component of the extracellular matrix is ​​a protein called collagen. Collagen proteins are modified with sugar chains, and when released from cells, they assemble into collagen fibers. Extracellular matrix bodies can adhere to the extracellular matrix, particularly to fibrils.

[0228] Bovine eyeballs were dissected, and orbital fat and extraocular muscles attached to the globe were removed. The globes were rinsed with 5 mL of ice-cold Tris-buffered saline (TBS) containing 50 mM Tris-HCl and 150 mM NaCl (pH 8.0) for 1 minute at 4°C. A 16-gauge needle was used to make a sclerotomy 4 or 8 mm posterior to the limbus, and a circumferential sagittal incision was made with scissors to separate the vitreous body into anterior and posterior cups. The formed vitreous body was cut and removed, and scissors were used to cut adhesions between the vitreous and ocular structures. Tissue samples were washed with TBS (pH 8.0) for 1 minute at 4°C. The vitreous specimens were collected in a 15 mL centrifuge tube and homogenized using an immersion blender. Aliquots of the homogenized bovine vitreous humor (BVH) were transferred to 1 mL centrifuge tubes. For further studies, bovine vitreous was resuspended in TBS buffer and frozen at −80° C. until use.

[0229] An aliquot of homogenized bovine vitreous diluted to 1 mL with buffered saline was loaded into a 1 mL syringe using a 22-gauge needle. The needle was replaced with a 5 μm cellulose acetate syringe filter, and even downward pressure was applied to force the bovine vitreous through the filter. The filtrate was collected in a new 1 mL tube, and an 80 μL aliquot of the filtrate was saved for imaging. Next, the filtrate recovered from the 5 μm filtration was loaded into a 1 μm syringe-tip filter using the same procedure as above, and an aliquot was saved for imaging. Next, the filtrate from the 1 μm filter was extruded through a 0.45 μm syringe-tip filter using the same procedure, and an aliquot was saved for imaging. Finally, the filtrate from the 0.45 μm filter was extruded through a 0.22 μm filter. Filtrate was collected from each filtration step.

[0230] The components of each fraction were visualized using wide-field microscopy. Each sample was imaged by placing the biofluid on a glass slide, crosslinking the sample with EDC, and staining the hyaluronic acid-containing material with Alcian blue. To stain the samples, each filtrate was incubated with 1% Alcian blue (Sigma 1% Alcian Blue in 3% Acetic Acid pH 2.5 B8483) at room temperature in a 1:1 v / v mixture for 30 minutes. After incubation, 40 μL of the stained filtrate was placed on a glass slide, covered with a coverslip, and imaged using a bright-field microscope. Color bright-field images were captured using an inverted phase-contrast microscope (Zeiss Axiovert 200) equipped with an Axiocam 105 color camera (Zeiss), and image processing was performed using Zen software (Zeiss, version 4.3).

[0231] Example 4. Isolation of extracellular matrix bodies from biofluids by continuous centrifugation This example demonstrates that EMBs can be isolated by centrifugation and distinguished from smaller particles, such as free exosomes or other small vesicles. To obtain cell-free vitreous specimens, the vitreous was first clarified by a series of low-speed centrifugations.

[0232] Figure 27 illustrates the isolation of extracellular matrix bodies from biofluids using centrifugation. Sequential centrifugation yielded four pellets: 9705, 9713, 9721, and 9729. Bovine vitreous was resuspended in 9701 and placed in a 1 ml tube. The tube was centrifuged (Sorvall Legend RT) at 350 g for 10 minutes at 4°C to form pellet 1 (9705). A 50 μl aliquot of the supernatant was saved for analysis and labeled Supernatant 1 (9709). The remaining supernatant was transferred to a new tube and centrifuged (Eppendorf, 5417R series, F45-30-11 Eppendorf rotor) at 2000 g for 10 minutes at 4°C to form pellet 2 (9713). A 50 μl aliquot of the supernatant was saved for analysis and labeled Supernatant 2 (9717), and the remaining supernatant was transferred to a new tube. The supernatant was centrifuged at 10,000 g for 10 minutes at 4°C to yield Pellet 3 (9721). A 50 μl aliquot of the supernatant was saved for analysis and labeled Supernatant 3 (9725), and the remaining supernatant was transferred to a new tube. The supernatant was centrifuged at 20,000 g for 10 minutes at 4°C to yield Pellet 4 (9729). A 50 μl aliquot of the supernatant was saved for analysis and labeled Supernatant 4, and the remaining supernatant was transferred to a new tube.

