Devices and methods for isolating extracellular matrix bodies

Abstract

Devices, methods, and systems for isolating extracellular matrix bodies are provided. More specifically, the present invention discloses devices, methods and systems for isolating extracellular matrix bodies from a biological sample for diagnosis and prognosis of a subject. The device may have a confinement channel for isolating extracellular matrix bodies and a uniform flow channel for accurate pressure measurements.

Description

[0001] This application is a divisional application of the application with the application date of March 24, 2021, application number 202180038203.8, and invention title "Device and Method for Separating Extracellular Matrix Bodies". Technical Field

[0002] The present invention relates to devices, methods, and systems for separating extracellular matrix bodies. More specifically, the present invention discloses devices, methods, and systems for separating extracellular matrix bodies from biological samples for the diagnosis and prognosis of a subject. Background Art

[0003] Conventional methods for disease diagnosis and prognosis may require the analysis or separation of extremely small fractions of biological samples. The corresponding analysis of components from extremely small fractions can be used for the diagnosis and prognosis of diseases. For example, individual cells or even individual nucleic acid molecules can be detected and analyzed. However, a disadvantage of such conventional methods is that they rely on very small amounts of biological material and corresponding small signals for decision-making. Useful signals may be lost in other unmeasured biological materials.

[0004] Conventional methods for disease diagnosis and prognosis often require obtaining the biological sample to be acquired and well-known structures that need to be separated from the sample for further analysis. For example, intact organ tissues, whole cells, exosomes, and other well-known structures can be separated and characterized. Disadvantages of such methods include the inability to detect or characterize diseases when the well-known structures do not easily reflect the disease state.

[0005] Other disadvantages of conventional methods for disease diagnosis and prognosis include the use of biomarkers that are only related to specific well-known structural elements of a subject's biological sample, such as exosomes. In these methods, biomarkers are often inherently limited because they are not directly related to the pathology of interest. Conventional methods often attempt to consider some biomarkers that may have a remote or partial association with the disease, hoping that statistical data can provide a diagnostic answer. Combinations of biomarkers are often required, and the results are highly unpredictable.

[0006] Other disadvantages of conventional devices include the inability to accurately measure the flow rate and pressure in complex fluids containing biological components. Due to the interaction of biological components with the device performing the measurement, fluids containing biological components may cause changes in the flow rate and pressure.

[0007] There is a need for devices and systems for separating particles from biological samples for diagnostic purposes that can provide increased sample isolates related to specific biology. More specifically, there is a need for devices, methods, and systems for separating important components from biological samples for the diagnosis and prognosis of diseases.

[0008] There is an urgent need for methods and devices for separating particles from biological samples that can separate relevant fractions of biological materials that more closely correspond to disease states and measure differential pressure and flow rates in relevant fluids. SUMMARY OF THE INVENTION

[0009] The present invention provides devices and systems for separating particles from biological samples applicable to a variety of diseases and medical conditions. Methods and systems for separating particles from biological samples are provided that can increase biologically relevant sample isolates. The devices and methods of the present invention can be used to separate important components from biological samples for the diagnosis and prognosis of diseases.

[0010] In some embodiments, the methods and devices of the present invention for separating particles from biological samples can be used for a variety of biological samples, including body fluids, tissues, and cells. In certain embodiments, the methods of the present disclosure can separate relevant fractions of biological materials that closely correspond to disease states.

[0011] The methods and devices of the present disclosure can be used to separate important fractions of biological samples and analyze their components relevant to the diagnosis and prognosis of diseases. In some embodiments, a significant amount of biological material and a correspondingly improved signal level can be obtained for decision-making. Aspects of the present invention include preserving the composition and properties of extracellular matrix bodies (EMBs) as disease indicators.

[0012] In some aspects, the present invention provides an enhanced ability to detect or characterize diseases using extracellular matrix bodies, which can more readily reflect disease states.

[0013] In additional aspects, the devices and methods of the present disclosure can provide increased signals for relevant biomarkers that associate specific biological fractions with disease states. The biomarkers of the present disclosure can be associated with diseases and provide diagnostic tools.

[0014] The devices of the present disclosure can provide precise measurements of the flow rate and pressure of fluids containing biological components by maintaining a continuous and substantial flow in the device, such that sensors can accurately measure differential pressure and flow rate. The devices and systems disclosed herein can provide improved measurements of the flow rate and pressure of fluids containing biological components through the arrangement of channels that maintain a continuous and substantial flow. In some embodiments, the devices of the present disclosure can have one or more channels that do not significantly restrict fluid flow, thereby maintaining a continuous and substantial flow in the system.

[0015] The devices and systems of the present disclosure can separate unique subpopulations or subset fractions of biological samples. In some embodiments, the unique subset fractions of biological samples can be disease-related. In certain embodiments, the unique subset fractions of biological samples can consist essentially of extracellular matrix bodies.

[0016] In a further aspect, the present disclosure provides devices and methods for separating, detecting, and / or analyzing the ultrastructural components of fluids containing biological materials or molecules. In certain embodiments, the ultrastructural components may be associated with a disease.

[0017] Embodiments of the present invention can be used to separate, extract, and utilize extracellular matrix vesicles (EMBs) as sources of biomarkers for a variety of specific diseases.

[0018] The present invention includes devices for separating, detecting, and analyzing extracellular matrix vesicles, biological particles, and complexes for various uses.

[0019] In certain aspects, extracellular matrix vesicles can serve as biomarkers by their morphological characteristics. In a further aspect, extracellular matrix vesicles can function by containing isolated biochemical markers that may be present in disease pathways.

[0020] Aspects of the present invention can further provide a diagnostic system including devices for detecting and measuring biomarkers by disease-associated extracellular matrix vesicles (EMBs) and / or biological particles or complexes.

[0021] In a further embodiment, the present disclosure describes methods and devices for preparing and analyzing biological material samples.

[0022] Biological material samples can include body fluids, tissues, and cells.

[0023] Embodiments of the present invention include the following:

[0024] A device for separating fractions of a biological sample, comprising:

[0025] One or more restricted channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel;

[0026] A plurality of spaced-apart obstacles located in the restricted channel to provide flow resistance, wherein the spacing between the obstacles decreases in the direction from the inlet end to the outlet end; and

[0027] An inlet reservoir for containing fluid, wherein the inlet fluid reservoir is in fluid communication with the inlet end of the restricted channel;

[0028] One or more uniform flow channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel, and wherein the inlet end is in fluid communication with the inlet reservoir.

[0029] The above device further includes 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 at the inlet reservoir.

[0030] The device may further include an outlet reservoir in fluid communication with the outlet ends of the restriction channel and the uniform flow channel.

[0031] The above device, wherein the restriction channel includes a barrier band having openings of at least about 1 micron, or at least about 2 microns, or at least about 4 microns, or at least about 10 microns, or at least about 25 microns, or at least about 50 microns, or at least about 100 microns, or at least about 200 microns, or at least about 500 microns.

[0032] The above device, wherein the restriction channel includes openings of about 1-4 microns, or about 1-15 microns, or about 4-35 microns, or about 4-100 microns, or about 4-200 microns.

[0033] The above device, wherein 1-90% of the flow in the device is within the uniform flow channel, or 1-75% of the flow in the device is within the uniform flow channel, or 1-50% of the flow in the device is within the uniform flow channel, or 1-25% of the flow in the device is within the uniform flow channel.

[0034] The restriction channel and the uniform flow channel can be integrated with the same chip or substrate. The restriction channel and the uniform flow channel can be located in different chips or substrates. The restriction channel can be a microfluidic channel.

[0035] The above device further includes means for analyzing a biological sample in the channel.

[0036] The above device further includes means for analyzing the proteomic composition, lipidomic composition, transcriptomic composition, or carbohydrate composition of a biological sample in the channel.

[0037] The above device further includes means for measuring the level of a separated fraction of a sample in the channel.

[0038] The above device further includes means for measuring the level of a biomarker in a separated fraction of a sample in the channel.

[0039] The above device, wherein the plurality of obstacles includes posts integrated with the channel.

[0040] The above-described device, wherein the plurality of obstacles includes one or more of the following: a part of the uveal meshwork of a human or animal, a part of the corneoscleral meshwork of a human or animal, or a part of the juxtacanalicular meshwork of a human or animal.

[0041] The plurality of obstacles may include glass beads, magnetic beads, gel particles, dextran particles, or polymer particles.

[0042] The biological sample may consist of human or animal body fluid, blood, tissue, or cells. The biological sample contains a carrier fluid.

[0043] The above-described device, wherein the biological sample contains one or more reagents.

[0044] The above-described device, wherein the restricted channel further includes a binding portion for binding a biomarker or biomolecule of the sample.

[0045] The biological sample can be from a subject undergoing diagnosis or prognosis.

[0046] The above-described device further includes a curved fluid mixing region in the restricted channel. The above-described device, wherein the restricted channel or the continuous flow channel has a fluorinated coating.

[0047] The present invention also relates to a method of extracting extracellular matrix bodies from a biological sample by flowing the biological sample from an inlet end of the device of claim 1 to an outlet end; and reversing the direction of fluid flow to be toward the inlet end of the device.

[0048] A microfluidic system for separating fractions of a biological sample, the system comprising:

[0049] A microfluidic device, which includes

[0050] One or more restricted channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel;

[0051] A plurality of spaced-apart obstacles located in the restricted channel to provide flow resistance, wherein the spacing between the obstacles becomes smaller in the direction from the inlet end to the outlet end; and

[0052] An inlet reservoir for containing fluid, wherein the fluid reservoir is in fluid communication with the inlet end of the restricted channel; and

[0053] One or more uniform flow channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel, and wherein the inlet end is in fluid communication with the inlet reservoir;

[0054] A drive unit including a pressure source;

[0055] A source unit including a fluid source, wherein the pressure source is in fluid communication with the fluid source and an inlet reservoir of the microfluidic device;

[0056] A sensor unit including a sensor in fluid communication with the inlet reservoir for measuring the flow rate and pressure of the fluid at the inlet reservoir and sending flow rate and pressure data to a processor; and

[0057] An on-chip analyzer unit including one or more appliances for analyzing separated fractions in the microfluidic device and sending analysis data to the processor; and

[0058] A processor for receiving and displaying the flow rate, pressure, and analysis.

[0059] A composition comprising a fraction of a biological sample extracted from the device of the present disclosure. The composition can be used for treating a human or animal body. The composition can be used for diagnosis or prognosis of a subject.

[0060] A method for preparing a biological sample, the method including separating extracellular matrix bodies from the biological sample. The extracellular matrix bodies can have a size of 0.5 to 5,000 microns, or 1 to 1,000 microns, or 1 to 200 microns, or 4 to 100 microns. Separating the extracellular matrix bodies can be carried out by ultrafiltration or centrifugation. Separating the extracellular matrix bodies can be carried out by the device of the present disclosure.

[0061] The above method further includes immobilizing the extracellular matrix bodies on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

[0062] A method for preparing a biological sample of extracellular matrix bodies by immobilizing the extracellular matrix bodies on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking. Brief Description of the Drawings

[0064] Figure 1 A top view showing an embodiment of the microfluidic chip of the present invention. In this form, a silicon wafer master 101 is printed with three microfluidic channel chip patterns 103. The silicon wafer 101 can be used as a substrate. A photoresist can be poured on the substrate and exposed to ultraviolet light, which forms the pattern of the microfluidic chip 103. The wafer and the photoresist together form a mold, and PDMS can be poured on the mold. Once solidified, the PDMS can be peeled off the mold, and each wafer can give three castings of the microfluidic chip. These castings can be adhered to a glass slide to form the final microfluidic chip.

[0065] Figure 2Shows a top view of the microfluidic chip insert in one embodiment of the device of the present invention. The chip has two confinement channels 203, each 2500 um wide and 25,000 um long in this example. The confinement channels 203 include posts of different diameters and spacings, as shown by the circles. The chip has a third uniform flow channel 205, which has posts of various sizes and spacings that do not significantly restrict flow. The chip has an inlet reservoir 201 and an outlet reservoir 207, which also contain larger posts. The dashed arrows show the direction of flow from the inlet reservoir towards the outlet reservoir.

[0066] Figure 3 Shows corresponding to Figure 2 of the top view. Figure 3 Shows PDMS polymer posts 301 of various sizes represented by circles. The dashed arrows show the flow of biofluid through the three channels.

[0067] Figure 4 Shows corresponding to Figure 2 of the inlet reservoir. Figure 4 Shows posts 401 represented by circles. The dashed arrows show the flow of biofluid through the three channels.

[0068] Figure 5 Shows corresponding to Figure 2 of the inlet reservoir area. Figure 5 Shows posts 501 represented by circles. The dashed arrows show the flow of biofluid through the three channels.

[0069] Figure 6 Shows corresponding to Figure 2 of the channel area. Figure 6 Shows posts 601 represented by circles. The dashed arrows show the flow of biofluid through the channel. The microfluidic channel device of the present invention can have areas of posts or obstacles of different sizes and / or spacings for generating turbulent or restricted flow.

