Biomimetic microfluidic devices for investigating ocular outflow and trabecular meshwork function

Abstract

Described herein are biomimetic microfluidic devices. In some embodiments, the devices may be a glaucoma eye organ-on-a-chip microfluidic device. Also described herein is a method of culturing and monitoring one or more cell types and a method of screening one or more anti-glaucoma therapeutic agents using the disclosed biomimetic microfluidic devices. In some embodiments, the disclosed devices may be used to investigate trabecular meshwork cell function and ocular outflow as they relate to glaucoma pathology.

Description

[0001] BIOMIMETIC MICROFLUIDIC DEVICES FOR INVESTIGATING OCULAR OUTFLOW AND TRABECULAR MESHWORK FUNCTION

[0002] CROSS-REFERENCE TO RELATED APPLICATION(S)

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 664,571 , filed on June 26, 2024, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Glaucoma affects over 80 million people worldwide, causing irreversible vision loss through progressive optic nerve degeneration. By 2040, this number is projected to exceed 111 million, with mostly Primary Open Angle Glaucoma (POAG). The disease originates from impaired aqueous humor outflow through the trabecular meshwork (TM), where pathological tissue stiffening creates a vicious cycle: increased stiffness elevates outflow resistance, raising intraocular pressure (IOP), which further stiffens the tissue through mechanotransduction pathways. In healthy eyes, the TM maintains a compliant structure with stiffness values of ~4.0 kPa, facilitating dynamic regulation of aqueous humor drainage. During glaucoma progression, tissue stiffness increases dramatically — up to 20-fold in advanced disease — reaching values exceeding 80 kPa. This pathological stiffening occurs heterogeneously, creating mechanical gradients that subject reside cells to complex, non-uniform stress fields during IOP fluctuations.

[0006] Beyond pathological stiffening, trabecular meshwork cells experience dynamic mechanical stretch due to IOP fluctuations and ciliary muscle contractions. However, physiological stretch magnitudes remain unknown due to the complex tissue architecture. Researchers have therefore employed 0-20% substrate elongation protocols, ranging from small amplitude cyclic stretch (0.45% at 1 Hz) mimicking ocular pulsations to high magnitudes (up to 20%) to study the trabecular meshwork cell mechanobiology and model pathological conditions. Mechanical forces transmitted through integrin-mediated attachments trigger mechanotransduction cascades involving phosphorylated focal adhesion kinase (pFAK) activation via ROCK-dependent pathways initially, then requiring ROCK and TRPV4 signaling during sustained stretch. Key stretch-induced responses include: myofibroblast differentiation with increased a-smooth muscle actin expression; upregulation of glaucoma-associated myocilin; enhanced matrix metalloproteinase-2 (MMP2) activity (31% increase after sustained stretch); altered collagen type I organization; and cytoskeletal reorganization with focal adhesion remodeling. Cyclic stretch primarily promotes matrix remodeling while static stretching induces myofibroblast differentiation and pathological ECM changes, demonstrating distinct mechanobiological responses dependent on loading characteristics.

[0007] Despite growing understanding of mechanobiology roles in glaucoma, a fundamental knowledge gap persists regarding how TM cells integrate multiple mechanical stimuli — substrate stiffness and dynamic stretch — to that are associated with disease progression. Current in vitro models examine these factors independently, failing to capture the synergistic effects that occur physiologically. Static culture on stiff substrates induces myofibroblast transformation, while cyclic stretch promotes extracellular matrix (ECM) remodeling, but their combined effects remain unexplored. This limitation has hindered the development of mechanically targeted therapeutics and the overall understanding of glaucoma pathophysiology. The inability to study coupled mechanical effects stems from significant technological barriers in microfluidic platform design. Existing organ chip systems face important limitations including: inability to integrate tunable hydrogel substrates with mechanical actuation systems, restriction to uniaxial or biaxial deformation that fails to replicate physiological triaxial stress states, poor hydrogel-device integration under dynamic loading conditions, and lack of optical transparency for real-time cellular imaging. Previous attempts using PDMS-based stretching devices achieved mechanical deformation but lacked substrate tunability, while hydrogel-based platforms provided stiffness control without dynamic mechanical capabilities.

[0008] What is needed are biomimetic microfluidic devices and methods for modeling and investigating ocular outflow and trabecular meshwork function. These devices and methods would be useful in a variety of applications including screening for anti-glaucoma therapeutic agents.

[0009] SUMMARY

[0010] One embodiment described herein is a biomimetic microfluidic device, comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer. In one aspect, the substrate base layer comprises silanized glass. In another aspect, each of the first microfluidic channel layer, second microfluidic channel layer, and membrane layer comprises a polymeric material of polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof. In another aspect, the membrane layer is porous and comprises an electrospun collagen nanofiber membrane. In another aspect, the membrane layer is porous and comprises a porosity of about 70% to about 80%. In another aspect, the membrane layer is porous and comprises an average pore size of about 1 pm to about 5 pm. In another aspect, the membrane layer comprises a flat, concave, or convex shaped curvature upon adjusting the pressure in the first microfluidic channel layer to stretch the population of cells ranging from 0% to 50% linear strain. In another aspect, the membrane layer comprises a uniform thickness across its surface of about 25 pm to about 250 pm. In another aspect, the second microfluidic channel layer comprises Hele-Shaw geometrical dimensions comprising a width of about 2 mm to about 15 mm, a length of about 25 mm to about 30 mm, and a height of about 0.05 mm to about 0.1 mm. In another aspect, the second microfluidic channel layer is configured to modulate shear stress on the population of cells from about 0.01 dyn / cm2to about 30 dyn / cm2. In another aspect, the second microfluidic channel layer is configured for fluid flow rates of about 1 pL / min to about 750 pL / min to modulate shear stress on the population of cells. In another aspect, the hydrogel layer comprises gelatin, collagen, elastin, alginate, or combinations thereof. In another aspect, the hydrogel layer comprises a photo-crosslinkable material comprising gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), or combinations thereof. In another aspect, the hydrogel layer comprises gelatin methacrylate (GelMA) at a concentration of about 1% w / v to about 20% w / v. In another aspect, the hydrogel layer comprises a Young’s modulus of about 1 kPa to about 25 kPa. In another aspect, the concentration and Young’s modulus of the hydrogel layer are associated with a stiffness of the population of cells. In another aspect, the hydrogel layer has a uniform thickness of about 0.05 mm to about 0.5 mm. In another aspect, the device further comprises one or more of a plurality of receiving channels, outlets, flow sensors, and pressure transducers to measure fluid flow rates of outflow from the population of cells. In another aspect, the population of cells is cultured on the first surface of the membrane layer, the second surface of the membrane layer, or a combination thereof. In another aspect, the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof. In another aspect, the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof. In another aspect, the TM cells comprise glaucomatous TM cells. In another aspect, TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer. In another aspect, the membrane layer mimics a juxtacanalicular tissue structure. In another aspect, the device is a glaucoma eye organ-on-a-chip microfluidic device. In another aspect, the device is capable of simultaneously and independently modulating stiffness, stretch, and shear stress conditions for the population of cells. In another aspect, the membrane layer is optically transparent for real-time cell imaging. In another aspect, the device is optically transparent for real-time cell imaging.

[0011] Another embodiment described herein is a method of culturing and monitoring one or more cell types, the method comprising: inserting a population of cells into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; modulating the hydraulic pressure in the first microfluidic channel layer to alter the shape of the membrane layer and stretch the population of cells; and analyzing the population of cells in the device. In one aspect, the method further comprises applying a fluid flow through the second microfluidic channel layer to modulate the shear stress on the population of cells prior to analyzing the population of cells in the device, wherein the shear stress and stretch are applied simultaneously to the population of cells. In another aspect, the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof. In another aspect, TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer. In another aspect, the TM cells are derived from a subject having glaucoma. In another aspect, the TM cells are isolated from juxtacanalicular and / or corneoscleral meshwork eye regions of a subject. In another aspect, the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof. In another aspect, the membrane layer of the biomimetic microfluidic device is surface treated with one or more extracellular matrix proteins comprising collagen type I, fibronectin, or a combination thereof prior to inserting the population of cells into the device. In another aspect, the population of cells is analyzed in the biomimetic microfluidic device using one or more imaging techniques. In another aspect, the method further comprises performing one or more biochemical assays on the population of cells. In another aspect, the method further comprises administering one or more anti-glaucoma therapeutic agents into the device.

[0012] Another embodiment described herein is a method of screening one or more antiglaucoma therapeutic agents, the method comprising: inserting a population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; administering one or more anti-glaucoma therapeutic agents into the device; and analyzing the population of cells in the device. In one aspect, TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer. In another aspect, the membrane layer is porous and comprises an electrospun collagen nanofiber membrane. In another aspect, the anti-glaucoma therapeutic agent comprises one or more ion channel agonists or antagonists for Piezol , TRPV4, TREK-1 , or combinations thereof. In another aspect, the anti-glaucoma therapeutic agent comprises an actin depolymerization agent, a Rho kinase inhibitor, or a combination thereof. In another aspect, the method further comprises modulating one or more of stiffness, stretch, and shear stress conditions for the population of cells. In another aspect, the method further comprises measuring fluid flow rates of outflow from the population of cells.

[0013] Another embodiment described herein is a kit for culturing and monitoring one or more cell types, the kit comprising: a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; optionally, cell culture buffers, reagents, and receptacles; and optionally, one or more of packaging or instruction for use.

[0014] Another embodiment described herein is the use of a biomimetic microfluidic device for culturing and monitoring one or more cell types in response to modulation of stiffness, stretch, and / or shear stress conditions, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

[0015] Another embodiment described herein is the use of a biomimetic microfluidic device for screening one or more anti-glaucoma therapeutic agents, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells, the population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

[0016] DESCRIPTION OF THE DRAWINGS

[0017] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0018] FIG. 1 shows a schematic of glaucoma chip using multilayered microfluidic system with nanofiber-based collagen, enabling high-throughput cellular response of TM.