[0233] Figure 28 shows representative transmission electron microscopy (TEM) images for the isolation of EMBs by sequential centrifugation. Figure 28a shows extracellular matrix bodies present in bovine vitreous humor. In Figure 28b, a representative TEM image of a sample taken from the pellet after centrifugation at 450 g shows the extracellular matrix bodies present in the pellet fraction. Similarly, in Figure 28c, a representative TEM image of a sample taken from the pellet after centrifugation at 2,000 g shows the extracellular matrix bodies present in the pellet fraction. In Figure 28d, a representative TEM image of a sample taken from the pellet after centrifugation at 10,000 g shows the extracellular matrix bodies present in the pellet fraction. In Figure 27e, a representative TEM image of a sample taken from the pellet after centrifugation at 20,000 g shows the extracellular matrix bodies present in the pellet fraction.

[0234] Example 5. Dose response in a microfluidic device to detect the activity of drugs in reducing intraocular pressure in a glaucoma model The dose-response behavior of bivalirudin TFA on intraocular pressure (IOP) was determined for use as an active agent in the treatment of glaucoma. Bivalirudin TFA had an EC50 of 1.2 nM in bovine vitreous humor. 50 showed.

[0235] The compound bivalirudin TFA was tested in bovine vitreous humor (BVH) using a microfluidic chip device. 25% homogenized BVH was prepared in PBS buffer and diluted with an equal volume of compound solution to achieve a total BVH concentration of 12.5%. Samples were vortexed and incubated at 37°C for 1 hour. As controls, PBS buffer or PBS plus 10% ethanol or DMSO were incubated under the same conditions as BVH.

[0236] The test compound-BVH solution was introduced into the reservoir of the microfluidic chip device, and the flow rate and pressure changes were recorded. Various concentrations of the compound were tested for their effects on the treatment of bovine vitreous humor. 7 μl of each test solution was injected into the microfluidic chip through the sample injector. After sample injection, the flow rate and pressure changes were continuously recorded for an additional 50 minutes. The relative changes in chip pressure throughout the entire experiment were determined.

[0237] Figure 29 shows the dose-dependent response curve for treatment of bovine vitreous humor with the compound bivalirudin TFA. EC 50 Values ​​were taken as the point on the x-axis where the logarithmic function of the micromolar concentration of the compound produced a half-maximal response. The logarithmic function of the micromolar concentration of the drug was plotted on the x-axis and the percent of maximal response on the y-axis. The maximal response was obtained by taking the value of the response for the highest drug concentration. Responses were calculated by taking the absolute value of the difference between the control and test values ​​at each concentration.

[0238] Example 6. Dose-Response in a Microfluidic Device to Detect the Activity of Drugs in Lowering Intraocular Pressure in a Glaucoma Model The dose-response behavior of colistin sulfate as an active agent in the treatment of glaucoma on intraocular pressure (IOP) was determined. Colistin sulfate had an EC of 0.36 nM in bovine vitreous humor. 50 showed.

[0239] Colistin sulfate was tested in bovine vitreous humor (BVH) using a microfluidic chip device. 25% homogenized BVH was prepared in PBS buffer and diluted with an equal volume of compound solution to achieve a total BVH concentration of 12.5%. Samples were vortexed and incubated at 37°C for 1 hour. As controls, PBS buffer or PBS plus 10% ethanol or DMSO was incubated with BVH under the same conditions.

[0240] The test compound-BVH solution was introduced into the reservoir of the microfluidic chip device, and the flow rate and pressure changes were recorded. Various concentrations of the compound were tested for their effects on the processing of bovine vitreous humor. 7 μl of each test solution was injected into the microfluidic chip through the sample injector. After sample injection, the flow rate and pressure changes were continuously recorded for an additional 50 minutes. The relative change in chip pressure during the experiment was calculated.

[0241] Figure 30 shows the dose-response curve for treatment of the bovine vitreous humor glaucoma model with the compound colistin sulfate. 50 Values ​​were taken as the point on the x-axis where the logarithmic function of the micromolar concentration of the compound produced a half-maximal response. The logarithmic function of the micromolar concentration of the drug was plotted on the x-axis and the percent of maximal response on the y-axis. The maximal response was obtained by taking the value of the response for the highest drug concentration. Responses were calculated by taking the absolute value of the difference between the control and test values ​​at each concentration.

[0242] Example 7. Dose-Response in a Microfluidic Device to Detect the Activity of Drugs in Lowering Intraocular Pressure in a Glaucoma Model The dose-response behavior of polymyxin B sulfate as an active agent in the treatment of glaucoma on intraocular pressure (IOP) was determined. Polymyxin B sulfate had an EC of 4.3 nM in a bovine vitreous glaucoma model. 50 showed.

[0243] Polymyxin B sulfate was tested in a bovine vitreous humor (BVH) glaucoma model using a microfluidic chip device. 25% homogenized BVH was prepared in PBS buffer and diluted with an equal volume of compound solution to achieve a total BVH concentration of 12.5%. The sample was vortexed and incubated at 37°C for 1 hour. As controls, PBS buffer or PBS plus 10% ethanol or DMSO was incubated with BVH under the same conditions.