[0070] Figure 7 Shows corresponding to Figure 2 of the enlarged top view of the channel area. Figure 7 Shows posts 701 represented by circles. The dashed arrows show the flow of biofluid through the channel. This view shows the transition from a 50 um gap between the posts to a 25 um gap in the confinement channel.

[0071] Figure 8 Shows corresponding to Figure 2 of the enlarged top view of the channel area. Figure 8The upright posts 801 are shown as circles. The dashed arrows show the flow of the biological fluid through the channels. This view shows the transition in the channels between the upright posts from a larger gap to a smaller gap being restricted.

[0072] Figure 9 Shows the enlarged top view corresponding to Figure 2 the channel area. Figure 9 The upright posts 901 are shown as circles. The dashed arrows show the flow of the biological fluid through the channels.

[0073] Figure 10 Shows the enlarged top view corresponding to Figure 2 the channel area. Figure 10 The upright posts 1001 are shown as circles. The dashed arrows show the flow of the biological fluid through the channels. This view shows the channels in the area with the blunt upright post obstacles 1001, which can generate turbulence.

[0074] Figure 11 Shows the enlarged top view corresponding to Figure 2 the outlet reservoir 1107. Figure 11 The upright posts 1101, 1103, and 1105 of various sizes are shown. The dashed arrows show the flow of the biological fluid through the channels. In this embodiment, each outer restricted channel contains a barrier 1102 formed by very small and closely spaced upright posts.

[0075] Figure 12 Shows the enlarged top view corresponding to Figure 2 the inlet reservoir 1201. Figure 12 The upright posts 1203 of various sizes are shown. The outer restricted channel 1207 includes upright posts of different sizes and spacings. The uniform flow channel 1205 includes upright posts of uniform size and spacing. The dashed arrows show the flow direction of the biological fluid through the outer channels.

[0076] Figure 13 Shows the top view of the microfluidic chip in one embodiment of the device of the present invention. Three microfluidic inserts are shown. The dashed arrows show the flow direction of the biological fluid.

[0077] Figure 14 Shows a perspective view of one embodiment of the microfluidic channel device of the present invention with restricted flow having blunt upright post obstacles 1401. Figure 14 is Figure 15 an enlargement. The dashed arrows show the flow direction of the biological fluid.

[0078] <> Figure 15 Shows a perspective view of one embodiment of the microfluidic channel device of the present invention. Figure 15 Shows the view corresponding to Figure 2 the channel area. Figure 15Show blunt post obstacles 1501 with different intervals in the confinement channel. In this embodiment, the confinement channel can have post obstacles 1501 that are organized into bands with different intervals between the posts. The dashed arrows show the flow direction of the biological fluid.

[0079] Figure 16 A front elevation side view of an embodiment of the microfluidic chip of the present invention is shown. The inlet reservoir 1605 is in fluid communication with the fluid line 1601 for introducing biological fluid and / or other fluids into the reservoir. The fluid line 1601 passes through the probe 1602, the probe adapter 1603, and the hole 1604 defined in the glass coverslip. The biological fluid passes through the inlet reservoir 1605 to reach the microfluidic channel 1606. The dashed arrows show the flow direction of the biological fluid.

[0080] Figure 17 Show corresponding to Figure 2 the inlet region and Figure 16 the enlarged top view of the position of the probe 1602. The dashed arrows show the flow direction of the biological fluid.

[0081] Figure 18 A front elevation side view of an embodiment of the microfluidic chip 1614 of the present invention is shown. The inlet reservoir is in fluid communication with the fluid line 1601 for introducing biological fluid into the reservoir. The fluid line 1601 passes through the probe 1602, the probe adapter 1603, and the hole 1604 defined in the glass coverslip 1613. The biological fluid passes through the inlet reservoir to reach the microfluidic channel 1606 and flows to the outlet reservoir 1607. A probe regulator 1612 can be provided to adjust the height of the probe 1602 to form a good seal with the probe adapter 1603 and the hole 1604. The dashed arrows show the flow direction of the biological fluid.

[0082] Figure 19 Show corresponding to Figure 2 the channel region. Figure 19 Show the posts 1701 represented by circles. For this embodiment, some representative lengths of the post band region in the outer channel are shown in microns.

[0083] Figure 20 Show corresponding to Figure 2 the enlarged top view of the channel region. Figure 20 Show the posts as dots. For this embodiment, some representative lengths of the post band region in the outer channel are shown in microns. The dashed arrows show the flow direction of the biological fluid.

[0084] Figure 21 Show corresponding to Figure 2Top view of an embodiment of a microfluidic device. A biological fluid can be introduced into the inlet region reservoir 2202 with a delivery probe 2201. The dashed arrow shows the direction of the biological fluid flow towards the outlet reservoir region 2203. The enlarged view of this embodiment shows some representative lengths of the post strip region in the external channel, in micrometers. For this embodiment, the dotted line in the enlarged view shows the possible tortuous path of the biological fluid between the obstacles.

[0085] Figure 22 Shows an embodiment of the microfluidic system of the present invention having a processor, a fluid driving unit, a fluid source unit, a sensor unit, an on-chip unit, and an off-chip unit.

[0086] Figure 23 Shows that the aqueous humor from patients with primary open-angle glaucoma increases the pressure in the microfluidic device. Figure 23 Shows the change in the relative pressure (mm Hg) within the artificial trabecular meshwork formed by the posts in the microfluidic channel when injecting human aqueous humor obtained from patients with severe primary open-angle glaucoma. The fluid flow rate is kept constant at 2 μl per minute, and the baseline system pressure is measured using an external pressure sensor. The human aqueous humor sample is injected at the time points indicated by the arrow and the letter "a." The pressure steadily rises to a maximum of about 41 mm Hg at 27 minutes. Figure 23 Shows that the aqueous humor from patients diagnosed with POAG glaucoma increases the pressure in the device.

[0087] Figure 24 (Upper figure) Shows a confocal micrograph of the microfluidic chip after capturing EMB from the human aqueous humor of a patient with primary open-angle glaucoma. The protein of EMB is labeled with the fluorescent marker carboxyfluorescein succinimidyl ester (CFSE, indicated by arrows). The circles are the posts in the confinement channel. Figure 24 (Lower figure) Shows the EMB separated in the microfluidic channel around the posts.

[0088] Figure 25 Illustrates the separation of extracellular matrix vesicles from a biological fluid using a size exclusion filter. Bovine vitreous humor is first filtered through a 5 μm cellulose acetate syringe filter, then through a 1 μm syringe-tip filter, then through a 0.45 μm syringe-tip filter, and then through a 0.22 μm filter. Each fraction is characterized using wide-field microscopy. Figure 25Unenriched bovine vitreous humor 4301 was aspirated into a 1 mL syringe 4305 with a 22g needle 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. The biofluid fractions were collected after each filtration step for light microscopy. Microscopic images showed a substantial decrease as the filtrate was sequentially passed through filters of smaller sizes.

[0089] Figure 26 Representative transmission electron microscopy (TEM) images showing the separation of EMB by size exclusion filters are presented. Figure 26 a and Figure 26 b show the presence of extracellular matrix bodies in the native biofluid of bovine vitreous humor. To separate and recover extracellular matrix bodies from complex biofluids, sequential syringe-based filtration using cellulose filters with pore sizes ranging from 5 μm to 0.22 μm was performed. The extracellular matrix bodies were stained with Alcian blue stain. Figure 26 c shows the bovine vitreous fraction separated by sequential filtration through a 5 μm syringe tip filter. Figure 26 d shows the bovine vitreous fraction separated by sequential filtration through a 1 μm syringe tip filter. Figure 26 e shows the bovine vitreous fraction separated by sequential filtration through a 0.45 μm syringe tip filter. Figure 26 f shows the bovine vitreous fraction separated by sequential filtration through a 0.22 μm syringe tip filter. The images show a relative decrease in larger ECM bodies as the filtrate was sequentially passed through smaller filter sizes.

[0090] Figure 27Illustrated is the separation of extracellular matrix vesicles from biological fluids using centrifugation. Four pellets 9705, 9713, 9721, and 9729 were obtained by sequential centrifugation. Bovine vitreous was resuspended 9701 and placed in a 1 ml tube and centrifuged at 350 g (Sorvall Legend RT) for 10 minutes at 4 °C to form pellet 19705. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 19709, and the remaining supernatant was transferred to a new tube and centrifuged at 2000 g (Eppendorf, 5417R series, F45-30-11 Eppendorf rotor) for 10 minutes at 4 °C to form pellet 29713. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 29717, and the remaining supernatant was transferred to a new tube. Then the supernatant was centrifuged at 10,000 g for 10 minutes at 4 °C to form pellet 39721. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 39725, and the remaining supernatant was transferred to a new tube. Then the supernatant was centrifuged at 20,000 g for 10 minutes at 4 °C to obtain pellet 49729. A 50 μl aliquot of the supernatant was saved for analysis and labeled as supernatant 4, and the remaining supernatant was transferred to a new tube.

[0091] Figure 28 Representative transmission electron microscopy (TEM) images showing the separation of EMB by sequential centrifugation are presented. Figure 28 a shows extracellular matrix vesicles present in bovine vitreous humor. In Figure 28 b, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 450 g shows extracellular matrix vesicles present in the pellet fraction. Similarly, in Figure 28 c, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 2,000 g shows extracellular matrix vesicles present in the pellet fraction. In Figure 28 d, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 10,000 g shows extracellular matrix vesicles present in the pellet fraction. In Figure 28 e, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 20,000 g shows extracellular matrix vesicles present in the pellet fraction.

[0092] Figure 29 Shows the dose-response behavior of the compound bivalirudin TFA on intraocular pressure (IOP) in bovine vitreous humor. The differential pressure was measured using the microfluidic device of the present invention.

[0093] Figure 30Show the dose - response behavior of the compound colistin sulfate on intraocular pressure (IOP) in bovine vitreous humor. Measure the differential pressure using the microfluidic device of the present invention.

[0094] Figure 31 Show the dose - response behavior of the compound polymyxin B sulfate on intraocular pressure (IOP) in a bovine vitreous humor glaucoma model. Measure the differential pressure using the microfluidic device of the present invention.

[0095] Figure 32 Show the separation and extraction of extracellular matrix vesicles in the restricted channels of the microfluidic device of the present disclosure. Figure 32 a and Figure 32 b show representative micrographs of a microfluidic chip perfused with the biological fluid of bovine vitreous humor. The letter "p" marks a post in the channel. After perfusion, extracellular matrix vesicles are separated between and around the posts. Figure 32 c and Figure 32 d show representative micrographs of the channel after extraction of extracellular matrix vesicles. The extracellular matrix vesicles are removed from the chip, showing significantly fewer extracellular matrix vesicles after extraction.

[0096] Figure 33 Show off - chip proteomic analysis of the separated and extracted bovine extracellular matrix vesicles by LC / MS. Biomarkers for the extracellular matrix vesicles are detected.

[0097] Figure 34 Show the separation and subsequent extraction of extracellular matrix vesicles in the restricted channels of the microfluidic device of the present disclosure. The image scale bar is 50 μm. Figure 34 a shows a representative wide - field micrograph (gray signal, bright field) of the microfluidic device perfused with bovine vitreous humor suspended in phosphate - buffered saline at pH 7.0 and counterstained with Alcian blue for hyaluronic acid. Figure 34 a shows the signal from extracellular matrix vesicles (arrows) captured between the posts (p) of the device. The chip is perfused with the biological fluid for at least 60 minutes. After perfusion, aggregates are separated between the posts and are observed as clump - like formations. Substances smaller than the extracellular matrix vesicle material have been discharged through the outlet port. Figure 34 b shows the extraction of extracellular matrix vesicles from the restricted channels of the microfluidic device. The device is perfused with the mild detergent 0.1% sodium dodecyl sulfate (SDS), and the flow direction is reversed from the outlet to the inlet. Figure 34 b shows significantly fewer extracellular matrix vesicles present in the channel after elution, indicating that the extracellular matrix vesicles have been extracted. Figure 34 c shows a higher - magnification image of extracellular matrix vesicles (arrows) captured between the posts (p) after perfusion. Figure 34Panel d shows the extraction with detergent and counterflow, again showing significantly fewer extracellular matrix bodies in the channel after extraction.

[0098] Figure 35 Shows on-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. The microfluidic device is injected with a fluid containing homogenized bovine vitreous suspended in a biological fluid. After perfusing the fluid into the device, the fluid flows through the inlet and out through the outlet. Larger extracellular matrix bodies (arrows) are captured between the posts (labeled Lp). The chip is perfused with a blocking solution to prevent non-specific antibody binding prior to antibody staining. Next, the protein fibronectin, a known extracellular matrix component and integrin-binding protein, is labeled by injecting a primary anti-fibronectin antibody, incubating the sample for 2 hours, and washing. Then, a secondary goat anti-rabbit FITC antibody is incubated for 1 hour and washed. The microfluidic chip is then imaged under wide-field fluorescence and bright-field microscopy. Figure 35 Shows a representative wide-field fluorescence micrograph. This image shows extracellular matrix bodies in the microfluidic channel. The distance between the large posts (Lp) is approximately 100 μm. The punctate signals within the extracellular matrix bodies represent fibronectin staining (anti-fibronectin Ab, secondary goat anti-rabbit antibody with Alexa 488, FITC, white signal).