[0019] FIG. 2A-B show a Hele-Shaw microfluidic channel enabling the generation of a wide range of shear stress in a single platform. FIG. 2A shows TM cells were cultured on the chip with 1 x 107cells / mL. FIG. 2B shows cells stained with cell tracker to visualize the distribution of the TM cells (10x).

[0020] FIG. 3 shows a photograph of two chambered Hele-Shaw microfluidic channel to perform outflow tests with respect to shear stress level. The microchannels are represented: the anterior portion of TM tissue (top channel, red) and posterior portion of TM (multiple bottom channel, dark red), separated by a porous membrane. FIG. 4 shows a schematic of an exemplary glaucoma chip fabrication procedure. The fabrication process includes three main steps: (1) hydrogel integration using silanized glass substrate, where the hydrogel is patterned within the designated area; (2) top channel bonding using uncured PDMS, where the microfluidic channels are formed and aligned over the hydrogel region; and (3) post-curing to complete the device assembly and ensure proper bonding between all components.

[0021] FIG. 5A-D show schematic illustrations of exemplary glaucoma chip architectures and operating principles. FIG. 5A shows one embodiment of the chip. The device includes multiple layers including a glass substrate, hydraulic pressure channel, membrane, hydrogel layer, and top PDMS channel with Hele-Shaw geometry. The hydrogel experiences multi-directional mechanical stimuli including tensile stress and shear stress. Cross-sectional views (right panels) show the device in relaxed state (top) and under applied hydraulic pressure (bottom), demonstrating membrane deflection and resulting hydrogel deformation. FIG. 5B shows an alternative embodiment of the chip. FIG. 5C and FIG. 5D show additional exemplary embodiments of the chips with alternative layer designs.

[0022] FIG. 6A-C show an exemplary hydrogel-integrated microfluidic device system concept and design. FIG. 6A shows a design of a hydrogel-integrated microfluidic system. FIG. 6B shows a cross-sectional view. Hydraulic pressure was applied in the microfluidic channel to apply tensile stretch on the hydrogel. FIG. 6C shows a photograph of a hydrogel-integrated microfluidic system. Color dye indicates the feasibility of the multi-experiment conditions.

[0023] FIG. 7 shows surface modification and hydrogel integration. PDMS surface was treated with oxygen plasma to form a hydroxyl group on the surface (OH-PDMS). 10% TMSPMA was treated to form a methacrylate functional group on the surface (TMSPMA-PDMS). Sigmacote- coated cover glass was placed on the TMSPMA-PDMS to make an instant microfluidic channel. A patterned Rubylith film was placed on the bottom of the microfluidic system. GelMA hydrogel was introduced from the center inlet to form four hydrogels simultaneously. UV was exposed from the bottom of the chip. Cover glasses were removed to create a flat hydrogel surface.

[0024] FIG. 8A-B show hydrogel surface area and UV exposure analysis. FIG. 8A shows a photograph of 5% GelMA hydrogel after exposure to UV for 150 seconds and incubated for 24 h. The line shows the original circumference of the hydrogel before incubation, and the dotted line shows the shrunk circumference of the hydrogel after incubation for 24 h. FIG. 8B shows total shrunk area of 5% GelMA hydrogel based on UV exposure time after incubation for 24 h. After UV exposure for 150 seconds, the total shrunk area was consistent after incubation for 24 h. FIG. 9A-B show Young’s Modulus of GelMA hydrogel with different concentrations using a rheometer. FIG. 9A shows representative data of each GelMA hydrogel. Storage modulus values were obtained by increasing the shear strain rate. FIG. 9B shows Young’s modulus of GelMA hydrogel with different GelMA Concentrations. Each Young’s modulus value was calculated based on the storage modulus values.

[0025] FIG. 10A-C show mechanical characterization of PDMS and GelMA hydrogels for the strain analysis and bursting strength test. FIG. 10A shows representative photographs of a PDMS substrate (left) and a GelMA hydrogel (right) within the experimental system. FIG. 10B shows linear strain percentage as a function of applied fluid volume (pL) for PDMS, 5% GelMA, 10% GelMA, and 20% GelMA substrates. Data points represent mean ± standard deviation (n = 3 for each material). FIG. 10C shows sequential images illustrating the bursting strength test, showing increasing membrane deformation at 0 pL, 50 pL, 90 pL, and 95 pL of applied fluid volume. The membrane ruptured at volumes exceeding 95 pL.

[0026] FIG. 11A-D show finite element simulation and experimental validation of dome deformation and mechanical responses of GelMA and PDMS hemispherical constructs under pressure loading. FIG. 11 A shows 3D finite element mesh model of a PDMS / 20% GelMA hemispherical composite showing x-direction displacement under applied pressure. FIG. 11 B shows peak height as a function of applied pressure for PDMS, 5% GelMA, and 20% GelMA domes, comparing experimental measurements (solid markers) with finite element simulations (dashed lines). FIG. 11C shows surface stress profiles across the x-axis for the different material systems, showing increasing stress magnitude with material stiffness. FIG. 11 D shows maximal membrane strain distribution along the x-axis, demonstrating material-dependent strain localization, with PDMS exhibiting significantly higher deformation compared to GelMA formulations.

[0027] FIG. 12A-B show finite element method (FEM) analysis of membrane deflection and stress under various pressure for three cases (PDMS only, 5% GelMA on PDMS, and 20% GelMA on PDMS). FIG. 12A shows cross-sectional views illustrating the displacement field of the membranes. Color scales indicate the magnitude of displacement (mm). FIG. 12B shows cross- sectional views showing the surface stress distribution of each case. Color scales indicate stress levels in kilopascals (kPa).

[0028] FIG. 13A-B show a cell viability test on GelMA hydrogel. FIG. 13A shows a large tile image of trabecular meshwork cells on the hydrogel in the well. FIG. 13B shows a close-up image of trabecular meshwork cells on the hydrogel. Trabecular meshwork cells were only seeded and proliferated on the hydrogel surface. FIG. 14A-C show morphology of TM and gTM on GelMA hydrogels under varying mechanical conditions. FIG. 14A shows immunofluorescence images of TM and gTM cells cultured on GelMA hydrogels for 48 hours in different microfluidic regions. Cells were stained for F-actin (red) and nuclei (Hoechst, blue). Images are shown for cells in the “Top,” “Mid,” and “Edge” regions with combined fluorescence. Scale bars are 50 pm. FIG. 14B shows normalized cell area ratio for TM and gTM cells on 5% and 20% GelMA hydrogels under 10% stretch conditions over a non-stretch 5% GelMA case. FIG. 14C shows normalized cell aspect ratio for TM and gTM cells on 5% and 20% GelMA hydrogel under 10% stretch conditions over a nonstretch 5% GelMA case. Cells were categorized based on their position (“Top,” “Mid,” “Edge”) within the HIMS. Data are presented as mean ± standard deviation.

[0029] FIG. 15 shows focal adhesion and ECM turnover biomarker of TM and gTM cells under matrix stiffness and mechanical stimulation. Each cell was cultured in 5% and 20% GelMA hydrogel with no stretch for 48 h and with 10% static stretch for 24 h after 24 h of cell stabilization. Representative immunofluorescence images show biomarker expression patterns indicative of mechanotransduction effects. Scale bar: 50 pm.

[0030] FIG. 16 shows phenotypic biomarker of TM and gTM Cells under matrix stiffness and mechanical stimulation. Each cell was cultured in 5% and 20% GelMA hydrogel with no stretch for 48 h and with 10% static stretch for 24 h after 24 h of cell stabilization. Representative immunofluorescence images show biomarker expression patterns indicative of mechanotransduction effects. Scale bar: 50 pm.

[0031] FIG. 17A-B show ECM and phenotypic protein expressions in TM and gTM cells under varying stiffness and stretch conditions. FIG. 17A-B show normalized mean fluorescent intensity of key proteins in TM and gTM cells related to focal adhesion and extracellular matrix remodeling (pFAK, MMP2, Coll) (FIG. 17A) phenotypic makers (a-SMA, MYOC) (FIG. 17B). Both cells were cultured on two different GelMA concentrations (5% GelMA and 20% GelMA) and subjected to either no stretch or 10% static stretch. Data are presented as mean ± standard deviation.

[0032] DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0034] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0035] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0036] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0037] As used herein, the term “or” can be conjunctive or disjunctive.

[0038] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0039] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0040] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.” All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0041] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0042] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0043] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0044] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0045] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0046] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0047] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0048] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0049] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0050] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifested.

[0051] As used herein, “microfluidic” refers to the behavior, precise control, and manipulation of fluids that are geometrically and dimensionally constrained to a small scale (typically submillimeter) at which surface forces dominate volumetric forces. A “microfluidic channel,” “microchannel,” “microfluidic chamber,” “channel,” “microfluidic channel layer,” or “flow channel” all generally refer to a micron-scale channel used for fluidically connecting various components of apparatuses, systems, and devices according to specific embodiments of the present disclosure. A microchannel typically has a rectangular, e.g., square, or a rounded cross-section, with side and depth dimensions of between about 10 and 500 pm. Fluids flowing in the microchannels may exhibit microfluidic behavior. When used to refer to a microfluidic channel layer within the biomimetic microfluidic devices of the present disclosure, the terms “microchannel,” “microfluidic chamber,” and “channel” are used interchangeably. In some aspects, perfusion channels or perfusion chambers generally denote channels designed for passage of media, reagents, or other fluids or gels, and in some embodiments, cells.