[0244] The test compound-BVH solution was introduced into the reservoir of the microfluidic chip device, and the flow rate and pressure changes were recorded. Various concentrations of the compound were tested for their effects on the treatment of bovine vitreous humor. 7 μl of each test solution was injected into the microfluidic chip through the sample injector. After sample injection, the flow rate and pressure changes were continuously recorded for an additional 50 minutes. The relative changes in chip pressure throughout the entire experiment were calculated.

[0245] Figure 31 shows the dose-response curve for treatment of a bovine vitreous humor glaucoma model with the compound polymyxin B sulfate. EC 50 Values ​​were taken as the point on the x-axis where the logarithm of the micromolar concentration of the compound produced a half-maximal response. The logarithm of the micromolar concentration of the drug was plotted on the x-axis and the percent of maximal response on the y-axis. The maximal response was obtained by taking the value of the response for the highest drug concentration. Responses were calculated by taking the absolute value of the difference between the control and test values ​​at each concentration.

[0246] Example 8. Isolation and extraction of extracellular matrix bodies using a microfluidic device A microfluidic device was used to isolate bovine vitreous extracellular matrix bodies, which were then extracted.

[0247] Figure 32 illustrates the isolation of extracellular matrix bodies in a restricted channel of a microfluidic device of the present disclosure. Figures 32a and 32b are representative micrographs of a microfluidic chip perfused with bovine vitreous humor. The letter "p" indicates pillars within the channel. After perfusion, extracellular matrix bodies were isolated between and around the pillars. Figures 32c and 32d are representative micrographs of the channel after extraction of the extracellular matrix bodies. The extracellular matrix bodies were extracted from the chip using mild detergent, 1% sodium dodecyl sulfate, SDS, and reversal of flow from the inlet port. The extracellular matrix bodies were detached from the chip and were significantly reduced after extraction.

[0248] Example 9. Detection of extracellular matrix biomarkers by proteomic profiling Bovine vitreous extracellular matrix bodies were isolated and their proteome profiles were analyzed off-chip using LC / MS.

[0249] 33 shows off-chip proteome analysis of bovine extracellular matrix bodies by LC / MS. Biomarkers of extracellular matrix bodies were detected.

[0250] The enriched bovine vitreous extracellular matrix aggregate pellet was resuspended in 50 μl of 1% sodium dodecyl sulfate (SDS, Sigma) and pelleted again at room temperature for 10 minutes at 25 kg. The pellet was solubilized in 20 μl of 2× SDS, 50 mM dithiothreitol (DTT) reducing agent, sonicated for 10 minutes, and incubated at 95°C for 5 minutes. The pellet and supernatant were electrophoresed onto NuPAGE 10% Bis-Tris Gels (1.5 mm x 10 well, Invitrogen). The gel was stained with Coomassie Brilliant Blue R250. The gel was photographed, stored, and then destained for further analysis.

[0251] Each gel band was reduced with 10 mM DTT at 60°C for 30 minutes and alkylated with 20 mM iodoacetamide at room temperature in the dark for 45 minutes. The bands were digested with 0.2 μg of trypsin (sequencing grade, Thermo Scientific Cat#90058) and incubated at 37°C for 16 hours. Peptides were extracted twice with 5% formic acid and 60% acetonitrile and dried under vacuum.

[0252] Samples were analyzed by LC-MS using a Nano LC-MS / MS (Dionex Ultimate 3000 RLSCanon System, ThermoFisher) interfaced with an Eclipse (ThermoFisher). Three μl of the 12.5 μl in-gel digested sample pellet was loaded onto a fused silica trap column (Acclaim PepMap 100, 75 μm x 2 cm, ThermoFisher). After washing with 0.1% trifluoroacetic acid (TFA) at 5 μl / min for 5 min, the trap column was inlined with an analytical column for LC-MS / MS (NanoeaseMZ peptide BEH C18, 130A, 1.7 μm, 75 μm x 250 mm, Waters). Peptides were fractionated at 300 nL / min using a segmented linear gradient (A: 0.2% formic acid, B: 0.16% formic acid, 80% acetonitrile) of 15-25% B for 40 min, 25-50% B for 44 min, and 50-90% B for 11 min. Then, solution B was returned to 4% for 5 min for the next run.