[0099] Figure 36 Shows on-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. Figure 36 Panel a shows a representative bright-field micrograph of stained extracellular matrix bodies (arrows) in the channel. The image scale bar is 20 μm. The control image has no fluorescence signal, indicating that Figure 36 the signal in a is specific for fibronectin. Figure 36 Panel b again shows stained extracellular matrix bodies (arrows) in the channel. The image scale bar is 50 μm. Again, the control image has no fluorescence signal, indicating that Figure 36 the signal in b is specific for fibronectin.

[0100] Figure 37 Shows on-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. Figure 37Representative micrographs of on-chip immunohistochemical staining of versican protein, a component of the chondrocyte extracellular matrix and a known cancer biomarker, in a biological fluid containing extracellular matrix bodies. The microfluidic chip was injected with a fluid containing homogenized bovine vitreous suspended in the biological fluid. After perfusion of the biological fluid into the device, the sample flowed through the inlet and out through the outlet. Larger extracellular matrix bodies (arrows) were captured between the posts (labeled "p"). The chip was perfused with a blocking solution to prevent non-specific antibody binding prior to antibody staining. Versican was labeled by injecting a versican primary antibody, incubating the sample for 2 hours, and washing. Then, a goat anti-rabbit TRITC secondary antibody was incubated for 1 hour and washed. The microfluidic chip was then imaged under wide-field fluorescence and bright-field microscopy. Figure 37 Shows extracellular matrix bodies in the microfluidic channel between the posts (p). The punctate signals represent versican staining (white signals). The control image has no fluorescent signal, indicating that Figure 37 the signals in

[0101] Figure 38 Show on-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. Figure 38 a Shows a bright-field image of a representative micrograph of on-chip immunohistochemical staining of versican. The control image has no fluorescent signal, indicating that Figure 38 the signals in a are specific for versican. The image scale bar is 10 μm. Figure 38 b Shows a bright-field image of a representative micrograph of on-chip immunohistochemical staining of versican. The control image has no fluorescent signal, indicating that Figure 38 the signals in b are specific for versican. The image scale bar is 10 μm. Figure 38 Also shows that Alcian blue, a marker for hyaluronic acid, can be used as a stain for EMB.

[0102] Figure 39 Show visualization of extracellular matrix bodies on the glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and staining of collagen with Sirius red dye. Figure 39 Show signals (dark staining) of extracellular matrix bodies, which show that EDC crosslinking retains the extracellular matrix bodies on the surface.

[0103] Figure 40 Show visualization of extracellular matrix bodies on the glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and staining of collagen with Sirius red dye. Figure 40Signals of extracellular matrix body collagen chains in vivo (dark staining), which show that EDC crosslinking retains extracellular matrix bodies on the surface. Figure 40 It also shows that Sirius red stain can be used to stain EMB.

[0104] Figure 41 Shows off-chip analysis of extracellular matrix bodies on a glass surface visualizing crosslinking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and staining of DNA with Hoechst dye. Figure 41 Shows DNA signals in extracellular matrix bodies (Hoechst dye, DAPI filter, white signal, DNA).

[0105] Figure 42 Shows on-chip separation and detection of extracellular matrix bodies. Figure 42 Shows a representative micrograph of a microfluidic chip perfused with bovine vitreous humor, suspended in phosphate-buffered saline at pH 7.0 and counterstained with Alcian blue for hyaluronic acid, dark staining, bright field. Figure 42 Shows signals from extracellular matrix bodies captured near the large posts (circles) of the device. The chip was perfused with biological fluid for at least 60 minutes. Extracellular matrix bodies with cluster-like formations were observed. The image scale bar is 50 μm.

[0106] Figure 43 Shows on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 43 Shows a representative low-magnification fluorescence micrograph of a channel after perfusion with extracellular matrix bodies of bovine vitreous humor, which were counterstained with carboxyfluorescein succinimidyl ester (CFSE) for proteins. The microfluidic chip was injected with a fluid containing homogenized bovine vitreous. After the fluid was perfused into the device, the fluid flowed through the inlet and out through the outlet. Larger extracellular matrix bodies were captured near the posts (labeled "p"). The image scale bar is 25 μm.

[0107] Figure 44 Shows on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 44 Shows a representative micrograph of a microfluidic chip perfused with bovine vitreous humor, suspended in phosphate-buffered saline at pH 7.0 and counterstained with Sirius red for collagen, dark staining, bright field. Figure 44 a Shows signals from extracellular matrix bodies between the posts (labeled "p") of the device. The chip was perfused with biological fluid for at least 60 minutes. Figure 44 b Shows the same image with a fluorescence filter and shows collagen detected with Sirius red (light gray signal). The image scale bar is 50 μm. Figure 44 c andFigure 44 d shows similar images at a higher magnification. The image scale bar is 10 μm.

[0108] Figure 45 Shows the frequency size distribution of human extracellular matrix vesicles present in human aqueous humor biofluids from healthy and pre-disease states, suspected glaucoma, and pre-glaucoma. Aqueous humor was obtained from 8 patients, healthy samples, or pre-glaucoma diagnoses, with intraocular pressure ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other means. The size of the extracellular matrix vesicles was determined by cross-linking the samples to slides using the carbodiimide EDC fixative, staining with uranyl acetate, and imaging with wide-field microscopy. Sizes were quantified using an automated program (ImageJ) in all eight samples. The size (area) of the extracellular matrix vesicles ranged from approximately 1.67 μm 2 to approximately 67×10 3 μm 2 . Figure 45 Shows the count of extracellular matrix vesicles in the range of 0 - 200 μm 2 .

[0109] Figure 46 Shows the frequency size distribution of human extracellular matrix vesicles present in human aqueous humor biofluids from healthy and pre-disease states, suspected glaucoma, and pre-glaucoma. Aqueous humor was obtained from 8 patients, healthy samples, or pre-glaucoma diagnoses, with intraocular pressure ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other means. The size of the extracellular matrix vesicles was determined by cross-linking the samples to slides using the carbodiimide EDC fixative, staining with uranyl acetate, and imaging with wide-field microscopy. Sizes were quantified using an automated program (ImageJ) in all eight samples. The size (area) of the extracellular matrix vesicles ranged from approximately 1.67 μm 2 to approximately 67×10 3 μm 2 . Figure 46 Shows the count of extracellular matrix vesicles in the range of 201 - 1000 μm 2 .

[0110] Figure 47 Shows the frequency size distribution of human extracellular matrix vesicles present in human aqueous humor biofluids from healthy and pre-disease states, suspected glaucoma, and pre-glaucoma. Aqueous humor was obtained from 8 patients, healthy samples, or pre-glaucoma diagnoses, with intraocular pressure ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other means. The size of the extracellular matrix vesicles was determined by cross-linking the samples to slides using the carbodiimide EDC fixative, staining with uranyl acetate, and imaging with wide-field microscopy. Sizes were quantified using an automated program (ImageJ) in all eight samples. The size (area) of the extracellular matrix vesicles ranged from approximately 1.67 μm2 to approximately 67×10 3 μm 2 。 Figure 47 shows the count of extracellular matrix bodies in the range of 1001 - 67,000 μm 2 range.

[0111] Figure 48 shows the size distribution of bovine vitreous extracellular matrix bodies separated and extracted from the microfluidic device of the present invention. Extracellular matrix bodies in bovine vitreous humor biofluid after separation and extraction from the microfluidic device of the present invention. The chip was perfused with biofluid for at least 60 minutes. After 60 minutes of perfusion, the ECM bodies were separated near the confinement channel posts. Next, the chip was treated with a detergent (0.1% sodium dodecyl sulfate, SDS) and the sample was extracted from the chip by reverse flow, which caused the extracellular matrix bodies to flow out of the inlet port. Fractions of the eluate were collected at 10 - minute intervals for a total of 80 minutes. The samples were fixed on slides and stained with Alcian blue and imaged by wide - field microscopy. The size was quantified using an automated program (ImageJ). The size (area) of the extracellular matrix bodies can reach up to approximately 16×10 3 μm 2 。 Figure 48 shows the count of extracellular matrix bodies in each eluate fraction, which increases with time. This experiment shows that the microfluidic device of the present invention can be used to separate and extract extracellular matrix bodies of various sizes.

[0112] Figure 49 shows that off - chip analysis of extracellular matrix bodies can be accomplished by retention through cross - linking with 1 - ethyl - 3 - (3 - dimethylaminopropyl) carbodiimide (EDC). Figure 49 a shows a representative TEM image of extracellular matrix bodies from native bovine vitreous humor obtained by cross - linking with EDC. The extracellular matrix bodies were observed with EDC fixation. Figure 49 b shows a similar image taken without EDC cross - linking. Without EDC fixation, no extracellular matrix bodies were observed.

[0113] Figure 50 shows the separation of extracellular matrix bodies from human plasma samples of patients with early pancreatic ductal adenocarcinoma (PDAC) compared to healthy controls. Figure 50 a shows a representative wide - field fluorescence micrograph image of the PDAC sample. Figure 50 a shows extracellular matrix bodies staying in the microfluidic channel. Figure 50a further shows that larger extracellular matrix bodies (arrows) remain between the pillars (labeled "p"). The width between the pillars is approximately 100 μm. The punctate signals from the extracellular matrix bodies that remain represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary Ab with Alexa 488, FITC, white signal). The staining shows abundant signals and punctate staining within the EMB ( Figure 50 a, arrow). Figure 50 b shows similarly obtained fluorescence micrographs of plasma samples from age-matched healthy controls. Figure 50 b shows a significantly reduced amount of fibronectin signal ( Figure 50 b, gray signal, arrow). When processed under the same conditions, the healthy control signal is much smaller than the disease signal. The image scale bar is 20 μm. DETAILED DESCRIPTION OF THE INVENTION

[0115] The present invention discloses devices and systems for separating particles from biological samples by separating and using organisms, materials, and / or molecules, which are applicable to various diseases and conditions. The devices and systems for separating particles from biological samples can be used to increase the level of sample isolates related to the biology of interest. The devices and methods of the present invention can be used to separate important components from biological samples for the diagnosis and prognosis of diseases.

[0116] The devices of the present invention for separating particles from biological samples can be used for various biological materials and samples, including body fluids, blood, tissues, cells, and tumors. In certain embodiments, the methods of the present disclosure can separate relevant fractions of biological materials corresponding to disease states.

[0117] The present disclosure provides devices that can be used to separate important fractions of biological samples and analyze components related to the diagnosis and prognosis of diseases. In some embodiments, a significant amount of biological material and a correspondingly increased signal level can be obtained.

[0118] Aspects of the present invention include separating 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) separated or extracted from biological samples, fluids, or materials, the EMBs can be used for the diagnosis or indication of diseases, or for monitoring chemical or biological processes or changes in sample materials.

[0119] In some aspects, the present invention provides an enhanced ability to detect or characterize extracellular matrix bodies, which can more easily reflect disease states.

[0120] In additional aspects, the devices and methods of the present disclosure can provide increased signals for relevant biomarkers that associate specific biological fractions with disease states. The biomarkers of the present disclosure can be associated with diseases and provide diagnostic tools.

[0121] The devices of the present disclosure can provide improved measurements of flow rate and / or pressure in fluids containing biological components by maintaining a continuous and substantial flow in the device. In some embodiments, when a substantial flow is maintained, the sensor can accurately measure the differential pressure and flow rate. The devices and systems disclosed herein can provide improved measurements of the flow rate and pressure of fluids containing biological components by a channel arrangement that maintains a continuous and substantial flow. In some embodiments, the devices of the present disclosure can have one or more channels that substantially do not restrict the flow of fluid, thereby maintaining a continuous and substantial flow in the system.

[0122] In addition to restricted channels, the devices disclosed herein can also provide improved measurements of the flow rate and pressure of fluids containing biological components by using one or more uniform flow channels that maintain a continuous and substantial flow.

[0123] In certain embodiments, the devices of the present invention will have 1 - 90% of the flow rate from the uniform flow channels, or 1 - 75% of the flow rate from the uniform flow channels, or 1 - 50% of the flow rate from the uniform flow channels, or 1 - 25% of the flow rate from the uniform flow channels.

[0124] In certain embodiments, the devices of the present invention will have at least 25% of the flow rate from the uniform flow channels, or at least 50% of the flow rate from the uniform flow channels, or at least 75% of the flow rate from the uniform flow channels, or at least 90% of the flow rate from the uniform flow channels.

[0125] In some embodiments, extracellular matrix bodies having a diameter size of about 0.5 to about 5,000 microns or greater can stay in the microfluidic channel, or can cause blockage in the channel, thereby increasing the pressure.

[0126] In additional embodiments, extracellular matrix bodies having a diameter size of about 1 to about 200 microns or greater can stay in the microfluidic channel, or can cause blockage in the channel, thereby increasing the pressure.

[0127] In a further aspect, the present disclosure provides devices and methods for separating, detecting, and / or analyzing the ultrastructural components of fluids containing biological materials or molecules. In certain embodiments, the ultrastructural components can be related to diseases.

[0128] Embodiments of the present invention can be used to separate, extract, and utilize extracellular matrix bodies (EMBs), which are sources of various specific biomarkers.