[0052] As used herein, “microfluidic device” or “microfluidic apparatus” refer to a device or apparatus comprising at least one microchannel or microchamber having a cross-sectional dimension of less than 1 millimeter (typically between about 10 and 500 pm). In some embodiments of the present disclosure, the described biomimetic microfluidic devices may comprise microfluidic layers being fabricated to create one or more of microfluidic chambers, channels, reservoirs, reaction areas, inlets, or outlets by one or more processes of laser cutting, injection molding, die cutting, milling, press cutting, layer-by-layer fabrication, 3D printing, lithography, or combinations thereof.

[0053] As used herein, “hydraulic actuation” is used to describe a system, device, or other external source in a microfluidic system that uses pressurized fluid (air, liquid, and / or gas fluid) to generate mechanical motion or deformation at the microscale, enabling the control of fluid flow and structural movement within a microfluidic device. In some embodiments described herein, discloses biomimetic microfluidic devices may comprise a first microfluidic channel layer configured for hydraulic pressure modulation, where the first microfluidic channel layer is fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer. In some aspects, modulating the hydraulic pressure in the first microfluidic channel layer can alter the shape of a membrane layer to provide a stretch / strain on a population of cells. For example, the membrane layer may comprise a flat, concave, or convex shaped curvature upon adjusting the pressure in the first microfluidic channel layer to stretch a population of cells ranging from 0% to 50% linear strain.

[0054] As used herein, “polymer” or “polymeric material” is intended to encompass a homopolymer, heteropolymer, block polymer, co-polymer, ter-polymer, etc., and blends, combinations, or mixtures thereof. Examples of polymers may include, but are not limited to, functionalized polymers. Polymers include, without limitation, polyesters, poly(meth)acrylamides, poly(meth)acrylates, polyethers, polystyrenes, polynorbornenes and monomers that have unsaturated bonds. Examples of other polymers include, but are not limited to, polyalkylenes such as polyethylene and polypropylene; polychloroprene; polyvinyl ethers; such as polyvinyl acetate); polyvinyl halides such as polyvinyl chloride); polysiloxanes; polystyrenes; polyurethanes; polyacrylates; such as poly(methyl (meth)acrylate), poly(ethyl (meth)acrylate), poly(n-butyl(meth)acrylate), poly(isobutyl (meth)acrylate), poly(tert-butyl (meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate); polyacrylamides such as poly(acrylamide), poly(methacrylamide), poly(ethyl acrylamide), polyethylene glycol diacrylate, poly(ethyl methacrylamide), poly( / V- isopropyl acrylamide), poly(n, iso, and tert-butyl acrylamide); and copolymers and mixtures thereof. These polymers may include useful derivatives, including polymers having substitutions, additions of chemical groups, for example, alkyl groups, alkylene groups, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art. The polymers may include zwitterionic polymers such as, for example, polyphosphorycholine, polycarboxybetaine, and polysulfobetaine.

[0055] In some embodiments described herein, biomimetic microfluidic devices may comprise one or more components comprising a polymeric material of polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), polyacrylic acid, polyester, nylon, polyvinyl chloride, polyethylene, polypropylene, polyethylene terephthalate glycol, polybutylene adipate terephthalate, ethylene tetrafluoroethylene, fluorinated ethylene propylene, perfluoro alkoxy alkane, polylactic acid, polycaprolactone, polyoxymethylene, cellulose, co-polymers thereof, or combinations thereof. In some aspects, biomimetic microfluidic devices may also comprise one or more components comprising a photo-crosslinkable material gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), or combinations thereof.

[0056] Polymeric materials such as PDMS are typically hydrophobic. In certain embodiments described herein, surface modification techniques including layer-by-layer (LBL) deposition, deposition of polyvinyl alcohol (PVA) following oxygen plasma bonding treatment, or polyethylene glycol) coating production can be performed on the polymeric material surfaces to finely control the hydrophilicity of the polymeric material and regulate overall fluid flow rates through channel layers of the biomimetic microfluidic device. Plasma oxidation treatment renders a PDMS surface more hydrophilic, allowing aqueous solutions to maintain surface wetness. For applications where long-term hydrophilicity is necessary, techniques such as hydrophilic polymer grafting, surface nano-structuring, and dynamic surface modification with embedded surfactants can also be used. As used herein, the terms “organ-chip” and “organ-on-a-chip” are used interchangeably and refer to a biomimetic microfluidic device comprising at least one cell type and physiological function of at least one mammalian (e.g., human) organ or tissue. While the organ-on-a-chip devices described herein mimic the physiological functions of a mammalian organ, it is to be understood that these devices can also be designed to mimic the functionality of any living organ from humans or other organisms (e.g., animals, insects, plants). As such, the devices and methods described herein can be used to model or study mammalian as well as non-mammalian (e.g., insects, plants, etc.) organs and physiological systems and the effect of active agents and mechanical stimuli on such organs and physiological systems. Organs are made up of different types of cells with various shaped backbone structures. To create realistic three-dimensional (3D) in vitro models, the diverse backbone structures are required to mimic the target organs and create organ-chips. Much effort has been devoted in the past to develop these 3D in vitro models. However, more investigations are needed in recapitulating biomechanical microenvironments associated with geometrical and dynamic aspects. In some embodiments described herein, biomimetic microfluidic devices are disclosed that can model certain eye cells and tissue structures. For example, in one nonlimiting exemplary aspect, the disclosed device is a glaucoma eye organ-on-a-chip microfluidic device comprising a porous membrane layer that mimics a juxtacanalicular tissue structure with cultured trabecular meshwork (TM) cells and Schlemm’s canal (SC) cells (see FIG. 1).

[0057] Biomimetic Microfluidic Devices

[0058] Described herein are biomimetic microfluidic devices useful for culturing and monitoring one or more cell types. The disclosed devices may also be useful for screening anti-glaucoma therapeutic agents when certain cell types are cultured and monitored. For example, when trabecular meshwork (TM) cells are cultured, the disclosed devices may be used to investigate ocular outflow and TM cell function in response to treatment with various agents. The disclosed devices are configured for simultaneous and independent control of substrate stiffness, mechanical stretch and strain, and shear stress to provide a dynamic biomimetic microenvironment. Other cell culture devices and platforms that provide dynamic biomimetic microenvironments have been described in U.S. Pat. App. Pub. No. US 2023 / 0365908 A1 , which is incorporated by reference herein in its entirety for such teachings.

[0059] In various embodiments, the disclosed biomimetic microfluidic devices may comprise a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer. FIG. 4-5 show exemplary biomimetic microfluidic devices as disclosed herein.

[0060] In certain embodiments, the substrate base layer may comprise silanized glass. In certain embodiments, the substrate base layer may be about 0.1 mm to about 2 mm thick (e.g., 1 mm glass substrate).

[0061] In certain embodiments, each of the first microfluidic channel layer, second microfluidic channel layer, and membrane layer may comprise a polymeric material of polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof.

[0062] In certain embodiments, the membrane layer may be porous and comprise an electrospun collagen nanofiber membrane with enhanced biocompatibility and cell adhesion properties. In certain embodiments, the membrane layer may be porous and comprise a porosity of about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 70% to about 80%, about 70% to about 90%, or about 80% to about 90%. In certain embodiments, the membrane layer may be porous and comprise a porosity of about 70% to about 80% and be configured to permit selective molecular transport while maintaining structural integrity.

[0063] In certain embodiments, the membrane layer may be porous and comprise an average pore size of about 0.1 pm to about 1 pm, about 0.1 pm to about 2.5 pm, about 0.1 pm to about 5 pm, about 0.1 pm to about 7.5 pm, about 0.1 pm to about 10 pm, about 1 pm to about 2.5 pm, about 1 pm to about 5 pm, about 1 pm to about 7.5 pm, about 1 pm to about 10 pm, about 2.5 pm to about 5 pm, about 2.5 pm to about 7.5 pm, about 2.5 pm to about 10 pm, about 5 pm to about 7.5 pm, about 5 pm to about 10 pm, or about 7.5 pm to about 10 pm. In certain embodiments, the membrane layer may be porous and comprise an average pore size of about 1 pm to about 5 pm and be optimized for cellular communication and nutrient transport.

[0064] In certain embodiments, the membrane layer may comprise a flat, concave, or convex shaped curvature upon adjusting the pressure in the first microfluidic channel layer to stretch the population of cells with controllable strain levels ranging from 0% to 50% linear strain. The hydraulic actuation system of the first microfluidic channel layer is configured for real-time pressure control capabilities.

[0065] In certain embodiments, the membrane layer may comprise a uniform thickness across its surface of about 25 pm to about 50 pm, about 25 pm to about 100 pm, about 25 pm to about 150 pm, about 25 pm to about 200 pm, about 25 pm to about 250 pm, about 50 pm to about 100 pm, about 50 pm to about 150 pm, about 50 pm to about 200 pm, about 50 pm to about 250 pm, about 100 pm to about 150 pm, about 100 pm to about 200 pm, about 100 pm to about 250 pm, about 150 pm to about 200 pm, about 150 pm to about 250 pm, or about 200 pm to about 250 pm. In certain embodiments, the membrane layer may comprise a uniform thickness across its surface of about 25 pm to about 250 pm to provide consistent mechanical properties and optical clarity.

[0066] In certain embodiments, the second microfluidic channel layer may comprise Hele-Shaw geometrical dimensions to modulate cellular shear stress. As used herein, “Hele-Shaw” geometry refers to a specific configuration in microfluidics between closely spaced parallel plates that is used to study and manipulate fluid flow in a quasi-two-dimensional environment. The fluid flow is laminar and controlled by a balance between viscous and pressure forces. In certain nonlimiting exemplary embodiments, the second microfluidic channel layer may comprise Hele-Shaw geometrical dimensions comprising a width of about 2 mm to about 15 mm, a length of about 25 mm to about 30 mm, and a height of about 0.05 mm to about 0.1 mm. In certain embodiments, the second microfluidic channel layer may be configured to modulate shear stress on a population of cells from about 0.01 dyn / cm2to about 30 dyn / cm2with programmable temporal control patterns. In certain embodiments, the second microfluidic channel layer may be configured for fluid flow rates of about 1 pL / min to about 750 pL / min to modulate shear stress on the population of cells. In certain embodiments, the fluid flow rates through the second microfluidic channel layer may be controlled by automated flow control systems.