[0253] The scan sequence began with MS1 spectra (Orbitrap analysis, resolution 120,000, scan range M / Z 375–1500, automatic gain control (AGC) target 1E6, maximum injection time 100 ms). The top-S (3 s) duty cycle method was used to determine the number of MSMS runs performed in each cycle. Parent ions with charges between 2 and 7 were selected for MSMS, and 60 s of dynamic exclusion was used to avoid repetitive sampling. Parent ion masses were isolated in the quadrupole with an isolation window of 1.2 m / z and an automatic gain control (AGC) target of 1E5. They were then fragmented by high-energy collisional dissociation with a normalized collision energy of 30%. The fragments were scanned at a resolution of 15,000 in the Orbitrap. The MSMS scan range was determined by the charge state of the parent ion, with the lower limit set at 110 amu.

[0254] Extracellular matrix-related proteins expressed in the bovine extracellular matrix fraction of vitreous were selectively identified by proteomic profiling and are shown in Table 1.

[0255] [Table 1]

[0256] The vitreous fraction was isolated by low-speed centrifugation and then isolated using a microfluidic device. Higher spectral counts indicate higher protein abundance.

[0257] Proteins known to be involved in protein aggregation found in the vitreous bovine extracellular matrix body fraction were selected by proteomic profiling and are shown in Table 2.

[0258] [Table 2]

[0259] Using the prototype microfluidic device, low-speed centrifugation was performed to obtain a vitreous ECM aggregate fraction. Proteins were classified by function, and highlighted proteins known to be involved in the extracellular matrix. For example, complement C3 (spectral count, 408), α-enolase (spectral count, 151), and clusterin (spectral count, 74) were detected with relatively high spectral counts.

[0260] Example 10. Isolation and extraction of extracellular matrix bodies in a microfluidic device Using a microfluidic device, bovine vitreous extracellular matrix bodies were isolated, followed by extraction of the bodies.

[0261] Figure 34 illustrates the isolation of extracellular matrix bodies in the restricted channel of a microfluidic device of the present disclosure and their subsequent extraction. The scale bar in the image is 50 μm. Figure 34a shows a representative wide-field photograph (gray signal, bright field) of a microfluidic device perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, counterstained for hyaluronic acid with Alcian blue. Figure 34a shows the signal from extracellular matrix bodies (arrows) trapped between the pillars (p) of the device. The chip was perfused with biofluid for at least 60 minutes. After perfusion, aggregates were isolated between the pillars and observed as clumps. Smaller materials than the extracellular matrix bodies exited through the outlet port. Figure 34b illustrates the extraction of extracellular matrix bodies from the restricted channel of the microfluidic device. The device was perfused with mild detergent, 0.1% sodium dodecyl sulfate, and SDS, and the flow direction was reversed from the outlet to the inlet. Figure 34b shows that substantially fewer bodies were present in the channel after elution, indicating that the bodies had been extracted. Figure 34c shows a high-power image of extracellular matrix bodies (arrows) trapped between pillars (p) after perfusion. Figure 34c shows extraction with detergent and backflow, again showing substantially fewer bodies in the channel after extraction.

[0262] Example 11. Isolation and extraction of extracellular matrix bodies in a microfluidic device This experiment demonstrated that on-chip staining can be used to detect the isolation of extracellular matrix bodies.

[0263] Figure 35 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. A fluid containing homogenized bovine vitreous suspended in a biofluid was injected into the microfluidic device. After perfusion of the fluid through the device, the fluid flowed through the inlet and exited through the outlet. Large extracellular matrix bodies (arrows) were captured between pillars (labeled Lp). Prior to antibody staining, the chip was perfused with a blocking solution to prevent nonspecific antibody binding. Next, fibronectin, an extracellular matrix component known as an integrin-binding protein, was labeled by injecting an anti-fibronectin primary antibody, incubating the sample for 2 hours, and washing. This was followed by incubation with a goat anti-rabbit FITC secondary antibody for 1 hour and washing. The microfluidic chip was then imaged using wide-field fluorescence and bright-field microscopes. Figure 35 shows a representative wide-field fluorescence micrograph. This image shows extracellular matrix bodies within the microfluidic channel. The distance between the large pillars (Lp) was approximately 100 μm. The punctate signals in the body represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary antibody with Alexa488, FITC, white signal).

[0264] Figure 36 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. Figure 36a is a representative photographic brightfield image of stained extracellular matrix bodies in the channel (arrow). The image scale bar is 20 μm. The control image had no fluorescent signal, indicating that the signal in Figure 36a is specific to fibronectin. Figure 36b again shows stained extracellular matrix bodies in the channel (arrow). The image scale bar is 50 μm. Again, the control image had no fluorescent signal, indicating that the signal in Figure 36b is specific to fibronectin.

[0265] Example 12. Isolation and extraction of extracellular matrix bodies in a microfluidic device This experiment demonstrated that on-chip staining can be used to detect the isolation of extracellular matrix bodies.