[0129] The present invention includes devices for separating, detecting, and analyzing the composition of extracellular matrix bodies, biological particles, and complexes for various uses.

[0130] In some aspects, extracellular matrix bodies can serve as biomarkers by their morphological characteristics. In further aspects, extracellular matrix bodies can function by containing isolated biochemical markers that may be involved in disease pathways.

[0131] Aspects of the present invention can further provide diagnostic systems that include means for detecting and measuring biomarkers by disease-related extracellular matrix bodies (EMBs) and / or biological particles or complexes.

[0132] In further embodiments, the present disclosure describes methods and devices for preparing and analyzing samples of biological materials.

[0133] Extracellular matrix bodies (EMBs) can be complexes and can be composed of proteins, lipids, carbohydrates, nucleic acid molecules, biological particles, small vesicles such as extracellular vesicles or exosomes, and combinations thereof.

[0134] Samples of biological materials can include body fluids, tissues, and cells.

[0135] Examples of body fluid samples include any body fluid, including whole blood, plasma, blood components, CSF, urine, semen, synovial fluid, pleural fluid, vaginal fluid, gastric fluid, pericardial fluid, peritoneal fluid, amniotic fluid, saliva, nasal fluid, ear fluid, breast milk, and any other body fluid, as well as combinations thereof.

[0136] In further aspects, the microfluidic devices and systems of the present invention can be used to separate and extract biological particles from samples.

[0137] The microfluidic devices and systems of the present invention can be used to purify or separate extracellular matrix bodies or complexes with diameters greater than about 0.5 microns up to particles with diameters of about 5,000 microns, or extracellular matrix bodies or complexes with diameters greater than about 2 microns up to particles with diameters of about 700 microns.

[0138] In further aspects, the microfluidic devices and systems of the present invention can be used to measure the relative viscosities and flow properties of biological and clinical fluids.

[0139] In certain aspects, the microfluidic devices and systems of the present invention can be used to separate and extract disease-related biological particles from samples.

[0140] In some aspects, the microfluidic devices and systems of the present invention can be used to measure intraocular pressure in eye fluid.

[0141] Apparatus and System

[0142] The present invention provides microfluidic devices and systems for measuring pressure and / or flow rate in fluids.

[0143] The present invention improves the measurement of fluid flow rate and pressure by providing a continuous and large flow rate and flow volume, thereby enabling accurate measurement of differential pressure and / or flow rate.

[0144] In a further aspect, the microfluidic devices and systems of the present invention can be used to detect, separate, and extract biological materials or biological particles or other components of fluids.

[0145] The microfluidic devices and systems of the present invention can be used to measure the relative viscosity and flow properties of biological fluids and clinical fluids.

[0146] The microfluidic devices and systems of the present invention can include a microfluidic chip that can be held in a substrate.

[0147] In some aspects, the microfluidic devices of the present invention can have channels with obstacles. The obstacles can affect and / or restrict the flow of fluid in the channels.

[0148] In some aspects, the microfluidic devices of the present invention can have channels with bands of obstacles. The bands of obstacles can span the width of the channel such that the bands of obstacles will affect the flow and flux of fluid along the channel.

[0149] In some embodiments, the spacing between the obstacles in the band can be constant. The spacing between the obstacles in the channel can be used to control the window size for fluid flow in the channel.

[0150] In additional embodiments, the microfluidic devices of the present invention can have channels with multiple continuous bands of obstacles. In certain embodiments, the spacing between the obstacles in the band can decrease in one direction along the length of the channel in multiple bands. Thus, the spacing between the obstacles in the band can increase in the opposite direction along the length of the channel in multiple bands.

[0151] For example, the microfluidic device of the present invention can have a restricted channel where multiple bands arranged in sequence in one direction have a spacing of 1,000 microns between the obstacles, which is adjacent to a band with a spacing of 500 microns, which is adjacent to a band with a spacing of 200 microns, which is adjacent to a band with a spacing of 100 microns, which is adjacent to a band with a spacing of 50 microns, which is adjacent to a band with a spacing of 25 microns, which is adjacent to a band with a spacing of 10 microns, which is adjacent to a band with a spacing of 4 microns.

[0152] In a further aspect, the microfluidic devices of the present invention can have channels with bands of obstacles, where the spacing between the obstacles in one band is the smallest. The band with the smallest spacing between the obstacles can be a barrier band.

[0153] In another aspect, the microfluidic device of the present invention may have a confinement channel, wherein the barrier band has a spacing between obstacles of at least 1 micron, 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 microns.

[0154] In certain aspects, the microfluidic device of the present invention may be a chip having a height of 7 to 25 microns.

[0155] In operation, the microfluidic devices and systems of the present invention can be used to separate, extract, and / or purify biological particles. In certain embodiments, the confinement channel may have a band with a gradually decreasing spacing between obstacles, where one band is a barrier band. The methods of the present invention can use such a band arrangement to separate larger particles from smaller particles and fluid by flowing a fluid containing the particles from an inlet in a direction of gradually decreasing spacing towards an outlet of the channel. In these methods, the larger particles can be separated and retained along the channel, while the smaller particles and fluid exit the channel at the outlet. The larger particles can be extracted from the channel at the inlet by reversing the flow direction of the extraction fluid to extract the larger particles from the inlet.

[0156] In operation, the microfluidic devices and systems of the present invention can be used to separate, extract, and / or purify biological particles that are extracted from the channel at the outlet.

[0157] In some embodiments, the larger particles can be extracted from the channel at the outlet by adding a detergent to break up the larger particles.

[0158] Figure 1 A top view showing an embodiment of the microfluidic chip of the present invention. In this form, a silicon wafer master 101 is printed with three microfluidic channel chip patterns 103. The silicon wafer 101 can be used as a substrate. A photoresist can be poured onto the substrate and exposed to UV light, which forms the pattern of the microfluidic chip 103. The wafer and the photoresist together form a mold onto which PDMS can be poured. Once solidified, the PDMS can be peeled off the mold, and each wafer can give three castings of the microfluidic chip. These castings can be adhered to a glass slide to form the final microfluidic chip.

[0159] The microfluidic chip of the present invention may have channels for confining fluid flow, as well as inlets and outlets for fluid flow. A pump can be used to apply a head pressure to the fluid at the inlet. In some embodiments, a reduced pressure or vacuum pressure can be used at the outlet to regulate the flow rate.

[0160] Figure 2Shows a top view of a microfluidic chip insert in one embodiment of the device of the present invention. The chip has two confinement channels 203, each 2,500 um wide and 25,000 um long in this example. The confinement channels 203 include posts of different diameters and spacings, as shown by the circles. The chip has a third uniform flow channel 205, which has posts 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 posts. The dashed arrows show the direction of flow from the inlet reservoir to the outlet reservoir. The confinement channels can have a point of maximum flow restriction, which is a flow restriction barrier 202. The barrier 202 can restrict flow and change the pressure in the channels and the system, such that the differential pressure and / or flow rate can be related to the composition of the fluid.

[0161] The microfluidic chip of the present invention can have one or more channels for restricting fluid flow, and one or more uniform or continuous flow channels. In some embodiments, the uniform flow channels do not restrict the fluid flow in the channels. The uniform continuous flow channels can include blunt obstacles for generating turbulence and / or a tortuous path for causing fluid flow.

[0162] Figure 3 Shows corresponding to Figure 2 of the top view. Figure 3 Shows PDMS polymer posts 301 of various sizes represented by circles. The dashed arrows show the flow of biofluid through the three channels.

[0163] In some embodiments, blunt or non-blunt obstacles can be provided in the confinement fluid channels to create a tortuous or eddy pattern of flow in certain regions. The obstacles in the channels can be formed as posts in a circular or other shape.

[0164] In some embodiments, the obstacles in the confinement channels can provide a Reynolds number greater than 500, or greater than 1000, or greater than 10,000, or even larger.

[0165] In additional embodiments, the continuous uniform flow channels can be located between various confinement channels. In some embodiments, the uniform flow channels and the confinement channels can have any arrangement order and be used in any number.

[0166] Figure 4 Shows corresponding to Figure 2 of the inlet reservoir. Figure 4 Shows posts 401 represented by circles. The dashed arrows show the flow of biofluid through the three channels.

[0167] Figure 5 Shows corresponding to Figure 2Top view of the inlet reservoir region. Figure 5 The posts 501 are shown as circles. The dashed arrows show the flow of the biological fluid through the three channels.

[0168] Figure 6 Shows corresponding to Figure 2 Top view of the channel region. Figure 6 The posts 601 are shown as circles. The dashed arrows show the flow of the biological fluid through the channel. The microfluidic channel device of the present invention has regions of posts or obstacles with different spacings and / or sizes, creating turbulence or restricted flow.

[0169] In certain embodiments, the microfluidic channel device of the present disclosure may have a region that mimics the trabecular meshwork of the eye.

[0170] The device of the present invention may include a reticular structure composition containing extracellular matrix bodies or complexes. The extracellular matrix bodies or complexes for the reticular structure composition can be extracted or purified from glaucomatous eye fluid. The eye fluid can be from an animal or clinical source.

[0171] In a further embodiment, the microfluidic chip of the present invention may have one or more channels for restricting fluid flow and one or more uniform flow channels. The uniform flow channels may include blunt obstacles for creating turbulence and / or tortuous paths for fluid flow.

[0172] In a further embodiment, the microfluidic chip of the present invention may have 1 - 20 channels for restricting fluid flow and 1 - 10 uniform flow channels, which are arranged on the substrate in any order. The uniform flow channels can be distributed relative to the restricting flow channels in any manner.

[0173] In certain embodiments, the uniform flow channels can alternate in a collinear or parallel position relative to the restricting flow channels. In additional embodiments, the uniform flow channels can be above or below the restricting flow channels. In some embodiments, the uniform flow channels can be arranged in a substrate separate from the chip containing the restricting flow channels.

[0174] In a further embodiment, the uniform flow channels can provide fluid communication from the inlet reservoir to the outlet reservoir. In certain embodiments, the uniform flow channels can provide fluid communication from the outlet reservoir to the fluid source entering the inlet reservoir.

[0175] In certain embodiments, the total cross-sectional area of the uniform flow channels can be greater than or less than the total cross-sectional area of the restricting flow channels in the microfluidic device of the present invention. In various embodiments, the uniform flow channels may not include obstacles and may not have a tortuous fluid flow. In such embodiments, the uniform flow channels may have laminar or turbulent fluid flow.

[0176] The microfluidic chip of the present invention may have one or more confinement channels for restricting fluid flow. The restricted flow may be attributed to various arrangements of blunt or non-blunt obstacles or posts in the channels. In some embodiments, the posts may assume the shape of the flowing fluid, such as circular, spherical, triangular, square, polygonal, rhomboidal, fin-shaped, and combinations thereof.

[0177] Figure 7 Showing a magnified top view of the channel region corresponding to Figure 2 thereof. Figure 7 The posts 701 are shown as circles. The dashed arrows show the flow of biofluid through the channel. This view shows the transition from a 50-μm gap between the posts to a 25-μm gap in the confinement channel.

[0178] Figure 8 Showing a magnified top view of the channel region corresponding to Figure 2 thereof. Figure 8 The posts 801 are shown as circles. The dashed arrows show the flow of biofluid through the channel. This view shows the transition from a larger gap to a smaller gap between the posts in the confinement channel.

[0179] Figure 9 Showing a magnified top view of the channel region corresponding to Figure 2 thereof. Figure 9 The posts 901 are shown as circles. The dashed arrows show the flow of biofluid through the channel.

[0180] In a further embodiment, the restricted flow in the channel may be attributed to various arrangements of blunt or non-blunt obstacles or posts in the channel, where the size and spacing of the obstacles vary with the distance along the channel.

[0181] In certain embodiments, the size and / or spacing of the blunt or non-blunt obstacles or posts in the confinement channel may change with the distance along the channel. The size and / or spacing of the blunt or non-blunt obstacles may decrease with the distance along the channel. At certain positions in the confinement channel, the size and / or spacing of the blunt or non-blunt obstacles may be reduced to a level that provides maximum flow restriction or barrier.

[0182] Figure 10 Showing a magnified top view of the channel region corresponding to Figure 2 thereof. Figure 10 The posts 1001 are shown as circles. The dashed arrows show the flow of biofluid through the channel. This view shows the channel having a region of blunt post obstacles 1001, which may generate turbulence.

[0183] Figure 11 Showing a magnified top view of the channel region corresponding to Figure 2An enlarged top view of the outlet reservoir 1107. Figure 11 Shows posts 1101, 1103, and 1105 of various sizes. The dashed arrows show the flow of biological fluid through the channels. In this embodiment, each outer confinement channel includes a barrier 1102 formed by very small and closely spaced posts.

[0184] In a further embodiment, various arrangements of blunt or non - blunt obstacles or posts in the confinement channels can be used to restrict the flow to any level. A wide range of spacings and / or patterns of blunt and / or non - blunt obstacles can be used in the confinement channels. The fluid can have a tortuous path in the restricted flow channels. The spacing of the obstacles in the confinement channels and / or the tortuosity of the fluid path can increase with distance along the channel in the flow direction.