[0067] In certain embodiments, the hydrogel layer may comprise gelatin, collagen, elastin, alginate, or combinations thereof. In certain embodiments, the hydrogel layer may comprise a photo-crosslinkable material comprising gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), or combinations thereof. In one nonlimiting exemplary embodiment, the hydrogel layer comprises gelatin methacrylate (GelMA) at a concentration of about 1% w / v to about 20% w / v. In certain embodiments, the hydrogel layer may comprise a Young’s modulus of about 1 kPa to about 25 kPa, providing tunable mechanical properties and stiffness conditions. In certain embodiments, the concentration and Young’s modulus of the hydrogel layer are associated with a stiffness of a population of cells. In certain embodiments, the hydrogel layer may have a uniform thickness of about 0.05 mm to about 0.5 mm, providing a uniform cell culture environment.

[0068] In certain embodiments, the disclosed biomimetic microfluidic devices may further comprise one or more of a plurality of receiving channels, outlets, flow sensors, and pressure transducers to measure fluid flow rates of outflow from a population of cells. In certain aspects, these receiving channels, outlets, flow sensors, and / or pressure transducers may be directly connected to the second microfluidic channel layer.

[0069] In certain embodiments, a population of cells is cultured on the first surface of the membrane layer, the second surface of the membrane layer, or a combination thereof. In certain embodiments, a population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof. In certain embodiments, a population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof. In certain embodiments, TM cells may comprise healthy normal TM cells and / or glaucomatous TM cells. In certain embodiments, TM cells may be cultured on the second surface of the membrane layer and SC cells may be cultured on the first surface of the membrane layer (see e.g., FIG. 1). In certain embodiments, the membrane layer may mimic a juxtacanalicular tissue structure. The juxtacanalicular tissue (JCT), also known as the cribriform region, is the outermost layer of the TM in the eye and lies directly adjacent to the SC, which is the final drainage pathway for aqueous humor exiting the anterior chamber of the eye. In one nonlimiting exemplary embodiment, the biomimetic microfluidic device is a glaucoma eye organ-on-a-chip microfluidic device.

[0070] In certain embodiments, the membrane layer may be optically transparent for real-time cell imaging. In certain embodiments, the entire biomimetic microfluidic device may be optically transparent for real-time cell imaging and analysis.

[0071] Methods of Use

[0072] Also described herein are methods of culturing and monitoring one or more cell types using the biomimetic microfluidic devices disclosed herein.

[0073] In various embodiments, the method may comprise inserting a population of cells into a biomimetic microfluidic device as disclosed herein, modulating the hydraulic pressure in the first microfluidic channel layer to alter the shape of the membrane layer and stretch the population of cells, and analyzing the population of cells in the device.

[0074] In certain embodiments, the method may further comprise applying a fluid flow through the second microfluidic channel layer to modulate the shear stress on the population of cells prior to analyzing the population of cells in the device, wherein the shear stress and stretch are applied simultaneously to the population of cells.

[0075] In certain embodiments, the population of cells may comprise trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof. In certain embodiments, the TM cells may be cultured on the second surface of the membrane layer and SC cells may be cultured on the first surface of the membrane layer. In certain embodiments, the TM cells may be derived from a subject having glaucoma and / or from a healthy normal subject. In certain embodiments, the TM cells may be isolated from juxtacanalicular and / or corneoscleral meshwork eye regions of a subject. In certain embodiments, the population of cells may be cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof.

[0076] In certain embodiments, the membrane layer of the biomimetic microfluidic device may be surface treated with one or more extracellular matrix proteins comprising collagen type I, fibronectin, or a combination thereof prior to inserting the population of cells into the device.

[0077] In certain embodiments, the population of cells may be analyzed in the biomimetic microfluidic device using one or more imaging techniques. In certain embodiments, the method may further comprise performing one or more biochemical assays on the population of cells. In certain embodiments, the method may further comprise administering one or more anti-glaucoma therapeutic agents into the device.

[0078] Also described herein are methods of screening one or more anti-glaucoma therapeutic agents using the biomimetic microfluidic devices disclosed herein.

[0079] In various embodiments, the method may comprise inserting a population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof into a biomimetic microfluidic device as disclosed herein, administering one or more antiglaucoma therapeutic agents into the device, and analyzing the population of cells in the device.

[0080] In certain embodiments, the TM cells may be cultured on the second surface of the membrane layer and the SC cells may be cultured on the first surface of the membrane layer. In certain embodiments, the membrane layer may be porous and comprise an electrospun collagen nanofiber membrane.

[0081] In certain embodiments, the anti-glaucoma therapeutic agent may comprise one or more ion channel agonists or antagonists for Piezol , TRPV4, TREK-1 , or combinations thereof. In certain embodiments, the anti-glaucoma therapeutic agent may comprise an actin depolymerization agent, a Rho kinase inhibitor, or a combination thereof.

[0082] In certain embodiments, the method may further comprise modulating one or more of stiffness, stretch, and shear stress conditions for the population of cells. In certain embodiments, the method may further comprise measuring fluid flow rates of outflow from the population of cells.

[0083] Kits

[0084] In some embodiments, this disclosure further relates to kits for culturing and monitoring one or more cell types. The kits may comprise any of the biomimetic microfluidic devices disclosed herein, optionally, cell culture buffers, reagents, and receptacles, and, optionally, one or more of packaging, a label, information, or instructions for use. In certain embodiments, the disclosed kits may also be useful for screening one or more anti-glaucoma therapeutic agents. The information and instructions of the disclosed kits may be in the form of words, pictures, internet addresses, internet viewable or downloadable media, and the like.

[0085] Embodiments

[0086] One embodiment described herein is a biomimetic microfluidic device, comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer. In one aspect, the substrate base layer comprises silanized glass. In another aspect, each of the first microfluidic channel layer, second microfluidic channel layer, and membrane layer comprises a polymeric material of polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof. In another aspect, the membrane layer is porous and comprises an electrospun collagen nanofiber membrane. In another aspect, the membrane layer is porous and comprises a porosity of about 70% to about 80%. In another aspect, the membrane layer is porous and comprises an average pore size of about 1 pm to about 5 pm. In another aspect, the membrane layer comprises a flat, concave, or convex shaped curvature upon adjusting the pressure in the first microfluidic channel layer to stretch the population of cells ranging from 0% to 50% linear strain. In another aspect, the membrane layer comprises a uniform thickness across its surface of about 25 pm to about 250 pm. In another aspect, the second microfluidic channel layer comprises Hele-Shaw geometrical dimensions comprising a width of about 2 mm to about 15 mm, a length of about 25 mm to about 30 mm, and a height of about 0.05 mm to about 0.1 mm. In another aspect, the second microfluidic channel layer is configured to modulate shear stress on the population of cells from about 0.01 dyn / cm2to about 30 dyn / cm2. In another aspect, the second microfluidic channel layer is configured for fluid flow rates of about 1 pL / min to about 750 pL / min to modulate shear stress on the population of cells. In another aspect, the hydrogel layer comprises gelatin, collagen, elastin, alginate, or combinations thereof. In another aspect, the hydrogel layer comprises a photo-crosslinkable material comprising gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), or combinations thereof. In another aspect, the hydrogel layer comprises gelatin methacrylate (GelMA) at a concentration of about 1% w / v to about 20% w / v. In another aspect, the hydrogel layer comprises a Young’s modulus of about 1 kPa to about 25 kPa. In another aspect, the concentration and Young’s modulus of the hydrogel layer are associated with a stiffness of the population of cells. In another aspect, the hydrogel layer has a uniform thickness of about 0.05 mm to about 0.5 mm. In another aspect, the device further comprises one or more of a plurality of receiving channels, outlets, flow sensors, and pressure transducers to measure fluid flow rates of outflow from the population of cells. In another aspect, the population of cells is cultured on the first surface of the membrane layer, the second surface of the membrane layer, or a combination thereof. In another aspect, the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof. In another aspect, the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof. In another aspect, the TM cells comprise glaucomatous TM cells. In another aspect, TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer. In another aspect, the membrane layer mimics a juxtacanalicular tissue structure. In another aspect, the device is a glaucoma eye organ-on-a-chip microfluidic device. In another aspect, the device is capable of simultaneously and independently modulating stiffness, stretch, and shear stress conditions for the population of cells. In another aspect, the membrane layer is optically transparent for real-time cell imaging. In another aspect, the device is optically transparent for real-time cell imaging.

[0087] Another embodiment described herein is a method of culturing and monitoring one or more cell types, the method comprising: inserting a population of cells into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; modulating the hydraulic pressure in the first microfluidic channel layer to alter the shape of the membrane layer and stretch the population of cells; and analyzing the population of cells in the device. In one aspect, the method further comprises applying a fluid flow through the second microfluidic channel layer to modulate the shear stress on the population of cells prior to analyzing the population of cells in the device, wherein the shear stress and stretch are applied simultaneously to the population of cells. In another aspect, the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof. In another aspect, TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer. In another aspect, the TM cells are derived from a subject having glaucoma. In another aspect, the TM cells are isolated from juxtacanalicular and / or corneoscleral meshwork eye regions of a subject. In another aspect, the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof. In another aspect, the membrane layer of the biomimetic microfluidic device is surface treated with one or more extracellular matrix proteins comprising collagen type I, fibronectin, or a combination thereof prior to inserting the population of cells into the device. In another aspect, the population of cells is analyzed in the biomimetic microfluidic device using one or more imaging techniques. In another aspect, the method further comprises performing one or more biochemical assays on the population of cells. In another aspect, the method further comprises administering one or more anti-glaucoma therapeutic agents into the device.