[0266] Figure 37 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. Figure 37 shows representative photographs of on-chip immunohistochemical staining of perlecan protein, a component of the extracellular matrix of cartilage, in a biofluid containing extracellular matrix bodies. A biofluid containing a suspension of homogenized bovine vitreous was injected into the microfluidic chip. After perfusion of the biofluid through the device, the sample flowed through the inlet and exited through the outlet. Large extracellular matrix bodies (arrows) were captured between pillars (marked "p"). Prior to antibody staining, the chip was perfused with blocking solution to prevent nonspecific antibody binding. Perlecan was labeled by injecting anti-perlecan primary antibody, incubating the sample for 2 hours, and washing. This was followed by incubation with goat anti-rabbit TRITC secondary antibody for 1 hour and washing. The microfluidic chip was then imaged with wide-field fluorescence and bright-field microscopes. Figure 37 shows extracellular matrix bodies in the microfluidic channel between pillars (p). The punctate signals represent perlecan staining (white signal). Control images had no fluorescent signal, indicating that the signal in Figure 37 is specific to perlecan.

[0267]

[0033] Figure 38 shows on-chip immunohistochemical staining of extracellular matrix bodies in a device channel of the present disclosure. Figure 38a shows a representative photographic brightfield image of on-chip immunohistochemical staining of perlecan. The control image had no fluorescent signal, indicating that the signal in Figure 38a is specific to perlecan. The scale bar in the image is 10 μm. Figure 38b shows a representative photographic brightfield image of on-chip immunohistochemical staining of perlecan. The control image had no fluorescent signal, indicating that the signal in Figure 38c is specific to perlecan. The scale bar in the image is 10 μm.

[0268] Example 13. Off-chip analysis of extracellular matrix bodies extracted from microfluidic devices 1 is an experiment showing off-chip analysis of extracellular matrix bodies as can be extracted from the device channels of the present disclosure.

[0269] Figure 39 shows that extracellular matrix bodies can be visualized on glass surfaces using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and Alcian blue staining of hyaluronic acid. Figure 39 shows the signal (dark staining) of extracellular matrix bodies, demonstrating that EDC crosslinking retains the extracellular matrix bodies on the surface.

[0270] Example 14. Off-chip analysis of extracellular matrix bodies extracted from microfluidic devices 1 is an experiment illustrating off-chip analysis of extracellular matrix bodies of the present disclosure.

[0271] Figure 40 shows off-chip analysis of extracellular matrix visualized on glass surfaces using crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and staining of collagen with picrosirius red dye. Figure 40 shows the signal (dark staining) of collagen strands within the extracellular matrix bodies, demonstrating that EDC crosslinking retains the extracellular matrix bodies on the surface. This also demonstrates that EMBs can be visualized by collagen staining.

[0272] Example 15. Off-chip analysis of extracellular matrix bodies extracted from microfluidic devices In this experiment, extracellular matrix bodies that can be visualized on glass surfaces were analyzed off-chip using nucleic acid markers.

[0273] Figure 41 shows off-chip analysis of extracellular matrix bodies that can be visualized on a glass surface using cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and staining of DNA with Hoechst dye. Figure 41 shows the signal of DNA in the extracellular matrix bodies.

[0274] Example 16. Isolation and detection of extracellular matrix bodies in a microfluidic device 10 is an experiment showing the isolation and detection of extracellular matrix bodies in a device channel of the present disclosure.

[0275] Figure 42 shows on-chip isolation and detection of extracellular matrix bodies. Figure 42 shows representative photographs, dark stain and bright field, of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and counterstained for hyaluronic acid with Alcian blue. Figure 42 shows signal from extracellular matrix bodies trapped near the large pillars (circles) of the device. The chip was perfused with biofluid for at least 60 minutes. Extracellular matrix bodies were observed forming clusters. The scale bar in the image is 50 μm.

[0276] Example 17. On-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device 1 is an experiment showing the on-chip isolation, detection, and analysis of extracellular matrix bodies in a device channel of the present disclosure.

[0277] Figure 43 illustrates the on-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 43 shows a representative low-power fluorescence micrograph of a channel after perfusion of bovine vitreous humor extracellular matrix bodies, counterstained for proteins with carboxyfluorescein succinimidyl ester (CFSE). A fluid containing homogenized bovine vitreous was injected into the microfluidic chip. After perfusion of the fluid through the device, the fluid flowed through the inlet and exited through the outlet. Large extracellular matrix bodies were trapped near the pillars (marked "p"). The scale bar in the image is 25 μm.

[0278] Example 18. On-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device 1 is an experiment showing the on-chip isolation, detection, and analysis of extracellular matrix bodies in a device channel of the present disclosure.