[0185] The fluid effluent from the channels of the microfluidic chip of the present invention can be collected in an outlet reservoir at the channel outlet end. The flow of fluid into or into the channels of the microfluidic chip of the present invention can be achieved through a reservoir at the channel inlet end.

[0186] Figure 12 Shows corresponding to Figure 2 An enlarged top view of the inlet reservoir 1201. Figure 12 Shows posts 1203 of various sizes. The outer confinement channel 1207 includes posts of different sizes and spacings. The uniform flow channel 1205 includes posts of uniform size and spacing. The dashed arrows show the direction of biological fluid flow through the outer channel.

[0187] Figure 13 Shows a top view of a microfluidic chip in an embodiment of the device of the present invention. Three microfluidic inserts are shown. The dashed arrows show the direction of biological fluid flow.

[0188] Figure 14 Shows a perspective view of an embodiment of a microfluidic channel device of the present invention with blunt post obstacles 1401 for restricted flow. Figure 14 Is Figure 15 An enlarged view. The dashed arrows show the direction of biological fluid flow.

[0189] Figure 15 Shows a perspective view of an embodiment of the microfluidic channel device of the present invention. Figure 15 Shows corresponding to Figure 2 A view of the channel area. Figure 15 Shows post obstacles 1501 with different spacings in the confinement channel. In this embodiment, the confinement channel can have post obstacles 1501 that are organized into bands of different spacings between the posts. The dashed arrows show the direction of biological fluid flow. The continuous uniform flow channel 1517 can be arranged separately from the confinement channel 1515.

[0190] Figure 16 Shows an elevation side view of an embodiment of the microfluidic chip of the present invention. The inlet reservoir 1605 is in fluid communication with the fluid line 1601 for introducing biological fluid and / or other fluids into the reservoir. The fluid line 1601 passes through the probe 1602, the probe adapter 1603, and the hole 1604 defined in the glass cover slip. The biological fluid reaches the microfluidic channel 1606 through the inlet reservoir 1605. The dashed arrow shows the flow direction of the biological fluid.

[0191] Figure 17 Shows corresponding to Figure 2 the inlet region and Figure 16 the enlarged top view of the position of the probe 1602. The dashed arrow shows the flow direction of the biological fluid.

[0192] Figure 18 Shows an elevation side view of an embodiment of the microfluidic chip 1614 of the present invention. The inlet reservoir is in fluid communication with the fluid line 1601 for introducing biological fluid into the reservoir. The fluid line 1601 passes through the probe 1602, the probe adapter 1603, and the hole 1604 defined in the glass cover slip 1613. The biological fluid reaches the microfluidic channel 1606 through the inlet reservoir and flows to the outlet reservoir 1607. A probe regulator 1612 can be provided to adjust the height of the probe 1602 to form a good seal with the probe adapter 1603 and the hole 1604. The dashed arrow shows the flow direction of the biological fluid.

[0193] Figure 19 Shows corresponding to Figure 2 the enlarged top view of the channel region. Figure 19 Shows the posts 1701 represented by circles. For this embodiment, some representative lengths of the post regions in the channel are shown in microns.

[0194] Figure 20 Shows corresponding to Figure 2 the micrograph of the enlarged top view of the channel region. Figure 20 The posts are shown as dots. For this embodiment, some representative spacings of the posts in the band in the channel are shown in microns. The dashed arrow shows the flow direction of the biological fluid.

[0195] Figure 21 Shows corresponding to Figure 2 the top view of an embodiment of the microfluidic device. Figure 21It is shown that a biological fluid can be introduced into the inlet zone reservoir 2202 using a delivery probe 2201. The dashed arrows indicate the direction of the biological fluid flow towards the outlet reservoir zone 2203. An enlarged view of this embodiment shows some representative spacings of the mid-posts in the channels, in micrometers. For this embodiment, the dotted lines in the enlarged view show the possible tortuous paths of the biological fluid between the obstacles.

[0196] Figure 22 An embodiment of the microfluidic system of the present invention is shown. The processor 102 can send control signals and / or receive signals from the fluid driving unit 101, which supplies driving fluid, such as compressed gas, to the fluid source unit 103. The fluid source unit 103 can contain a fluid of interest, a biological fluid, a carrier, and / or a reagent. The fluid of interest, biological fluid, carrier, and / or reagent can flow towards the sensor unit 105, which can monitor the flow rate and / or pressure of the fluid. The fluid of interest, biological fluid, carrier, and / or reagent can flow towards the on-chip unit 107, which can include the microfluidic device of the present invention. The fluid of interest, biological fluid, carrier, and / or reagent can enter the inlet reservoir of the microfluidic chip of the on-chip unit 107. The fluid of interest, biological fluid, carrier, and / or reagent can reach the outlet reservoir of the microfluidic chip of the on-chip unit 107 and flow towards the off-chip unit 109. The processor 102 can receive data from the sensor unit 105 and record the flow rate and / or pressure. The on-chip unit 107 can include analytical tools for spectrometry, such as irradiation and a light detector. The off-chip unit 109 can include various analytical tools, such as microscopy tools, imagers and analyzers, chromatographs, mass spectrometers, 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.

[0197] In some aspects, the fluid composition in the system or device of the present invention can be analyzed by various techniques. For example, the fluid composition can be analyzed by imaging techniques.

[0198] Examples of imaging techniques include electron microscopy, stereomicroscopy, wide-field microscopy, polarization microscopy, phase-contrast microscopy, multiphoton microscopy, differential interference contrast microscopy, fluorescence microscopy, laser scanning confocal microscopy, multiphoton excitation microscopy, x-ray microscopy, and ultrasonic microscopy.

[0199] Examples of imaging techniques include positron emission tomography, computed tomography, and magnetic resonance imaging.

[0200] Examples of assay techniques include colorimetric assays, chemiluminescent assays, spectrophotometry, immunofluorescence assays, and light scattering.

[0201] In some embodiments, the present invention can provide an apparatus for measuring the pressure and flow rate of a fluid composition. In certain embodiments, the apparatus may have a reticular structure composition that stays in the channel to provide flow resistance. The reticular structure composition can have any one or more of a uveal reticular structure, a corneoscleral reticular structure, and a juxtacanalicular reticular structure. For example, these reticular structures can be simulated with obstacles in the channel or provided from the extraction of ocular fluid, body fluid, or clinical samples.

[0202] The extracellular matrix bodies or complexes for the reticular structure composition can be composed of various biomolecules or composite particles, and the diameter range can be from about 0.5 to about 5,000, 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 microns.

[0203] The diameter range of the extracellular matrix bodies or complexes separable in the apparatus of the present invention is 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 microns.

[0204] In some embodiments, the channel may include obstacles, such as glass beads, microbeads, magnetic beads, gel particles, dextran particles, or polymer particles. The obstacles can also be composed of glass fibers, polymer fibers, inorganic fibers, organic fibers, or metal fibers.

[0205] In further embodiments, the apparatus or channel of the present invention may include a binder, an affinity detector, or an immunizing agent attached to a device element in fluid communication with the sample fluid. The apparatus may have a reagent for internal capture and / or detection of biomolecules from the sample. In certain embodiments, the apparatus may have a reagent for internal capture and / or detection of biomarkers of the sample fluid.

[0206] In certain embodiments, the uveal reticular structure or the restricted channel may have a fenestration of about 25 microns. The corneoscleral reticular structure or the restricted channel may have a fenestration of about 2 - 15 microns. The juxtacanalicular reticular structure or the restricted channel may have a fenestration of about 1 to 4 microns or less.

[0207] The apparatus may further include a fluid reservoir for containing the fluid composition such that the fluid reservoir is in fluid communication with the inlet of the channel to introduce the fluid composition into the inlet of the channel.

[0208] The apparatus of the present disclosure may have a driving source or a pressure source for applying pressure to the driving fluid composition. The driving fluid can enter the fluid reservoir to drive the fluid composition into the inlet of the microfluidic channel.

[0209] The device of the present invention may have a sensor unit in fluid communication with the fluid composition for measuring the flow rate and pressure of the fluid composition at the channel inlet and transmitting the flow rate and pressure to the processor.

[0210] Signals and data from the system device unit 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.

[0211] The processor of the present disclosure can be a general-purpose or special-purpose computer. The processor can execute instructions stored in a machine-readable storage device or medium. The processor can include an integrated circuit chip, a microprocessor, a controller, a digital signal processor, any of which can be used to receive and / or transmit data and execute the stored instructions. The processor can also perform calculations and transform data, and / or store data in a memory, medium or file. The processor can receive and execute instructions, which can include one or more steps of performing the method of the present invention. The device of the present invention can 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 can have an integrated display for displaying data or transforming data.

[0212] In some aspects, the system of the present disclosure can have a device with a microfluidic channel. One or more channels can be arranged in a microfluidic chip.

[0213] The system of the present disclosure can include an on-chip unit having one or more detectors for analyzing the fluid composition within the channel or at the channel inlet or exiting the outlet. The detector can also be arranged to detect the fluid composition within the channel.

[0214] The system 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.

[0215] In certain embodiments, the extracellular matrix body or complex of the reticular structure composition for use in the system or device of the present disclosure can include a fixing agent, a stabilizing component or a cross-linking component, which can transform the structure into a stable and uniform composition.

[0216] Examples of stabilizing components include the fixing agents described herein, the cross-linking compounds described herein, organic solvents, polypeptides, and pharmaceutically acceptable organic salts.

[0217] The cross-linked extracellular matrix body or complex can be reversibly cross-linked or irreversibly cross-linked.

[0218] In some embodiments, the devices of the present invention may comprise an extracellular matrix body or complex as a reticular structure composition that can be used to identify or screen for active agents. The reticular structure composition may include a drug delivery excipient.

[0219] In additional embodiments, the devices of the present invention can be used to measure the quantity or level of an extracellular matrix body or complex in a test sample. Measuring the quantity or level of an extracellular matrix body or complex in a test sample can provide a diagnostic marker level for the test sample. The devices of the present invention can be used to identify glaucoma or pre-glaucoma in a subject.

[0220] In further embodiments, the devices of the present invention can be used to measure the pressure associated with the quantity or level of an extracellular matrix body or complex in a test sample. The pressure value in the channel can be directly related to the quantity or level of an extracellular matrix body or complex in the test sample.

[0221] In certain embodiments, the devices of the present invention can be used to measure a measured value that can be associated with the quantity or level of an extracellular matrix body or complex in a test sample. The measured value of the composition in the channel can be directly related to the quantity or level of an extracellular matrix body or complex in the test sample.

[0222] Examples of assays include colorimetric assays, chemiluminescent assays, spectrophotometric assays, immunoassays, or light scattering assays.

[0223] Apparatus for analyzing a sample in a microfluidic device includes analytical tools such as an irradiation source and a light detector for spectrometry and spectroscopy, as well as immuno-labeling and detection, as further shown in the examples herein.

[0224] Apparatus for analyzing a sample in a microfluidic device includes imaging tools such as an irradiation source and microscopy, as further shown in the examples herein.

[0225] Extracted Composition and Method

[0226] In some embodiments, the composition may comprise a fraction of a biological sample extracted from a microfluidic device.

[0227] In certain embodiments, a composition extracted from a microfluidic device can be used to treat a human or animal body.

[0228] In additional embodiments, a composition extracted from a microfluidic device can be used for the diagnosis or prognosis of a subject.

[0229] A composition of isolated and / or extracted extracellular matrix bodies can be combined with a drug carrier and one or more drug excipients.

[0230] The morphology of the isolated and / or extracted extracellular matrix vesicles can be modified by the isolation and / or extraction process.

[0231] The morphology of the isolated and / or extracted extracellular matrix vesicles can be chemically modified.

[0232] In some embodiments, a composition of extracellular matrix vesicles can be isolated and / or extracted for use in treating a human or animal body.

[0233] In further embodiments, the composition can comprise a sample from which extracellular matrix vesicles have been removed by an isolation and / or extraction process for use in treating a 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 vesicles of the sample have been removed by the isolation and / or extraction process for use in treating a human or animal body.

[0234] In some aspects, extracting a composition from a microfluidic device can be a method for preparing a biological sample for diagnosis or prognosis in a subject.

[0235] In certain embodiments, methods for isolating extracellular matrix vesicles can be by ultrafiltration or centrifugation or by the microfluidic devices of the present disclosure.

[0236] Embodiments of the present invention further include immobilizing extracellular matrix vesicles on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

[0237] For all purposes, all publications cited in this specification, including patents, patent application publications, and non-patent publications, are hereby expressly incorporated by reference in their entirety.

[0238] Although the foregoing disclosure has been described in detail for purposes of clarity of understanding by way of example, it will be apparent to those skilled in the art that certain changes and modifications are covered within the present disclosure and can be practiced within the scope of the appended claims, which are presented by way of illustration and not limitation. The present invention includes all such additional embodiments, equivalents, and modifications. The present invention includes any combination or mixture of the various exemplary components, examples, and features, materials, elements, or limitations of the claimed embodiments.