[0088] Another embodiment described herein is a method of screening one or more antiglaucoma therapeutic agents, the method comprising: inserting a population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; administering one or more anti-glaucoma therapeutic agents into the device; and analyzing the population of cells in the device. In one aspect, TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer. In another aspect, the membrane layer is porous and comprises an electrospun collagen nanofiber membrane. In another aspect, the anti-glaucoma therapeutic agent comprises one or more ion channel agonists or antagonists for Piezol , TRPV4, TREK-1 , or combinations thereof. In another aspect, the anti-glaucoma therapeutic agent comprises an actin depolymerization agent, a Rho kinase inhibitor, or a combination thereof. In another aspect, the method further comprises modulating one or more of stiffness, stretch, and shear stress conditions for the population of cells. In another aspect, the method further comprises measuring fluid flow rates of outflow from the population of cells.

[0089] Another embodiment described herein is a kit for culturing and monitoring one or more cell types, the kit comprising: a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; optionally, cell culture buffers, reagents, and receptacles; and optionally, one or more of packaging or instruction for use.

[0090] Another embodiment described herein is the use of a biomimetic microfluidic device for culturing and monitoring one or more cell types in response to modulation of stiffness, stretch, and / or shear stress conditions, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

[0091] Another embodiment described herein is the use of a biomimetic microfluidic device for screening one or more anti-glaucoma therapeutic agents, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells, the population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

[0092] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0093] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0094] Clause 1 . A biomimetic microfluidic device, comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

[0095] Clause 2. The device of clause 1 , wherein the substrate base layer comprises silanized glass.

[0096] Clause 3. The device of clause 1 or 2, wherein each of the first microfluidic channel layer, second microfluidic channel layer, and membrane layer comprises a polymeric material of polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof.

[0097] Clause 4. The device of any one of clauses 1-3, wherein the membrane layer is porous and comprises an electrospun collagen nanofiber membrane.

[0098] Clause 5. The device of any one of clauses 1-4, wherein the membrane layer is porous and comprises a porosity of about 70% to about 80%.

[0099] Clause 6. The device of any one of clauses 1-5, wherein the membrane layer is porous and comprises an average pore size of about 1 pm to about 5 pm.

[0100] Clause 7. The device of any one of clauses 1-6, wherein the membrane layer comprises a flat, concave, or convex shaped curvature upon adjusting the pressure in the first microfluidic channel layer to stretch the population of cells ranging from 0% to 50% linear strain.

[0101] Clause 8. The device of any one of clauses 1-7, wherein the membrane layer comprises a uniform thickness across its surface of about 25 pm to about 250 pm.

[0102] Clause 9. The device of any one of clauses 1-8, wherein the second microfluidic channel layer comprises Hele-Shaw geometrical dimensions comprising a width of about 2 mm to about 15 mm, a length of about 25 mm to about 30 mm, and a height of about 0.05 mm to about 0.1 mm.

[0103] Clause 10. The device of any one of clauses 1-9, wherein the second microfluidic channel layer is configured to modulate shear stress on the population of cells from about 0.01 dyn / cm2to about 30 dyn / cm2.

[0104] Clause 11. The device of any one of clauses 1-10, wherein the second microfluidic channel layer is configured for fluid flow rates of about 1 pL / min to about 750 pL / min to modulate shear stress on the population of cells.

[0105] Clause 12. The device of any one of clauses 1-11 , wherein the hydrogel layer comprises gelatin, collagen, elastin, alginate, or combinations thereof.

[0106] Clause 13. The device of any one of clauses 1-12, wherein the hydrogel layer comprises a photo-crosslinkable material comprising gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), or combinations thereof.

[0107] Clause 14. The device of any one of clauses 1-13, wherein the hydrogel layer comprises gelatin methacrylate (GelMA) at a concentration of about 1% w / v to about 20% w / v.

[0108] Clause 15. The device of any one of clauses 1-14, wherein the hydrogel layer comprises a Young’s modulus of about 1 kPa to about 25 kPa.

[0109] Clause 16. The device of any one of clauses 1-15, wherein the concentration and Young’s modulus of the hydrogel layer are associated with a stiffness of the population of cells.

[0110] Clause 17. The device of any one of clauses 1-16, wherein the hydrogel layer has a uniform thickness of about 0.05 mm to about 0.5 mm.

[0111] Clause 18. The device of any one of clauses 1-17, further comprising one or more of a plurality of receiving channels, outlets, flow sensors, and pressure transducers to measure fluid flow rates of outflow from the population of cells.

[0112] Clause 19. The device of any one of clauses 1-18, wherein the population of cells is cultured on the first surface of the membrane layer, the second surface of the membrane layer, or a combination thereof.

[0113] Clause 20. The device of any one of clauses 1-19, wherein the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof.

[0114] Clause 21. The device of any one of clauses 1-20, wherein the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof.

[0115] Clause 22. The device of any one of clauses 1-21, wherein the TM cells comprise glaucomatous TM cells. Clause 23. The device of any one of clauses 1-22, wherein TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer.

[0116] Clause 24. The device of any one of clauses 1-23, wherein the membrane layer mimics a juxtacanalicular tissue structure.

[0117] Clause 25. The device of any one of clauses 1-24, wherein the device is a glaucoma eye organ-on-a-chip microfluidic device.

[0118] Clause 26. The device of any one of clauses 1-25, wherein the device is capable of simultaneously and independently modulating stiffness, stretch, and shear stress conditions for the population of cells.

[0119] Clause 27. The device of any one of clauses 1-26, wherein the membrane layer is optically transparent for real-time cell imaging.

[0120] Clause 28. The device of any one of clauses 1-27, wherein the device is optically transparent for real-time cell imaging.

[0121] Clause 29. A method of culturing and monitoring one or more cell types, the method comprising: inserting a population of cells into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; modulating the hydraulic pressure in the first microfluidic channel layer to alter the shape of the membrane layer and stretch the population of cells; and analyzing the population of cells in the device.

[0122] Clause 30. The method of clause 29, further comprising applying a fluid flow through the second microfluidic channel layer to modulate the shear stress on the population of cells prior to analyzing the population of cells in the device, wherein the shear stress and stretch are applied simultaneously to the population of cells.

[0123] Clause 31. The method of clause 29 or 30, wherein the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof.

[0124] Clause 32. The method of any one of clauses 29-31 , wherein TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer.

[0125] Clause 33. The method of any one of clauses 29-32, wherein the TM cells are derived from a subject having glaucoma.

[0126] Clause 34. The method of any one of clauses 29-33, wherein the TM cells are isolated from juxtacanalicular and / or corneoscleral meshwork eye regions of a subject.

[0127] Clause 35. The method of any one of clauses 29-34, wherein the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof.

[0128] Clause 36. The method of any one of clauses 29-35, wherein the membrane layer of the biomimetic microfluidic device is surface treated with one or more extracellular matrix proteins comprising collagen type I, fibronectin, or a combination thereof prior to inserting the population of cells into the device.

[0129] Clause 37. The method of any one of clauses 29-36, wherein the population of cells is analyzed in the biomimetic microfluidic device using one or more imaging techniques.

[0130] Clause 38. The method of any one of clauses 29-37, further comprising performing one or more biochemical assays on the population of cells.

[0131] Clause 39. The method of any one of clauses 29-38, further comprising administering one or more anti-glaucoma therapeutic agents into the device.

[0132] Clause 40. A method of screening one or more anti-glaucoma therapeutic agents, the method comprising: inserting a population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; administering one or more anti-glaucoma therapeutic agents into the device; and analyzing the population of cells in the device.

[0133] Clause 41. The method of clause 40, wherein TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer.

[0134] Clause 42. The method of clause 40 or 41 , wherein the membrane layer is porous and comprises an electrospun collagen nanofiber membrane.

[0135] Clause 43. The method of any one of clauses 40-42, wherein the anti-glaucoma therapeutic agent comprises one or more ion channel agonists or antagonists for Piezol , TRPV4, TREK-1 , or combinations thereof.

[0136] Clause 44. The method of any one of clauses 40-43, wherein the anti-glaucoma therapeutic agent comprises an actin depolymerization agent, a Rho kinase inhibitor, or a combination thereof. Clause 45. The method of any one of clauses 40-44, further comprising modulating one or more of stiffness, stretch, and shear stress conditions for the population of cells.

[0137] Clause 46. The method of any one of clauses 40-45, further comprising measuring fluid flow rates of outflow from the population of cells.

[0138] Clause 47. A kit for culturing and monitoring one or more cell types, the kit comprising: a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; optionally, cell culture buffers, reagents, and receptacles; and optionally, one or more of packaging or instruction for use.

[0139] Clause 48. Use of a biomimetic microfluidic device for culturing and monitoring one or more cell types in response to modulation of stiffness, stretch, and / or shear stress conditions, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

[0140] Clause 49. Use of a biomimetic microfluidic device for screening one or more anti-glaucoma therapeutic agents, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells, the population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer. EXAMPLES

[0141] Example 1

[0142] Glaucoma Chip using A Dual-Chamber Hele-Shaw Microfluidic System for Glaucoma Modeling and Drug Testing

[0143] The biomimetic microfluidic device technology disclosed herein represents a breakthrough microfluidic platform designed to revolutionize glaucoma research and drug development. The device includes a dual-chamber / channel trabecular meshwork chip (TM chip) that utilizes Hele- Shaw microchannel design and geometrical dimensions to precisely control and study the effects of shear stress on human eye cells involved in glaucoma pathophysiology.

[0144] The disclosed devices provide a comprehensive platform that can simultaneously study individual cellular responses and important cell-cell interactions between trabecular meshwork (TM) cells and Schlemm’s canal (SC) cells under precisely controlled mechanical conditions. This addresses a fundamental gap in current glaucoma research, where existing treatments target only secondary outflow pathways handling 5-15% of fluid drainage, while ignoring the primary pathway responsible for 85-95% of aqueous humor outflow.