[0279] Figure 44 illustrates the on-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 44 shows representative photographs of a microfluidic chip perfused with bovine vitreous humor suspended in phosphate-buffered saline, pH 7.0, and counterstained for collagen with picrosirius red, dark staining, and bright field. Figure 44a shows the signal from extracellular matrix bodies between the pillars (labeled "p") of the device. The chip was perfused with biofluid for at least 60 minutes. Figure 44b shows the same image using a fluorescent filter, demonstrating that collagen was detected with picrosirius red (light gray signal). The scale bar in the image was 50 μm. Figures 44c and 44d show images at higher power. The scale bar in the image was 10 μm.

[0280] Example 19. Isolation, detection, and analysis of extracellular matrix bodies using a microfluidic device 1 is an experiment demonstrating the isolation, detection, and analysis of extracellular matrix bodies using the device of the present disclosure.

[0281] Figure 45 shows the frequency size distribution of human extracellular matrix bodies present in human aqueous humor biofluids from healthy and pre-disease states, glaucoma suspects, and pre-glaucoma states. Aqueous humor was obtained from eight healthy or pre-diagnosed glaucoma patients with intraocular pressures ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other methods. Extracellular matrix body size was determined by crosslinking the samples to glass slides using carbodiimide EDC fixative, staining with uranyl acetate, and photographing them under a wide-field microscope. Size was quantified for all eight samples using an automated program (ImageJ). The size (area) of the extracellular matrix bodies was approximately 1.67 μm. 2 From about 67 x 10 3 μm 2 Figure 45 shows the results for the range of 0 to 200 μm. 2 FIG. 1 shows counts of extracellular matrix bodies in a range of 100-1500 nm.

[0282] Example 20. Isolation, detection, and analysis of extracellular matrix bodies using a microfluidic device 1 is an experiment demonstrating the isolation, detection, and analysis of extracellular matrix bodies using the device of the present disclosure.

[0283] Figure 46 shows the frequency and size distribution of human extracellular matrix bodies present in aqueous humor fluid from healthy, glaucoma-suspected, and pre-glaucoma patients. Aqueous humor was obtained from eight healthy or pre-diagnosed glaucoma patients with intraocular pressures ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other methods. The size of extracellular matrix bodies was determined by crosslinking the samples to glass slides using carbodiimide EDC fixative, staining with uranyl acetate, and photographing them under a wide-field microscope. Size was quantified for all eight samples using an automated program (ImageJ). The size (area) of the extracellular matrix bodies was approximately 1.67 μm. 2 From about 67 x 10 3 μm 2 Figure 46 shows the results for the range of 201 to 1000 μm. 2 Counts of extracellular matrix bodies in the range of 100-1500 are shown.

[0284] Example 21. Isolation, detection, and analysis of extracellular matrix bodies using a microfluidic device 1 is an experiment demonstrating the isolation, detection, and analysis of extracellular matrix bodies using the device of the present disclosure.

[0285] Figure 47 shows the frequency size distribution of human extracellular matrix bodies present in human aqueous humor biofluids from healthy and pre-disease states, glaucoma suspects, and pre-glaucoma states. Aqueous humor was obtained from eight healthy or pre-diagnosed glaucoma patients with intraocular pressures ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other methods. Extracellular matrix body size was determined by crosslinking the samples to glass slides using carbodiimide EDC fixative, staining with uranyl acetate, and photographing them under a wide-field microscope. Size was quantified for all eight samples using an automated program (ImageJ). The size (area) of the extracellular matrix bodies was approximately 1.67 μm. 2 From about 67 x 10 3 μm 2 Figure 47 shows the results for 1001 to 5000 μm 2 Counts of extracellular matrix bodies in the range of 100-1500 are shown.

[0286] Example 22. Isolation, detection, and analysis of extracellular matrix bodies using a microfluidic device 1 is an experiment demonstrating the isolation, detection, and analysis of extracellular matrix bodies using the device of the present disclosure.

[0287] Figure 48 shows the size distribution of bovine vitreous extracellular matrix bodies isolated and extracted using the microfluidic device of the present invention. Extracellular matrix bodies from bovine vitreous humor after isolation and extraction using the microfluidic device of the present invention. The chip was perfused with biofluid for over 60 minutes. After 60 minutes of perfusion, ECM bodies were isolated near the restrictor channel pillars. The chip was then treated with detergent (0.1% sodium dodecyl sulfate, SDS), and the sample was extracted from the chip by backflow, allowing the bodies to flow out of the inlet port. Fractions of the eluate were collected at 10-minute intervals for a total of 80 minutes. Samples were mounted on glass slides, stained with Alcian blue, and imaged using a wide-field microscope. Size was quantified using an automated program (ImageJ). The size (area) of the extracellular matrix bodies was up to approximately 16 x 10 3 μm 2Figure 48 shows the counts of extracellular matrix bodies in each eluate fraction, which increased over time. This experiment demonstrated that the microfluidic device of the present invention can be used to isolate and extract extracellular matrix bodies of various sizes.