[0239] The terms "a / an", "the", and similar terms as used in the description of the present invention and the claims should be construed to include the singular and the plural. Examples

[0240] Example 1. Pressure and flow rate measurements were performed in a microfluidic device using a disease-related biological fluid. Extracellular matrix vesicles were isolated in the microfluidic device.Figure 23 The aqueous humor from patients with primary open-angle glaucoma was shown to increase the pressure in the microfluidic device. Figure 23 Shows the change in relative pressure (mm Hg) within the trabecular meshwork of the microfluidic model when injecting human aqueous humor obtained from patients with severe primary open-angle glaucoma. The microfluidic channel flow rate was kept constant at 2 μl per minute, and the baseline system pressure was measured using an external pressure sensor. Human aqueous humor samples were injected at the time points indicated by the arrow and the letter "a". The pressure steadily rose to a maximum of approximately 41 mm Hg at 27 minutes. Figure 23 The aqueous humor from patients diagnosed with POAG glaucoma was shown to increase the pressure in the device.

[0241] Example 2. Isolation of disease-related extracellular matrix bodies in a microfluidic device. Figure 24 (Upper panel) Shows a confocal micrograph of the microfluidic chip after isolation of EMB from human aqueous humor of a patient with primary open-angle glaucoma at the end of the experiment shown in Figure 23 The protein content in the aqueous humor was labeled with the fluorescent marker carboxyfluorescein succinimidyl ester (arrow). The circles are the posts in the restriction channel. Figure 24 (Lower panel) Shows the EMB isolated in the microfluidic channel being captured between the posts (arrow). Figure 24 (Lower panel) Shows the EMB isolated in the microfluidic channel being captured between the posts (arrow).

[0242] Example 3. Isolation of extracellular matrix bodies from biological fluids using size exclusion filters. This example demonstrates that EMB can be isolated by size exclusion. EMB can be separated by size exclusion and distinguished from smaller particles (such as free extracellular vesicles or other small vesicles).

[0243] Figure 25 Illustrates the isolation of extracellular matrix bodies from biological fluids using size exclusion filters. Bovine vitreous humor was first filtered through a 5 μm cellulose acetate syringe filter, then through a 1 μm needle filter, then through a 0.45 μm needle filter, and then through a 0.22 μm filter. Each fraction was characterized using wide-field microscopy.

[0244] Figure 26 Shows representative wide-field microscopy images of the separation of EMB by size exclusion filters. Figure 26 a and Figure 26 b show the presence of extracellular matrix bodies in the native biological fluid of bovine vitreous humor. To isolate and recover extracellular matrix bodies from complex biological fluids, sequential syringe-based filtration using cellulose filters with pore sizes ranging from 5 μm to 0.22 μm was used. The extracellular matrix bodies were fixed on slides with EDC and stained with Alcian blue stain. Figure 26Panel c shows bovine vitreous fractions separated by filtration through a 5-μm syringe filter series. Figure 26 Panel d shows bovine vitreous fractions separated by filtration through a 1-μm syringe filter series. Figure 26 Panel e shows bovine vitreous fractions separated by filtration through a 0.45-μm syringe filter series. Figure 26 Panel f shows bovine vitreous fractions separated by filtration through a 0.22-μm syringe filter series. The images show that as the filtrate series passes through smaller filter sizes, the larger ECM bodies are relatively reduced.

[0245] This example demonstrates that EMB can be separated by size exclusion. Analysis of the separated EMB shows that it consists of DNA, RNA, and proteins, as well as hyaluronic acid or collagen, which are components of the extracellular matrix.

[0246] Vitreous humor is a highly hydrated tissue, with a water content between 98 - 99.7%, and is mainly composed of the extracellular matrix. The main component of the extracellular matrix is the protein collagen. Collagen is modified with carbohydrates and, once released from cells, assembles into collagen fibrils. Extracellular matrix bodies can attach to the fibrils, as well as other locations within the extracellular matrix.

[0247] Anesthetize the bovine eye to remove orbital fat and extraocular muscles attached to the eyeball. At 4 °C, rinse the eyeball for 1 minute with 5 ml of ice-cold Tris-buffered saline (TBS) containing 50 mM Tris-HCl, 150 mM NaCl (pH 8.0). Separate the vitreous humor by making a sclerotomy incision 4 or 8 mm posterior to the limbus using a 16-gauge needle and then cutting a circumferential sagittal incision with scissors to divide the eyeball into an anterior cup and a posterior cup. Use scissors to cut and remove the formed vitreous humor and sever the adhesions between the vitreous humor and the eye structures. Rinse the tissue samples with TBS (pH 8.0) for 1 minute at 4 °C. Collect the vitreous specimens in 15 mL centrifuge tubes and homogenize using an immersion blender. Transfer aliquots of the homogenized bovine vitreous humor (BVH) to 1 mL centrifuge tubes. Resuspend the bovine vitreous humor in TBS buffer for further study and freeze at -80 °C until use.

[0248] Load aliquots of homogenized bovine vitreous humor diluted to 1 mL with buffered saline into 1 mL syringes using a 22-gauge needle. Replace the needle with a 5 μm cellulose acetate syringe filter and extrude the bovine vitreous humor through the filter by applying uniform downward pressure. Collect the filtrate into a new 1 mL tube and save an aliquot of 80 μL of the filtrate for imaging. Then, following the same steps as above, load the filtrate collected from the 5 μm filtration into a 1 μm tip filter and save an aliquot for imaging. Next, following the same procedure, extrude the filtrate from the 1 μm filter through a 0.45 um tip filter and save an aliquot for imaging. Finally, extrude the filtrate from the 0.45 μm filter through a 0.22 μm filter. Recover the filtrate from each filtration step.

[0249] Use a wide-field microscope to visualize the components of each fraction. Image each sample by placing the biological fluid on a slide, crosslinking the sample with EDC, and staining the material containing hyaluronic acid with Alcian blue. For staining of the samples, incubate each filtrate with 1% Alcian blue (Sigma 1% Alcian blue in 3% acetic acid pH 2.5 B8483) at a 1:1 v / v ratio for 30 minutes at room temperature. After incubation, place 40 μL of the stained filtrate on a slide and cover with a coverslip, and image using bright-field microscopy. Capture color bright-field images on an inverted phase contrast microscope (Ziess Axiovert 200) equipped with an Axiocam 105 color camera (Zeiss) and process the images using Zen software (Zeiss, version 4.3).

[0250] Example 4. Isolation of extracellular matrix bodies from biological fluids using sequential centrifugation. This example demonstrates that EMBs can be separated by centrifugation. EMBs can be separated by centrifugation and distinguished from smaller particles such as free exosomes or other small vesicles. To obtain a cell-free vitreous sample, first clarify the vitreous by a series of low-speed centrifugations.

[0251] Figure 27Illustrated is a method for separating extracellular matrix vesicles from biological fluids using centrifugation. Four pellets 9705, 9713, 9721, and 9729 are obtained by sequential centrifugation. Bovine vitreous is resuspended 9701 and placed in a 1 ml tube and centrifuged at 350 g (Sorvall Legend RT) for 10 minutes at 4 °C to form pellet 19705. A 50 μl aliquot of the supernatant is saved for analysis and labeled as supernatant 19709, and the remaining supernatant is transferred to a new tube and centrifuged at 2000 g (Eppendorf, 5417R series, F45 - 30 - 11 Eppendorf rotor) for 10 minutes at 4 °C to form pellet 29713. A 50 μl aliquot of the supernatant is saved for analysis and labeled as supernatant 29717, and the remaining supernatant is transferred to a new tube. Then the supernatant is centrifuged at 10,000 g for 10 minutes at 4 °C to form pellet 39721. A 50 μl aliquot of the supernatant is saved for analysis and labeled as supernatant 39725, and the remaining supernatant is transferred to a new tube. Then the supernatant is centrifuged at 20,000 g for 10 minutes at 4 °C to obtain pellet 49729. A 50 μl aliquot of the supernatant is saved for analysis and labeled as supernatant 4, and the remaining supernatant is transferred to a new tube.

[0252] Figure 28 Representative transmission electron microscopy (TEM) images showing the separation of EMB by sequential centrifugation are presented. Figure 28 a shows extracellular matrix vesicles present in bovine vitreous humor. In Figure 28 b, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 450 g shows extracellular matrix vesicles present in the pellet fraction. Similarly, in Figure 28 c, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 2,000 g shows extracellular matrix vesicles present in the pellet fraction. In Figure 28 d, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 10,000 g shows extracellular matrix vesicles present in the pellet fraction. In Figure 28 e, a representative TEM micrograph of a sample collected from the pellet after centrifugation at 20,000 g shows extracellular matrix vesicles present in the pellet fraction.

[0253] Example 5. Dose - response for detecting the activity of a drug in reducing intraocular pressure in a glaucoma model in a microfluidic device. To use bivalirudin TFA as an active agent for treating glaucoma, the dose - response behavior of bivalirudin TFA on intraocular pressure (IOP) was determined. Bivalirudin TFA exhibited an EC50 of 1.2 nM for treating bovine vitreous humor.

[0254] The compound bivalirudin TFA was tested in bovine vitreous humor (BVH) in a microfluidic chip device. A PBS buffer solution of 25% homogenized BVH was prepared and diluted with an equal volume of the compound solution to give a total BVH concentration of 12.5%. The samples were vortexed and incubated at 37 °C for 1 hour. PBS buffer or PBS containing 10% ethanol or DMSO was used as a control and incubated with BVH under the same conditions.

[0255] The test compound - BVH solution was introduced into the reservoir of the microfluidic chip device and the flow rate and pressure changes were recorded. The effects of various concentrations of the compound on treating bovine vitreous humor were tested. 7 μl of each test solution was injected into the microfluidic chip by a syringe. After sample injection, the flow rate and pressure changes were recorded for an additional 50 minutes. The relative change in chip pressure during the entire experiment was obtained.

[0256] Figure 29 A dose - response curve for treating bovine vitreous humor with the compound bivalirudin TFA was shown. The EC50 value was taken as the point on the logarithmic function of the compound micromolar concentration on the x - axis that produced half - maximal response. The logarithmic function of the drug micromolar concentration was plotted on the x - axis and the percentage of maximal response was plotted on the y - axis. The maximal response was obtained by taking the value of the response to the highest drug concentration. The response was calculated by taking the absolute difference between the control value and the test value at each concentration.

[0257] Example 6. Dose - response for detecting the intraocular pressure - lowering activity of an agent in a glaucoma model in a microfluidic device. To use colistin sulfate as an active agent for treating glaucoma, the dose - response behavior of colistin sulfate on intraocular pressure (IOP) was determined. Colistin sulfate exhibited an EC50 of 0.36 nM for treating bovine vitreous humor.

[0258] The compound colistin sulfate was tested in bovine vitreous humor (BVH) in a microfluidic chip device. A PBS buffer solution of 25% homogenized BVH was prepared and diluted with an equal volume of the compound solution to give a total BVH concentration of 12.5%. The samples were vortexed and incubated at 37 °C for 1 hour. PBS buffer or PBS containing 10% ethanol or DMSO was used as a control and incubated with BVH under the same conditions.

[0259] The test compound - BVH solution was introduced into the reservoir of the microfluidic chip device and the flow rate and pressure changes were recorded. The effects of various concentrations of the compound on treating bovine vitreous humor were tested. 7 μl of each test solution was injected into the microfluidic chip by a syringe. After sample injection, the flow rate and pressure changes were recorded for an additional 50 minutes. The relative change in chip pressure during the entire experiment was obtained.

[0260] Figure 30Shows the dose - dependent response curve of treating the bovine vitreous humor glaucoma model with the compound colistin sulfate. The EC50 value is taken as the point on the logarithmic function of the micromolar concentration of the compound on the x - axis that produces half - maximal response. The logarithmic function of the drug micromolar concentration is plotted on the x - axis, and the percentage of the maximal response is plotted on the y - axis. The maximal response is obtained by taking the value of the response to the highest drug concentration. The response is calculated by taking the absolute difference between the control value and the test value at each concentration.

[0261] Example 7. Dose - response for detecting the activity of an agent in reducing intraocular pressure in a glaucoma model in a microfluidic device. To use polymyxin B sulfate as an active agent for treating glaucoma, the dose - response behavior of polymyxin B sulfate on intraocular pressure (IOP) was determined. Polymyxin B sulfate showed an EC50 of 4.3 nM for treating the bovine vitreous humor glaucoma model.

[0262] The compound polymyxin B sulfate was tested in a microfluidic chip device in a bovine vitreous humor (BVH) glaucoma model. A PBS - buffered solution of 25% homogenized BVH was prepared and diluted with an equal volume of the compound solution to give a total BVH concentration of 12.5%. The samples were vortexed and incubated at 37 °C for 1 hour. PBS buffer or PBS containing 10% ethanol or DMSO was used as a control and incubated with BVH under the same conditions.

[0263] The test compound - BVH solution was introduced into the reservoir of the microfluidic chip device and the flow rate and pressure changes were recorded. The effects of various concentrations of the compound on treating bovine vitreous humor were tested. 7 μl of each test solution was injected into the microfluidic chip by a syringe. After sample injection, the flow rate and pressure changes were recorded for an additional 50 minutes. The relative change in chip pressure during the entire experiment was obtained.