[0145] Technical Problem Addressed

[0146] Current glaucoma treatments face several important limitations that this technology directly addresses, as further discussed below.

[0147] Limited Therapeutic Targets

[0148] Existing medications primarily target the ciliary muscle pathway, which handles only a small fraction of eye fluid drainage, leaving the major drainage pathway (TM-SC interface) completely untargeted.

[0149] Inadequate Research Models

[0150] Previous in vitro approaches only test discrete shear stress levels and study individual cell types in isolation, preventing comprehensive understanding of dose-response relationships and the crucial interactions between different cell types that occur in the living eye.

[0151] Lack of physiologically Relevant Testing Platforms

[0152] No existing system enables direct measurement of how mechanosensitive ion channels regulate fluid outflow under controlled conditions while properly modeling the TM-SC tissue interface. Incomplete Understanding of Disease Mechanisms

[0153] The precise mechanisms by which mechanical forces trigger cellular responses that lead to elevated eye pressure and glaucoma progression remain poorly understood, hindering development of effective treatments.

[0154] Technical Solution

[0155] Core Platform Design

[0156] The disclosed device comprises a sophisticated dual-chamber microfluidic system featuring: Hele-Shaw Microchannel Architecture.

[0157] As illustrated in FIG. 1 and FIG. 2, the platform employs parallel microchannels with precisely engineered dimensions (widths ranging 2-8 mm, 20 mm length, 100 pm height) that generate continuous, linearly varying shear stress profiles from 0.01 to 30 dyn / cm2. FIG. 1 demonstrates the fundamental Hele-Shaw channel design principles, while FIG. 2 shows the validated fabrication and cell culture viability within the microchannel system. This eliminates experimental variations from using multiple devices and enables comprehensive dose-response mapping on a single chip.

[0158] Dual-Chamber Configuration

[0159] The system features separate but connected top and bottom chambers / channels, allowing for three distinct experimental configurations that provide unprecedented insight into cellular behavior:

[0160] Configuration 1 : TM cells only (top chamber active)

[0161] Configuration 2: SC cells only (bottom chamber active)

[0162] Configuration 3: TM-SC co-culture (both chambers active with cellular interaction)

[0163] Biomimetic Membrane Interface

[0164] A specialized porous collagen membrane fabricated using near-field electrospinning separates the chambers while enabling paracrine communication and flow measurement. This membrane mimics the juxtacanalicular tissue structure with controlled porosity (70-80%), pore size (1-5 pm), and mechanical properties matching native tissue.

[0165] Advanced Monitoring and Measurement Systems

[0166] Integrated Sensor Technology The disclosed glaucoma chips can incorporate a micro-flow sensor for real-time monitoring of outflow facility and pressure changes across cellular barriers, enabling direct correlation between cellular responses and functional outcomes. FIG. 3 specifically illustrates the dual-chambered microfluidic platform showing the integration of Hele-Shaw microchannels in both top and bottom chambers with the porous membrane interface.

[0167] Multi-Modal Analysis Capabilities

[0168] The disclosed cell culture devices support comprehensive cellular analysis including:

[0169] Live calcium imaging for ion channel activation monitoring (performed at 1 Hz acquisition for 30 minutes per experiment)

[0170] Immunofluorescence mapping for protein expression analysis

[0171] Gene expression profiling for molecular response characterization

[0172] Nitric oxide production measurement for endothelial function assessment using fluorescent NO indicators (DAF-FM)

[0173] Cytoskeletal remodeling analysis for mechanotransduction studies including F-actin visualization with fluorescent phalloidin

[0174] Mechanosensitive Ion Channel Investigation

[0175] The platform specifically targets three important mechanosensitive ion channels known to regulate eye pressure, as discussed below.

[0176] Piezol Channel Analysis

[0177] Rapid pressure-responsive channel activation (within 30 seconds) that dramatically increases fluid drainage through the conventional outflow pathway. Validated using agonist Yodal (1-10 pM) and antagonist GsMTx4 (100 nM).

[0178] TRPV4 Channel Characterization

[0179] Slower-activating channel (2-5 minutes timescale) involved in calcium-dependent cellular remodeling and contractility regulation. Tested using agonist GSK1016790A (10-100 nM) and antagonist HC-067047 (1-10 pM).

[0180] TREK-1 Channel Study Intermediate-kinetics channel functioning as a mechanical threshold regulator and stretch- induced contractility modulator. Investigated using agonist BL-1249 (1-10 pM) and antagonist Spadin (1-10 pM).

[0181] Methodological Approaches

[0182] Three-Configuration Experimental Design

[0183] This approach enables, for the first time, direct comparison of individual cellular contributions versus tissue-tissue interactions.

[0184] Individual Cell Type Analysis

[0185] Configurations 1 and 2 establish baseline mechanosensitive responses for TM and SC cells independently. TM cells are characterized by expression of myocilin, SPARC, and a-smooth muscle actin markers, while SC cells are validated through CD31 , VE-cadherin, and eNOS expression.

[0186] Interactive System Analysis

[0187] Configuration 3 reveals how TM-SC interactions modulate individual cellular responses, creating coordinated tissue-level responses that differ significantly from isolated cell behaviors. This configuration uses electrospun collagen nanofiber membranes to separate chambers while allowing paracrine communication.

[0188] Comparative Analysis Framework

[0189] By testing identical conditions across all three configurations, the system can quantify individual contributions, identify synergistic effects, map paracrine signaling pathways, and characterize disease-specific differences.

[0190] Advanced Pharmacological Testing Capabilities

[0191] The platform enables sophisticated drug testing through:

[0192] Channel-Specific Modulation

[0193] Systematic testing of specific agonists and antagonists for each ion channel type, with perfusion chambers operating at 2, 4, 8, and 16 pL / min flow rates to generate varying shear stress conditions.

[0194] Dose-Response Mapping Continuous shear stress generation enables precise determination of activation thresholds. Expected results include Piezol activation around 2-5 dyn / cm2, TRPV4 activation around 1-3 dyn / cm2, and TREK-1 activation around 5-10 dyn / cm2.

[0195] Temporal Dynamics Analysis

[0196] Extended monitoring capabilities (24-72 hours) reveal whether therapeutic effects are maintained, adapted, or dysregulated over time, providing crucial information for chronic versus acute treatment strategies.

[0197] Fabrication and Manufacturing Process

[0198] Microfabrication Technology

[0199] Soft Lithography Process

[0200] FIG. 4 illustrates the complete microfabrication process including soft lithography for PDMS microchannels and collagen-fiber formation for mimicking the cribriform meshwork structure.

[0201] Porous Membranes

[0202] A porous polycarbonate or polystyrene membrane was used in this glaucoma organ-on- a-chip. When a high-porosity membrane is needed, it can be fabricated through near-field electrospinning of various polymer materials, enabling precise control of porosity, pore size distribution, and mechanical properties to match native tissue characteristics.

[0203] Quality Control Systems

[0204] Each chip undergoes comprehensive testing including fluorescent dye leak testing for bonding integrity, mechanical property verification for the membrane interface, and sterilization validation for cell culture applications.

[0205] Standardized Cell Culture Protocols

[0206] Primary Cell Isolation and Characterization

[0207] Protocols for isolating and expanding human TM and SC cells from donor tissue (supplied by Utah Eye Bank), with seeding densities of 1x10scells per membrane and 7-10 days cultivation for proper barrier formation.

[0208] ECM Functionalization Surface treatment protocols using cell-specific extracellular matrix proteins (collagen type I for TM cells, fibronectin for SC cells) to optimize cell attachment and maintain physiological phenotypes.

[0209] Co-Culture Optimization

[0210] Specialized protocols for achieving balanced confluent cell layers in co-culture configurations while maintaining distinct cellular populations and enabling proper tissue-tissue interactions.

[0211] Applications and Commercial Potential

[0212] Pharmaceutical Drug Development

[0213] Primary Screening Platform

[0214] The technology provides the first comprehensive platform for testing glaucoma drug candidates that target the primary outflow pathway, representing a massive untapped therapeutic market. Current glaucoma medications include prostaglandin analogs, beta blockers, alpha agonists, carbonic anhydrase inhibitors, and Rho kinase inhibitors, but none effectively target the TM-SC interface.

[0215] Combination Therapy Development

[0216] The three-configuration approach enables identification of compounds that work synergistically by targeting different cell types or enhancing beneficial cell-cell interactions. Testing includes actin depolymerization agents (latrunculin) and Rho kinase inhibitors (Y-27632) to observe agonistic and antagonistic effects.

[0217] Personalized Medicine Applications

[0218] Patient-derived cells can be tested across all configurations to identify optimal therapeutic strategies based on individual cellular responses and interaction patterns. The platform enables comparison of normal versus glaucomatous cell combinations in multiple configurations.

[0219] Disease Mechanism Research

[0220] Glaucoma Pathophysiology Studies

[0221] The platform enables investigation of how disease progression affects individual cell types and their interactions differently. Glaucomatous cells show elevated [Ca2+]i, generate more actin stress fibers, secrete more ECM, and appear more contractile compared to normal cells. Comorbidity Factor Analysis

[0222] The system can study how systemic conditions (diabetes, hypertension, aging) affect outflow pathway function at the cellular level using cells from donors with these conditions.

[0223] Circadian Regulation Investigation

[0224] Both TM and SC cells exhibit circadian rhythms affecting eye pressure. The platform can investigate temporal patterns of shear stress to understand circadian mechanics of outflow regulation.

[0225] Broader Biomedical Applications

[0226] Tissue Interface Modeling

[0227] The dual-chamber approach with biomimetic membrane interface has potential applications beyond glaucoma research for studying other tissue-tissue interactions in disease and health.