[0288] Example 13. On-chip isolation, detection, and analysis of extracellular matrix bodies in a microfluidic device This experiment demonstrated the off-chip isolation, detection, and analysis of extracellular matrix bodies.

[0289] Figure 49 demonstrates that off-chip analysis of extracellular matrix bodies can be performed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinking. Figure 49a shows a representative TEM image of extracellular matrix bodies from native bovine vitreous humor obtained with EDC crosslinking. Extracellular matrix bodies were observed with EDC fixation. Figure 49b shows a similar image taken without EDC crosslinking. Without EDC fixation, no extracellular matrix bodies were observed.

[0290] Example 24. Isolation and detection of extracellular matrix bodies of early stage pancreatic cancer from human plasma samples using a microfluidic device This experiment demonstrated the isolation and detection of extracellular matrix bodies of early-stage pancreatic cancer from human plasma samples using a microfluidic device. This experiment demonstrated that extracellular matrix bodies of early-stage pancreatic cancer can be isolated and detected from human plasma samples using the microfluidic device of the present invention. This experiment further demonstrated that extracellular matrix bodies are a useful marker for distinguishing the plasma of early-stage pancreatic ductal adenocarcinoma from healthy controls.

[0291] FIG. 50 shows the isolation of extracellular matrix bodies from human plasma samples from patients with early stage pancreatic ductal adenocarcinoma (PDAC) compared to healthy controls.

[0292] In this experiment, human plasma derived from early-stage pancreatic ductal adenocarcinoma (PDAC) was injected into a microfluidic chip. After perfusion through the device, the biofluid flowed through the inlet and exited through the outlet. Results were compared with those of age-matched healthy controls.

[0293] Prior to antibody staining, the chip was perfused with a blocking solution to prevent nonspecific antibody binding. Next, the extracellular matrix component fibronectin and integrin-binding proteins were labeled by on-chip immunohistochemistry by injecting anti-fibronectin primary antibodies, incubating for 2 hours, and washing. This was followed by incubating with goat anti-rabbit FITC secondary antibodies for 1 hour and washing. The microfluidic chip was then imaged using a fluorescence microscope.

[0294] Figure 50a shows a representative wide-field fluorescence microscopy image of a PDAC sample. Figure 50a shows an extracellular matrix body placed in a microfluidic channel. Figure 50a also shows that larger extracellular matrix bodies (arrows) were lodged between pillars (marked "p"). The width between pillars was approximately 100 μm. Punctate signals from the lodged extracellular matrix bodies represent fibronectin staining (anti-fibronectin Ab, Alexa488, goat anti-rabbit secondary Ab with FITC, white signal). Staining showed abundant signal and punctate staining within the EMB (Figure 50a, arrowheads).

[0295] Figure 50b is a similarly obtained fluorescence micrograph of an age-matched healthy control human plasma sample. Figure 50b shows a significantly reduced amount of fibronectin signal (Figure 50b, gray signal, arrow). When treated under identical conditions, the signal in the healthy control was much smaller than that in the diseased control. The scale bar in the image is 20 μm.

Claims

1. 1. An apparatus for isolating a fraction of a biological sample, comprising: one or more restricted channels having an inlet end and an outlet end, said inlet end and outlet end being in fluid communication via the channel; a plurality of spaced apart obstacles disposed in the restriction channel to provide resistance to flow, the spacing between obstacles decreasing in a direction from the inlet end to the outlet end; and an inlet reservoir for holding a fluid, the inlet fluid reservoir being in fluid communication with the inlet end of the restriction channel; one or more uniform flow channels having an inlet end and an outlet end, the inlet end and the outlet end being in fluid communication via a channel, the inlet end being in fluid communication with the inlet reservoir; An apparatus comprising:

2. a pressure source for applying pressure to fluid in the inlet reservoir; a flow sensor in fluid communication with the inlet reservoir for measuring the flow rate and pressure of fluid in the inlet reservoir; The apparatus of claim 1 further comprising:

3. The device may further comprise an outlet reservoir in fluid communication with the outlet ends of the restriction channel and the uniform flow channel.

10. The apparatus of claim 1.

4. 10. The device of claim 1, wherein the restriction channel comprises a barrier band having perforations of at least about 1 micrometer, or at least about 2 micrometers, or at least about 4 micrometers, or at least about 10 micrometers, or at least about 25 micrometers, or at least about 50 micrometers, or at least about 100 micrometers, or at least about 200 micrometers, or at least about 500 micrometers.

5. 10. The device of claim 1, wherein the restriction channel comprises perforations of about 1 to 4 micrometers, or about 1 to 15 micrometers, or about 4 to 35 micrometers, or about 4 to 100 micrometers, or about 4 to 200 micrometers.