[0264] Figure 31 Shows the dose - dependent response curve of treating the bovine vitreous humor glaucoma model with the compound polymyxin B sulfate. The EC50 value is taken as the point on the logarithmic function of the micromolar concentration of the compound on the x - axis that produces half - maximal response. The logarithmic function of the drug micromolar concentration is plotted on the x - axis, and the percentage of the maximal response is plotted on the y - axis. The maximal response is obtained by taking the value of the response to the highest drug concentration. The response is calculated by taking the absolute difference between the control value and the test value at each concentration.

[0265] Example 8. Isolation and extraction of extracellular matrix vesicles in a microfluidic device. The microfluidic device was used to isolate extracellular matrix vesicles from bovine vitreous cells. After isolation, the extracellular matrix vesicles were extracted.

[0266] Figure 32 Shows the isolation of extracellular matrix vesicles in the restricted channels of the microfluidic device of the present disclosure. Figure 32 a andFigure 32 b shows a representative micrograph of the microfluidic chip perfused with bovine vitreous humor. The letter "p" marks a post in the channel. After perfusion, the extracellular matrix bodies were separated between and around the posts. Figure 32 c and Figure 32 d show representative micrographs of the channel after extraction of the extracellular matrix bodies. The extracellular matrix bodies were extracted from the chip using a mild detergent, 1% sodium dodecyl sulfate (SDS), and a reverse flow from the inlet port outwards. The extracellular matrix bodies were removed from the chip, which showed significantly fewer extracellular matrix bodies after extraction.

[0267] Example 9. Detection of biomarkers for extracellular matrix bodies by proteomic profiling. Bovine vitreous extracellular matrix bodies were isolated and their proteomic profiles were analyzed using LC / MS off-chip.

[0268] Figure 33 Shows off-chip proteomic analysis of bovine extracellular matrix bodies by LC / MS. Biomarkers for extracellular matrix bodies were detected.

[0269] The precipitated bovine vitreous extracellular matrix aggregates were resuspended in 50 μl of 1% sodium dodecyl sulfate (SDS, Sigma) and centrifuged again at 25 Kg for 10 minutes at room temperature. The pellet was dissolved in 20 μl of 2X 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 into a NuPAGE 10% Bis-Tris gel (1.5 mm X 10 wells, Invitrogen). The gel was stained with Coomassie Brilliant Blue R250. Photographs of the gel were captured and stored, and then the gel was decolorized for further analysis.

[0270] 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 samples were digested with 0.2 μg of trypsin (sequencing grade, Thermo Scientific Cat#90058) and incubated at 37 °C for 16 hours. The peptides were extracted twice with 5% formic acid, 60% acetonitrile and dried under vacuum.

[0271] Samples were analyzed by LC-MS using a Nano LC-MS / MS (Dionex Ultimate 3000 RLS Canon system, ThermoFisher) interfaced with Eclipse (ThermoFisher). 3 μl of the 12.5 μl in-gel digested sample precipitate was loaded onto a fused silica trapping column (Acclaim PepMap 100, 75 um x 2 cm, ThermoFisher). After washing with 0.1% trifluoroacetic acid (TFA) at 5 μl / min for 5 minutes, the trapping column was connected in-line with the analytical column (Nanoease MZ Peptide BEH C18, 130A, 1.7 μm, 75 μm x 250 mm, Waters) for LC-MS / MS. A segmented linear gradient was used: 4 - 15% B in 30 minutes (where A: 0.2% formic acid, and B: 0.16% formic acid, 80% acetonitrile), 15 - 25% B in 40 minutes, 25 - 50% B in 44 minutes, and 50 - 90% B in 11 minutes, with peptides fractionated at 300 nL / min. Then solution B was returned to 4% for 5 minutes for the next run.

[0272] The scan sequence started with an MS1 spectrum (Orbitrap analysis, resolution 120,000, scan range from m / z 375–1500, automatic gain control (AGC) target 1E6, maximum injection time 100 ms). A top S (3 seconds) duty cycle scheme was used to determine the number of MSMS performed per cycle. Parent ions with a charge of 2 - 7 were selected for MSMS, and a 60-second dynamic exclusion was used to avoid repeated sampling. The parent ion mass was isolated in the quadrupole with an isolation window of 1.2 m / z, an automatic gain control (AGC) target of 1E5, and fragmented by high-energy collision dissociation with 30% normalized collision energy. These fragments were scanned in the Orbitrap at a resolution of 15,000. The MSMS scan range was determined by the charge state of the parent ion, but the lower limit was set to 110 amu.

[0273] Selected extracellular matrix-related proteins expressed in the vitreous bovine extracellular matrix body fraction were identified by proteomic profiling and are shown in Table 1.

[0274] Table 1: Extracellular matrix proteins in the vitreous bovine extracellular matrix body

[0275]

[0276]

[0277] Vitreous fractions were obtained by low-speed centrifugation and separation using a microfluidic device. Higher spectrometer values represent larger amounts of protein.

[0278] Selected proteins known to be involved in protein aggregates found in the vitreous bovine extracellular matrixome fraction were identified by proteomic profiling and are shown in Table 2.

[0279] Table 2: Protein Aggregate Proteins in Vitreous Bovine Extracellular Matrixome

[0280]

[0281] Vitreous ECM aggregate fractions were obtained by low-speed centrifugation and separation using a prototype microfluidic device. These proteins were classified by function, and the highlighted proteins are known to play a role in the extracellular matrix. For example, relatively high spectral counts were found for complement C3 (spectral count, 408), α-enolase (spectral count, 151), and clusterin (spectral count, 74).

[0282] Example 10. Isolation and extraction of extracellular matrixome in a microfluidic device. A microfluidic device was used to isolate bovine vitreous extracellular matrixome. After isolation, the extracellular matrixome was extracted.

[0283] Figure 34 Isolation of extracellular matrixome in the confinement channels of the microfluidic device of the present disclosure and its subsequent extraction are shown. The image scale bar is 50 μm. Figure 34 a shows a representative wide-field micrograph (gray signal, bright field) of the microfluidic device perfused with bovine vitreous humor suspended in phosphate-buffered saline at pH 7.0 and counterstained with Alcian blue for hyaluronic acid. Figure 34 a shows the signal from extracellular matrixome (arrows) trapped between the posts (p) of the device. The chip was perfused with biological fluid for at least 60 minutes. After perfusion, the aggregates were separated between the posts and were observed as clump-like formations. Substances smaller than extracellular matrixome material had been discharged through the outlet ports. Figure 34 b shows the extraction of extracellular matrixome from the confinement channels of the microfluidic device. The device was perfused with a mild detergent, 0.1% sodium dodecyl sulfate (SDS), and the flow direction was reversed from the outlet to the inlet. Figure 34 b shows that there are significantly fewer extracellular matrixome present in the channels after elution, indicating that the extracellular matrixome has been extracted. Figure 34 c shows a higher magnification image of extracellular matrixome (arrows) trapped between the posts (p) after perfusion. Figure 34 c shows the extraction with detergent and reverse flow, again showing significantly fewer extracellular matrixome in the channels after extraction.

[0284] Example 11. Isolation and extraction of extracellular matrixome in a microfluidic device. This experiment demonstrates that on-chip staining can be used to detect the isolation of extracellular matrixome.

[0285] Figure 35 On-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. The microfluidic device is injected with a fluid containing homogenized bovine vitreous suspended in a biological fluid. After perfusion of the fluid into the device, the fluid flows through the inlet and outflows via the outlet. Larger extracellular matrix bodies (arrows) are captured between the posts (labeled Lp). The chip is perfused with a blocking solution to prevent non-specific antibody binding prior to antibody staining. Next, the protein fibronectin, a known extracellular matrix component and integrin-binding protein, is labeled by injecting a primary anti-fibronectin antibody, incubating the sample for 2 hours, and washing. Then, a secondary goat anti-rabbit FITC antibody is incubated for 1 hour and washed. The microfluidic chip is then imaged under wide-field fluorescence and bright-field microscopy. Figure 35 A representative wide-field fluorescence micrograph is shown. This image shows extracellular matrix bodies in the microfluidic channel. The distance between the large posts (Lp) is approximately 100 μm. The punctate signals within the extracellular matrix bodies represent fibronectin staining (anti-fibronectin Ab, secondary goat anti-rabbit antibody with Alexa488, FITC, white signal).

[0286] Figure 36 On-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. Figure 36 a shows a representative bright-field image of the micrograph of the stained extracellular matrix bodies (arrows) in the channel. The image scale bar is 20 μm. The control image has no fluorescence signal, indicating that Figure 36 the signal in a is specific for fibronectin. Figure 36 b shows again the stained extracellular matrix bodies (arrows) in the channel. The scale bar of the image is 50 μm. Again, the control image has no fluorescence signal, indicating that Figure 36 the signal in b is specific for fibronectin.

[0287] Example 12. Isolation and extraction of extracellular matrix bodies in a microfluidic device. This experiment demonstrates that on-chip staining can be used to detect the isolation of extracellular matrix bodies.

[0288] Figure 37 On-chip immunohistochemical staining of extracellular matrix bodies in the device channels of the present disclosure. Figure 37Representative micrographs of on-chip immunohistochemical staining of aggrecan protein in a biological fluid containing extracellular matrix bodies, where aggrecan protein is a component of the chondrocyte extracellular matrix. The microfluidic chip is injected with a fluid containing homogenized bovine vitreous suspended in the biological fluid. After perfusing the biological fluid into the device, the sample flows through the inlet and out through the outlet. Larger extracellular matrix bodies (arrows) are captured between the posts (labeled "p"). The chip is perfused with a blocking solution to prevent non-specific antibody binding prior to antibody staining. Aggrecan is labeled by injecting anti-aggrecan primary antibody, incubating the sample for 2 hours, and washing. Then, goat anti-rabbit TRITC secondary antibody is incubated for 1 hour and washed. The microfluidic chip is then imaged under wide-field fluorescence and bright-field microscopy. Figure 37 Shows extracellular matrix bodies in the microfluidic channel between the posts (p). The punctate signals represent aggrecan staining (white signals). The control image has no fluorescent signal, indicating that Figure 37 the signals in

[0289] Figure 38 Show on-chip immunohistochemical staining of extracellular matrix bodies in the channels of the device of the present disclosure. Figure 38 a shows a representative micrograph bright-field image of on-chip immunohistochemical staining of aggrecan. The control image has no fluorescent signal, indicating that Figure 38 the signals in a are specific to aggrecan. The image scale bar is 10 μm. Figure 38 b shows a representative micrograph bright-field image of on-chip immunohistochemical staining of aggrecan. The control image has no fluorescent signal, indicating that Figure 38 the signals in b are specific to aggrecan. The image scale bar is 10 μm.

[0290] Example 13. Off-chip analysis of extracellular matrix bodies extracted from the microfluidic device. This experiment shows an off-chip analysis of extracellular matrix bodies that can be extracted from the channels of the device of the present disclosure.

[0291] Figure 39 Show that extracellular matrix bodies can be visualized on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking and staining with Alcian blue dye for hyaluronic acid. Figure 39 Show the signal of extracellular matrix bodies (dark staining), which shows that EDC crosslinking retains the extracellular matrix bodies on the surface.

[0292] Example 14. Off-chip analysis of extracellular matrix bodies extracted from the microfluidic device. This experiment shows an off-chip analysis of the extracellular matrix bodies of the present disclosure.

[0293] Figure 40Off-chip analysis of extracellular matrix bodies, which can be crosslinked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and visualized on a glass surface by staining collagen with Sirius red dye. Figure 40 Showing signals (dark staining) of collagen chains in extracellular matrix bodies in vivo, which shows that EDC crosslinking retains extracellular matrix bodies on the surface. This also shows that EMB can be visualized by collagen staining.

[0294] Example 15. Off-chip analysis of extracellular matrix bodies extracted from a microfluidic device. This experiment shows the off-chip analysis of extracellular matrix bodies, which can be visualized on a glass surface with nucleic acid markers.

[0295] Figure 41 Showing off-chip analysis of extracellular matrix bodies, which can be crosslinked using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and visualized on a glass surface by staining DNA with Hoechst dye. Figure 41 Showing DNA signals in extracellular matrix bodies.

[0296] Example 16. Isolation and detection of extracellular matrix bodies in a microfluidic device. This experiment shows the isolation and detection of extracellular matrix bodies in the device channels of the present disclosure.

[0297] Figure 42 Showing on-chip separation and detection of extracellular matrix bodies. Figure 42 Showing a representative micrograph of a microfluidic chip perfused with bovine vitreous humor, which is suspended in phosphate-buffered saline at pH 7.0 and counterstained with Alcian blue for hyaluronic acid, dark staining, bright field. Figure 42 Showing signals from extracellular matrix bodies captured near the large pillars (circles) of the device. The chip was perfused with biological fluid for at least 60 minutes. Extracellular matrix bodies with cluster formations were observed. The image scale bar is 50 μm.

[0298] Example 17. On-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. This experiment shows the on-chip separation, detection, and analysis of extracellular matrix bodies in the device channels of the present disclosure.