[0228] Mechanobiology Platform

[0229] The precise shear stress control system has applications in studying mechanosensitive cellular responses in other organ systems where fluid flow and mechanical forces drive pathophysiology.

[0230] Organ-on-a-Chip Technology

[0231] The platform represents an advancement in organ-on-a-chip technology specifically designed for studying complex tissue interfaces with controlled mechanical environments.

[0232] Competitive Advantages and Technical Superiority

[0233] High Precision and Control

[0234] Continuous Stress Profiling

[0235] Unlike existing discrete-level testing approaches, the Hele-Shaw design generates continuous shear stress gradients linearly increasing with channel length, enabling precise threshold determination and comprehensive dose-response mapping impossible with current methods. As demonstrated in FIG. 1—2, shear stress profiles increase linearly with channel length in both top and bottom chambers. Elimination of Experimental Variation

[0236] Single-chip testing across full stress ranges eliminates variations introduced by multiple chip preparations and enables direct statistical comparison of responses across the complete 0.1-30 dyn / cm2range.

[0237] Real-Time Functional Measurement

[0238] Integration of micro-flow sensors and pressure transducers provides immediate functional readouts correlating cellular responses with physiologically relevant outcomes.

[0239] Physiologically Relevant Modeling

[0240] Complete Pathway Representation

[0241] The platform is the first to model the complete primary outflow pathway including both major cell types and their important interactions. The filtering portion of the TM includes uveal meshwork, corneoscleral meshwork, and juxtacanalicular tissue (JCT), with the largest resistance generated by JCT interfacing with the inner wall of SC.

[0242] Native Tissue Mimicry

[0243] The electrospun collagen membrane interface closely replicates native juxtacanalicular tissue structure with appropriate pore sizes (target 1-5 pm), porosity (target 70-80%), and elastic moduli matching native tissue properties.

[0244] Scalable Complexity

[0245] The three-configuration approach enables systematic building from simple individual cell responses to complex tissue-level interactions, providing complete mechanistic understanding.

[0246] Performance Outcomes

[0247] Outflow Facility Measurements

[0248] Anticipated results include Configuration 1 (TM-only) baseline outflow facility of 0.05-0.15 pL / min / mmHg with 20-40% enhancement following ion channel agonist treatment; Configuration 2 (SC-only) higher baseline outflow facility of 0.15-0.25 pL / min / mm Hg; and Configuration 3 (TM- SC co-culture) combined resistance with enhanced responses potentially achieving 40-60% enhancement with optimized ion channel modulation.

[0249] Ion Channel Activation Thresholds Expected distinct activation patterns include Piezol activation around 2-5 dyn / cm2with rapid kinetics (<30 seconds), TRPV4 activation around 1-3 dyn / cm2with slower kinetics (2-5 minutes), and TREK-1 activation around 5-10 dyn / cm2with intermediate kinetics.

[0250] Enhanced Mechanical Control Capabilities

[0251] Integrating Stiffness Modulation with Hele-Shaw Channel Technology

[0252] The disclosed platform’s mechanical control capabilities extend beyond shear stress manipulation to include comprehensive biomechanical conditioning that more completely recapitulates the complex mechanical environment of the native outflow pathway. By forming a hydrogel layer on the porous membrane interface (FIG. 4), the system enables independent control of substrate stiffness while maintaining precise shear stress regulation through the Hele- Shaw channel design. This dual-parameter control allows for systematic investigation of how substrate mechanical properties interact with fluid shear forces to modulate cellular responses and outflow regulation.

[0253] Mechanical Stretch Integration

[0254] The disclosed platform incorporates an additional dimension of mechanical stimulation through controlled chip deformation to achieve physiologically relevant mechanical stretch effects (FIG. 5). By implementing controlled bending of the entire chip assembly, the system can apply uniform or gradient mechanical stretch to the cultured cell layers while simultaneously maintaining controlled shear stress and substrate stiffness conditions. This tri-modal mechanical control (stiffness, shear stress, and mechanical stretch) provides unprecedented capability to study the complex mechanobiology of the outflow pathway under conditions that closely mimic the dynamic mechanical environment experienced by TM and SC cells in vivo.

[0255] This enhanced mechanical control system enables investigation of candidate glaucoma drugs under the full spectrum of biomechanical conditions present in both healthy and diseased eyes, providing more physiologically relevant drug screening and mechanism-of-action studies than any currently available platform.

[0256] Example 2

[0257] Investigating the Coupled Effects of Stiffness and Stretch on Trabecular Meshwork Cells using a Hydrogel- Integrated Microfluidic System

[0258] Glaucoma, the leading cause of irreversible blindness globally, is characterized by increased TM cell stiffness and impaired aqueous humor outflow. While TM biomechanics are important for intraocular pressure (IOP) regulation, how substrate stiffness and mechanical stretch interact to influence TM cell behavior remains poorly understood.

[0259] In this example, hydrogel-integrated microfluidic system (HIMS) devices were developed to address this problem. These HIMS devices are capable of independently and simultaneously modulating substrate stiffness and triaxial mechanical stretch - capabilities not available in current platforms. The disclosed HIMS devices combine tunable-stiffness gelatin methacrylate (GelMA) hydrogels with a hydraulically actuated PDMS microfluidic platform. Optimization of UV exposure time revealed that 150 seconds minimizes hydrogel shrinkage (~1.5%) while maintaining dimensional stability important for reproducible cell culture. The disclosed platform supports up to 50% linear strain with excellent reproducibility (CV < 5%) and mechanical integrity across extended culture durations. Finite element simulations closely matched experimental deformation profiles and revealed stress and strain localization patterns that vary with material stiffness.

[0260] Using these HIMS devices, it was found that mechanical stretch amplifies stiffnessdependent mechanotransduction in human TM (TM) and glaucomatous TM (gTM) cells. On stiff substrates (e.g., 20% GelMA), gTM cells exhibited a ~2-fold increase in a-smooth muscle actin (a-SMA) expression and a ~2.5-fold rise in phosphorylated focal adhesion kinase (pFAK) activation compared to softer stiffness conditions (5% GelMA). Notably, applying 10% stretch for 24 hours partially reversed the pathological phenotype in gTM cells on stiff substrates, reducing cell size by -20% and a-SMA expression by -30%. Additionally, normal TM cells exposed to both stretch and pathological stiffness exhibited a 1.9-fold increase in myocilin expression - the highest observed across all conditions - suggesting the presence of a mechanical threshold for disease initiation. Together, the results of this example demonstrate that stiffness and stretch synergistically reinforce a pathological signaling in TM cells, establishing a feed-forward loop that may drive glaucoma progression. Beyond glaucoma, these HIMS devices provide a versatile platform for exploring mechanobiology in mechanically dynamic tissue systems including cardiovascular, musculoskeletal, and cancer microenvironments.

[0261] Hydraulically Actuated Microfluidic Device and System

[0262] The HIMS was designed to enable simultaneous testing of four different conditions with precise mechanical control. First, an inflatable microfluidic system was designed using AutoCAD (Autodesk, USA) and fabricated using five distinct polydimethylsiloxane (PDMS) layers mounted on microscope slides (Fisherbrand 12-550-A3, Fisher Scientific, USA) (FIG. 6A). The five-layer architecture comprised: (1) hydraulic pressure channel layer, (2) PDMS membrane layer, (3) hydrogel channel layer, (4) well layer, and (5) port layer. The hydraulic pressure channel layer, hydrogel channel layer, and well layer were fabricated from 250 pm-thick PDMS sheets (BISCO HT-6240, Rogers Corp., USA) and cut using a vinyl cutter (CAMM-1 GX-24 24", Roland DGA Corp., USA). The PDMS membrane (GASKET-UT-100, SiMPore Inc., USA) was procured commercially to ensure uniformity across experiments. The port layer was cast from PDMS (Sylgard 184, Dow Inc., USA) mixed at a 10:1 ratio (base:curing agent). Glass slides were precleaned sequentially with acetone, isopropyl alcohol (I PA), and deionized water. The five PDMS layers were sequentially bonded to the glass substrate using oxygen plasma treatment (PE-25, Plasma Etch Inc., USA; 100 W, 30 s, 200 mTorr O2). Following assembly, the complete microfluidic system was cured at 70 °C overnight to enhance interlayer bonding. The assembled device was sterilized by sequential treatment with 70% ethanol, deionized water rinse, and UV exposure (30 min at 254 nm). Quality control measurements confirmed dimensional tolerances within ± 5% and inter-device reproducibility with coefficient of variation <10%.

[0263] To enable GelMA hydrogel integration, the hydrogel channel layer surface was functionalized with 3-(trimethoxysilyl)propyl methacrylate (TMSPMA) (M6514, Sigma-Aldrich, USA). The microfluidic system was treated with oxygen plasma (PE-25; 100 W, 60 s, 200 mTorr O2) to generate hydroxyl functional groups on the PDMS surface. A 10% TMSPMA solution in ethanol / deionized water (v / v, 1 :1) was applied to the activated PDMS surface and incubated for 1 h at 50 °C. Following treatment, the surface was rinsed with deionized water to remove excess TMSPMA and dried in a biosafety cabinet with 1 h UV exposure (254 nm) for sterilization. Glass coverslips (8 mm diameter) were silanized for hydrogel integration. Coverslips were cleaned sequentially with acetone, I PA, and deionized water, followed by oxygen plasma treatment (PE- 25; 100 W, 60 s, 200 mTorr O2). Sigmacote® (SL2, Sigma-Aldrich, USA) was applied overnight at 50°C, excess solution was removed with deionized water, and samples were dried in a biosafety cabinet with 1 h UV exposure (254 nm).