6. 10. The device of claim 1, wherein 1-90% of the flow in the device is in the uniform flow channel, or 1-75% of the flow in the device is in the uniform flow channel, or 1-50% of the flow in the device is in the uniform flow channel, or 1-25% of the flow in the device is in the uniform flow channel.

7. The device of claim 1 , wherein the restriction channel and the uniform flow channel are integrated on the same chip or substrate.

8. The device of claim 1 , wherein the restriction channel and the uniform flow channel are on different chips or substrates.

9. The device of claim 1 , wherein the restricted channel is a microfluidic channel.

10. The device of claim 1 further comprising means for analyzing the biological sample in the channel.

11. The device of claim 1 , further comprising means for analyzing the proteomic, lipidomic, transcriptomic, or carbohydrate composition of the biological sample in the channel.

12. The device of claim 1 , further comprising a means for measuring the level of the isolated fraction of the sample in the channel.

13. The device of claim 1 , further comprising a means for measuring the level of a biomarker in the isolated fraction of the sample in the channel.

14. The apparatus of claim 1 , wherein the plurality of obstacles comprise pillars integral with the channel.

15. The plurality of obstacles are: a part of the uvea of ​​a human or animal, Part of the cornea or retina of a human or animal, and Part of the human or animal chorioretina The apparatus of claim 1 , comprising one or more of:

16. The device of claim 1 , wherein the plurality of obstacles are comprised of glass beads, magnetic beads, gel particles, dextran particles, or polymer particles.

17. The device of claim 1 , wherein the biological sample comprises a human or animal body fluid, blood, tissue, or cells.

18. The device of claim 1 , wherein the biological sample comprises a carrier liquid.

19. The device of claim 1 , wherein the biological sample comprises one or more reagents.

20. The device of claim 1 , wherein the restricted channel further comprises binding sites for binding biomarkers or biomolecules of the sample.

21. The device of claim 1 , wherein the biological sample is from a subject undergoing diagnosis or prognosis.

22. The device of claim 1 , further comprising a serpentine fluid mixing region in the restriction channel.

23. The device of claim 1 , wherein the restricted channel or continuous flow channel has a fluorinated coating.

24. Flowing a biological sample from the inlet end to the outlet end of the device of claim 1; and reversing the direction of fluid flow toward the inlet end of the device; A method for extracting extracellular matrix bodies from a biological sample by

25. 1. A microfluidic system for isolating a fraction of a biological sample, comprising:

1. A microfluidic device comprising: one or more restricted channels having an inlet end and an outlet end, said inlet end and outlet end being in fluid communication via the channel; a plurality of spaced apart obstacles disposed in the restriction channel to provide resistance to flow, the spacing between the obstacles decreasing in a direction from the inlet end to the outlet end; and an inlet reservoir for holding a fluid, said fluid reservoir in fluid communication with the inlet end of said restriction channel; and one or more uniform flow channels having an inlet end and an outlet end, the inlet end and the outlet end being in fluid communication through the flow channel, the inlet end being in fluid communication with an inlet reservoir; a microfluidic device comprising: a drive unit including a pressure source; a source unit including a fluid source, the pressure source being in fluid communication with the fluid source and the inlet reservoir of the microfluidic device; a sensor unit including a sensor in fluid communication with the inlet reservoir for measuring a flow rate and a pressure of fluid at the inlet reservoir and transmitting the flow rate and pressure data to a processor; and an on-chip analyzer unit, comprising one or more means, for analyzing the isolated fraction in the microfluidic device and sending the analysis data to a processor; and Processor for receiving and displaying flow, pressure and analysis A system including:

26. A composition comprising a fraction of a biological sample extracted from a device according to any one of claims 1 to 23.

27. 27. The composition of claim 26 for use in human or animal treatment.

28. 27. The composition of claim 26 for use in the diagnosis or prognosis of a subject.

29. A method of preparing a biological sample, the method comprising isolating extracellular matrix bodies from the biological sample.

30. 30. The method of claim 29, wherein the biological sample is comprised of human or animal body fluids, blood, tissue, or cells.

31. 30. The method of claim 29, wherein the extracellular matrix body has a size of 0.5 to 5,000 micrometers, or 1 to 1,000 micrometers, or 1 to 200 micrometers, or 4 to 100 micrometers.

32. The isolation of extracellular matrix bodies can be carried out by ultrafiltration or centrifugation.

33. 30. The method of claim 29, wherein said isolation of extracellular matrix bodies is performed by an apparatus of the present disclosure.

34. 30. The method of claim 29, further comprising anchoring the extracellular matrix body to a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

35. A method for preparing a biological sample in which an extracellular matrix body is fixed to a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

Images