[0299] Figure 43 Showing on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 43Shows a representative low-magnification fluorescence micrograph of channels after perfusion with bovine vitreous extracellular matrix bodies, which were restained for proteins with carboxyfluorescein succinimidyl ester (CFSE). The microfluidic chip was injected with a fluid containing homogenized bovine vitreous. After the fluid was perfused into the device, the fluid flowed through the inlet and out through the outlet. Larger extracellular matrix bodies were captured near the posts (labeled "p"). The image scale bar is 25 μm.

[0300] Example 18. On-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. This experiment shows the on-chip separation, detection, and analysis of extracellular matrix bodies in the channels of the device of the present disclosure.

[0301] Figure 44 Shows on-chip separation, detection, and analysis of extracellular matrix bodies in a microfluidic device. Figure 44 Shows a representative micrograph of a microfluidic chip perfused with bovine vitreous humor, which was suspended in phosphate-buffered saline at pH 7.0 and restained for collagen with Sirius red, dark staining, bright field. Figure 44 a shows the signal of extracellular matrix bodies between the posts (labeled "p") from the device. The chip was perfused with biological fluid for at least 60 minutes. Figure 44 b shows the same image with a fluorescence filter and shows collagen detected with Sirius red (light gray signal). The image scale bar is 50 μm. Figure 44 c and Figure 44 c shows a higher-magnification image. The image scale bar is 10 μm.

[0302] Example 19. Separation, detection, and analysis of extracellular matrix bodies using a microfluidic device. This experiment shows the separation, detection, and analysis of extracellular matrix bodies using the device of the present disclosure.

[0303] Figure 45 Shows the frequency size distribution of human extracellular matrix bodies present in human aqueous humor biological fluids from healthy and pre-disease states, suspected glaucoma, and pre-glaucoma. Aqueous humor was obtained from 8 patients, healthy samples, or pre-glaucoma diagnoses, with intraocular pressure ranging from 9 to 25 mmHg. Human samples were not processed by centrifugation or other means. The size of the extracellular matrix bodies was determined by cross-linking the samples to slides using a carbodiimide EDC fixative, staining with uranyl acetate, and imaging with wide-field microscopy. The size (area) of the extracellular matrix bodies was quantified using an automated program (ImageJ) in all eight samples. The size of the extracellular matrix bodies ranged from approximately 1.67 μm 2 to approximately 67×10 3 μm 2 . Figure 45 Shows 0 - 200 μm 2Counting of extracellular matrix vesicles within a range.

[0304] Example 20. Isolation, detection, and analysis of extracellular matrix vesicles using a microfluidic device. This experiment demonstrated the isolation, detection, and analysis of extracellular matrix vesicles using the device of the present disclosure.

[0305] Figure 46 Showing the frequency size distribution of human extracellular matrix vesicles present in human aqueous humor biofluids from healthy and pre-disease states, suspected glaucoma, and pre-glaucoma. Aqueous humor was obtained from 8 patients, healthy samples, or pre-glaucoma diagnoses, with intraocular pressure ranges from 9 to 25 mmHg. Human samples were not processed by centrifugation or other means. The size of the extracellular matrix vesicles was determined by cross-linking the samples to slides using carbodiimide EDC fixative, staining with uranyl acetate, and imaging with wide-field microscopy. Sizes were quantified using an automated program (ImageJ) in all eight samples. The size (area) of the extracellular matrix vesicles ranged from approximately 1.67 μm 2 to approximately 67×10 3 μm 2 . Figure 46 Showing the count of extracellular matrix vesicles within the range of 201 - 1000 μm 2 Counting of extracellular matrix vesicles within a range.

[0306] Example 21. Isolation, detection, and analysis of extracellular matrix vesicles using a microfluidic device. This experiment demonstrated the isolation, detection, and analysis of extracellular matrix vesicles using the device of the present disclosure.

[0307] Figure 47 Showing the frequency size distribution of human extracellular matrix vesicles present in human aqueous humor biofluids from healthy and pre-disease states, suspected glaucoma, and pre-glaucoma. Aqueous humor was obtained from 8 patients, healthy samples, or pre-glaucoma diagnoses, with intraocular pressure ranges from 9 to 25 mmHg. Human samples were not processed by centrifugation or other means. The size of the extracellular matrix vesicles was determined by cross-linking the samples to slides using carbodiimide EDC fixative, staining with uranyl acetate, and imaging with wide-field microscopy. Sizes were quantified using an automated program (ImageJ) in all eight samples. The size (area) of the extracellular matrix vesicles ranged from approximately 1.67 μm 2 to approximately 67×10 3 μm 2 . Figure 47 Showing the count of extracellular matrix vesicles within the range of 1001 - 5000 μm 2 Counting of extracellular matrix vesicles within a range.

[0308] Example 22. Isolation, detection, and analysis of extracellular matrix vesicles using a microfluidic device. This experiment demonstrated the isolation, detection, and analysis of extracellular matrix vesicles using the device of the present disclosure.

[0309] Figure 48 Shows the size distribution of bovine vitreous extracellular matrix vesicles isolated and extracted from the microfluidic device of the present invention. Extracellular matrix vesicles in bovine vitreous humor biofluid after isolation and extraction from the microfluidic device of the present invention. The chip was perfused with biofluid for at least 60 minutes. After 60 minutes of perfusion, ECM vesicles were separated near the confinement channel posts. Next, the chip was treated with a detergent (0.1% sodium dodecyl sulfate, SDS), and the sample was extracted from the chip by reverse flow, which caused the extracellular matrix vesicles to flow out of the inlet port. Fractions of the eluate were collected at 10-minute intervals for a total of 80 minutes. The samples were fixed on glass slides and stained with Alcian blue, and imaged by wide-field microscopy. The size was quantified using an automated program (ImageJ). The size (area) of the extracellular matrix vesicles can reach about 16×10 3 μm 2 。 Figure 48 Shows the count of extracellular matrix vesicles in each eluate fraction, which increases with time. This experiment demonstrates that the microfluidic device of the present invention can be used to isolate and extract extracellular matrix vesicles of various sizes.

[0310] Example 23. On-chip separation, detection and analysis of extracellular matrix vesicles in a microfluidic device. This experiment shows the off-chip separation, detection and analysis of extracellular matrix vesicles.

[0311] Figure 49 Shows that off-chip analysis of extracellular matrix vesicles can be accomplished by retention through cross-linking with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Figure 49 a shows a representative TEM image of extracellular matrix vesicles from native bovine vitreous humor obtained by cross-linking with EDC. The extracellular matrix vesicles were observed after fixation with EDC. Figure 49 b shows a similar image taken without EDC cross-linking. In the absence of EDC fixation, no extracellular matrix vesicles were observed.

[0312] Example 24. Isolation and detection of extracellular matrix vesicles in early pancreatic cancer using a microfluidic device with human plasma samples. This experiment shows the isolation and detection of extracellular matrix vesicles in early pancreatic cancer using a microfluidic device with human plasma samples. This experiment demonstrates that the microfluidic device of the present invention can be used to isolate and detect extracellular matrix vesicles in early pancreatic cancer from human plasma samples. This experiment further demonstrates that extracellular matrix vesicles are useful markers for differentiating plasma of early pancreatic ductal adenocarcinoma from healthy controls.

[0313] [[ID=28 Shows the isolation of extracellular matrix vesicles from human plasma samples of patients with early pancreatic ductal adenocarcinoma (PDAC) compared to healthy controls.

[0314] In this experiment, a microfluidic chip was infused with human plasma from early pancreatic ductal adenocarcinoma (PDAC). After perfusion into the device, the biofluid flowed through the inlets and out through the outlets. The results were compared with age-matched healthy controls.

[0315] The chip was perfused with a blocking solution to prevent non-specific antibody binding prior to antibody staining. Next, the protein fibronectin (a known extracellular matrix component and integrin-binding protein) was labeled by on-chip immunohistochemical staining by injecting an anti-fibronectin primary antibody, incubating the sample for 2 hours, and washing. Then, a goat anti-rabbit FITC secondary antibody was incubated for 1 hour and washed. The microfluidic chip was then imaged under a fluorescence microscope.

[0316] ​ Panel a shows a representative wide-field fluorescence microscopy image of a PDAC sample. ​ Panel a shows extracellular matrix bodies that remained in the microfluidic channels. ​ Panel a further shows larger extracellular matrix bodies (arrows) that remained between the posts (labeled "p"). The width between the posts was approximately 100 μm. The punctate signals from the remaining extracellular matrix bodies represent fibronectin staining (anti-fibronectin Ab, goat anti-rabbit secondary Ab with Alexa488, FITC, white signal). The staining showed abundant signals and punctate staining within the EMB ( ​ a, arrows).

[0317] ​ Panel b shows a similarly obtained fluorescence micrograph of an age-matched healthy control human plasma sample. ​ Panel b shows a significantly reduced amount of fibronectin signal ( ​ b, grey signal, arrow). When processed under the same conditions, the healthy control signal was much smaller than the disease signal. The image scale bar was 20 μm.

Claims

1. A method for preparing a biological sample, the method comprising isolating extracellular matrix vesicles from the biological sample.

2. The method according to claim 1, wherein the biological sample comprises a body fluid, blood, tissue or cell of a human or an animal.

3. The method according to claim 1, wherein the extracellular matrix vesicles have a size of 0.5 to 5,000 microns, or 1 to 1,000 microns, or 1 to 200 microns, or 4 to 100 microns.

4. The method according to claim 1, wherein the isolation of the extracellular matrix vesicles is carried out by ultrafiltration or centrifugation.

5. The method according to claim 1, further comprising immobilizing the extracellular matrix vesicles on a glass surface using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide crosslinking.

6. The method according to claim 1, wherein the isolation of the extracellular matrix vesicles is carried out by a device for separating fractions of a biological sample, the device comprising: one or more confinement channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel; a plurality of spaced-apart obstacles located in the confinement channel to provide flow resistance, wherein the spacing between the obstacles decreases in the direction from the inlet end to the outlet end; an inlet reservoir for containing fluid, wherein the inlet fluid reservoir is in fluid communication with the inlet end of the confinement channel; and one or more uniform flow channels having an inlet end and an outlet end, wherein the inlet end and the outlet end are in fluid communication through the channel, wherein the inlet end is in fluid communication with the inlet reservoir.

7. The method according to claim 6, wherein the device further comprises: a pressure source for applying pressure to the fluid in the inlet reservoir; and a flow sensor in fluid communication with the inlet reservoir for measuring the flow rate and pressure of the fluid at the inlet reservoir.

8. The method according to claim 6, wherein the device further comprises an outlet reservoir in fluid communication with the outlet ends of the confinement channel and the uniform flow channel.

9. The method according to claim 6, wherein the confinement channel comprises a barrier strip having windows with at least about 1 micron, or at least about 2 microns, or at least about 4 microns, or at least about 10 microns, or at least about 25 microns, or at least about 50 microns, or at least about 100 microns, or at least about 200 microns, or at least about 500 microns.

10. The method according to claim 6, wherein the confinement channel comprises windows of about 1-4 microns, or about 1-15 microns, or about 4-35 microns, or about 4-100 microns, or about 4-200 microns.

11. The method according to claim 6, wherein 1-90% of the flow in the device is within the uniform flow channel, or 1-75% of the flow in the device is within the uniform flow channel, or 1-50% of the flow in the device is within the uniform flow channel, or 1-25% of the flow in the device is within the uniform flow channel.

12. The method according to claim 6, wherein the confinement channel and the uniform flow channel are integrated with the same chip or substrate.

13. The method according to claim 6, wherein the restriction channel and the uniform flow channel are located in different chips or substrates.

14. The method according to claim 6, wherein the restriction channel is a microfluidic channel.

15. The method according to claim 6, wherein the device further comprises an instrument for analyzing a biological sample in the channel.

16. The method according to claim 6, wherein the device further comprises an instrument for analyzing the proteomic composition, lipidomic composition, transcriptomic composition, or carbohydrate composition of a biological sample in the channel.

17. The method according to claim 6, wherein the device further comprises an instrument for measuring the level of a separated fraction of a sample within the channel.

18. The method according to claim 6, wherein the device further comprises an instrument for measuring the level of a biomarker in a separated fraction of a sample within the channel.

19. The method according to claim 6, wherein the plurality of obstacles comprises posts integrated with the channel.

20. The method according to claim 6, wherein the plurality of obstacles comprises one or more of the following: a part of the uveal meshwork of a human or animal; a part of the corneoscleral meshwork of a human or animal; and a part of the juxtatubular meshwork of a human or animal.

21. The method according to claim 6, wherein the plurality of obstacles comprises glass beads, magnetic beads, gel particles, dextran particles, or polymer particles.

22. The method according to claim 1, wherein the biological sample comprises a carrier fluid.

23. The method according to claim 1, wherein the biological sample comprises one or more reagents.

24. The method according to claim 6, wherein the restriction channel further comprises a binding portion for binding a biomarker or biomolecule of the sample.

25. The method according to claim 1, wherein the biological sample is from a subject undergoing diagnosis or prognosis.

26. The method according to claim 6, wherein the device further comprises a curved fluid mixing region in the restriction channel.

27. The method according to claim 6, wherein the restriction channel or the continuous flow channel has a fluorinated coating.

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