[0264] Hydrogel Preparation and Integration

[0265] Hydrogel Preparation

[0266] Gelatin methacrylate (GelMA) hydrogels were prepared at three concentrations (5%, 10%, and 20% w / v) following the manufacturer’s protocol. Photogel® 50% DS (VL3500000502, Cellink, Sweden) was dissolved at room temperature in photo-initiator containing buffer solution. Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) (#5269, Advanced Biomatrix, Sweden) served as the photo-initiator for crosslinking at 365 nm wavelength, while phosphate-buffered saline (PBS, pH 7.4) (10010031 , Thermo Fisher Scientific Inc., USA) maintained physiological pH. The photo-initiator solution was prepared by dissolving LAP in PBS to achieve 0.5% (w / v) concentration, mixed thoroughly using a vortex mixer at 50 °C. The solution was sterile filtered using a 0.22pm syringe filter. To prepare final GelMA concentrations, specific volumes of 0.5% LAP in PBS (10 mL, 5 mL, and 2.5 mL for 5%, 10%, and 20% GelMA, respectively) were added directly to PhotoGel 50% bottles and stirred at 100 rpm at 60 °C for 1 h using a magnetic stir bar.

[0267] Hydrogel Integration

[0268] Strong hydrogel-PDMS integration was achieved through sequential surface modification, where PDMS surfaces were first activated via oxygen plasma treatment to generate hydroxyl groups (OH-PDMS), followed by functionalization with 10% (v / v) 3-(trimethoxysilyl) propyl methacrylate (TMSPMA) in ethanol for 2 hours at room temperature to introduce methacrylate groups (TMSPMA-PDMS), enabling covalent bonding with gelatin methacrylate (GelMA) during subsequent photocrosslinking (FIG. 7). Prior to hydrogel integration, deionized water was introduced into the hydraulic channel to equilibrate and level the PDMS membrane surface, after which a patterned Rubylith® photomask (RU3, Ulano Corp., USA) was positioned at the bottom of the microfluidic device to define crosslinking regions and silanized glass coverslips (8 mm diameter) were placed with the treated surface facing downward onto the hydrogel channel layers, creating temporary sealed microfluidic channels. GelMA solutions at concentrations of 5%, 10%, and 20% (w / v) containing 0.5% (w / v) lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) photoinitiator were prepared in PBS, preheated to 60 °C to reduce viscosity, and introduced into the temporary channels via the center inlet using gentle pipetting to ensure homogeneous distribution and eliminate air bubbles. The hydrogel-filled system was positioned over a UV light source (365 nm) and exposed at 8 J / cm2through the patterned photomask to selectively crosslink hydrogel only in designated well regions, followed by transfer to a cooling plate and maintenance at 4 °C for 5 minutes to complete crosslinking reactions. Glass coverslips were carefully removed, uncured hydrogel from UV-blocked regions was aspirated using a micropipette, and crosslinked hydrogel surfaces were immediately rinsed with PBS and maintained hydrated prior to cell seeding. The 10% TMSPMA concentration was optimized to balance sufficient crosslinking density with surface uniformity, as over-silanization can compromise cell compatibility, and complete removal of uncured GelMA from UV-blocked regions was important for maintaining consistent hydrogel geometry and ensuring uniform stress distribution during mechanical loading assays.

[0269] UV Exposure Optimization To determine optimal UV exposure conditions for uniform GelMA hydrogel formation without swelling, 5% GelMA hydrogels were exposed to UV for varying durations: 60, 90, 120, 150, 180, and 300 s. Following exposure, samples were rested on a cooling plate for 5 min and subsequently submerged in PBS (pH 7.4) for 24 h at 37 °C. The hydrogel surface area was assessed using color dye-enhanced PBS and imaged using a Nikon Eclipse Ti-E inverted microscope to evaluate shrinkage.

[0270] Young’s Modulus of GelMA

[0271] Young’s modulus of GelMA hydrogels was determined using shear rheological measurements (Anton Paar MCR 302). Storage and loss moduli were measured using amplitude sweep rheology with strain increasing from 0 to 200% at constant frequency (1 Hz) in a parallel plate configuration (25 mm diameter). Prior to measurement, hydrogel samples were equilibrated in PBS at room temperature for 20 min to maintain consistent humidity and temperature. Excess PBS was carefully removed using laboratory wipes before analysis.

[0272] Mechanical Characterization

[0273] Experimental Setup: The mechanical testing apparatus consisted of a 1 ml_ BD™ reusable syringe (512027, BD, USA) connected via plastic barbed tube fittings and polyurethane tubing to the hydrogel-integrated microfluidic system. The syringe was actuated using a precision syringe pump (Fusion 200, Chemyx, Inc., USA). Barbed fittings were inserted into inlets with slight interference fit to create pressure- tight seals. Prior to each experiment, constant flow was applied to remove air bubbles from the hydraulic pressure channels and to level the PDMS membrane by confirming zero internal pressure. Following membrane leveling, a plastic sliding clamp was applied to the outlet tubing to prevent leakage during testing. All experiments were conducted at room temperature (22 ± 2°C).

[0274] For Strain Analysis and Burst Testing, optical measurements were performed using an optical microscope equipped with a 4.2 MP near-infrared enhanced CMOS camera (MQ042RG- CM, Ximea, Inc., USA). Image analysis was conducted using open-source software (Fiji / / mageJ2, NIH Image, USA). Membrane strain was calculated assuming semicircular deflection geometry. The deflected arc length (L) was determined from the deflected membrane height h) and original membrane diameter ( / _0) using: The change in arc length (AL) was calculated as:

[0275] AL = L - LQ

[0276] Linear strain (E) was determined from: 100

[0277] Finite Element Analysis

[0278] PDMS Membrane Model

[0279] The PDMS membrane was modeled as a cylindrical geometry centered at (0,0,0) with 2 mm radius and 0.1 mm height. Mesh optimization employed a ratio of 0.1 with 8 slices, 16 segments, and 8 stacks, with Z-bias and R-bias set to 0.8. The wedge center mesh type with Hex8 elements resulted in 4,617 nodes, 1 ,280 faces, and 4,096 elements. Material properties were characterized using the Mooney-Rivlin hyperelastic model fitted to equi-biaxial tension data. PDMS properties included density of 9.7 x 10’7kg / mm3, bulk modulus of 20,000 kPa, and Mooney-Rivlin constants Ci = 38.479 kPa and C2 = 2.9317 kPa derived from experimental curve fitting. Boundary conditions assumed hydraulic pressure application exclusively to the bottom membrane surface. Zero displacement constraints were applied to sidewalls (fixed boundaries), while pressure loading was applied to the bottom surface using the pressure load function.

[0280] PDMS / GelMA Composite Model

[0281] The composite model incorporated a 5% GelMA hydrogel layer (2 mm radius, 0.25 mm height) positioned at (0,0,0.1) above the PDMS membrane. Identical meshing parameters were employed, and components were merged using the Merge Objects function, resulting in 8,725 nodes, 2,048 faces, and 8,192 elements. The PDMS component retained identical Mooney-Rivlin properties. The hydrogel was modeled using a biphasic approach combining Veronda-Westmann hyperelastic behavior for the solid phase with water as the fluid phase. For 5% GelMA, solid phase properties included density of 5 x 1O-8kg / mm3, bulk modulus of 51.33 kPa, and Veronda- Westmann constants Ci = 50 kPa and C2 = 0.415. Fluid phase characteristics included 90% porosity ( o = 0.1), water density of 1 x io-6kg / mm3, zero stabilization parameter (r = 0) for free- draining conditions, and isotropic permeability of 5 x 1O-12mm4 / mN s. For 20% GelMA, enhanced solid phase properties included density of 2 x 10-7kg / mm3, bulk modulus of 125 kPa, and Veronda-Westmann constants Ci = 60 kPa and C2= 0.467, with reduced permeability of 2 xio-13 mm4 / mN s. Boundary conditions included zero displacement constraints at membrane sidewalls, hydraulic pressure loading at the bottom membrane surface, and zero fluid pressure boundary conditions at hydrogel sidewalls and top surface to simulate free-draining behavior in aqueous media.

[0282] Biological Characterization

[0283] Trabecular Meshwork Cell Culture

[0284] Normal trabecular meshwork (TM) cells were isolated from de-identified postmortem eyes of a 53-year-old male donor with no glaucoma history (Utah Lions Eye Bank). Glaucomatous trabecular meshwork (gTM) cells were obtained from a 79-year-old male donor with documented glaucoma, intraocular lenses, hypermetropia, astigmatism, amblyopia, and blepharitis (Utah Lions Eye Bank). Primary TM and gTM cells were isolated from juxtacanalicular and corneoscleral meshwork regions. Both cell types were cultured in collagen type l-coated (Advanced Biomatrix #5005, USA) T75 flasks at 10,000 cells / cm2seeding density under standard conditions (37 °C, 5% CO2). Passages 2-5 were used for all experiments, cultured in Trabecular Meshwork Cell Medium (TMCM) (#6591 , ScienCell Research Laboratories Inc., USA). Cells were passaged at 80-90% confluency using TrypLE™ Express Enzyme (1 x), phenol red-free (#12604013, Thermo Fisher Scientific Inc., USA). Culture medium was exchanged daily following cell seeding on GelMA hydrogels. For static culture conditions, cells were cultured for 48 h post-seeding. For mechanical stimulation experiments, 10% strain was applied for 24 h following an initial 24 h adaptation period on hydrogels.

[0285] Cell Viability Assessment

[0286] Cell viability was assessed using CellTracker™ Green CM FDA fluorescent probes (C7025, Invitrogen Co., USA). CellTracker™ was diluted 1:1000 in TMCM. Existing culture medium was removed and replaced with CellTracker™-containing medium, followed by 30 min incubation at 37 °C. After incubation, the staining solution was replaced with fresh TMCM and cells were imaged using a Nikon Eclipse Ti-E inverted microscope with FITC filter (excitation / emission: 492 / 517 nm).

[0287] Immunofluorescence Analysis

[0288] Cells were washed once with PBS, then fixed with 4% paraformaldehyde in PBS (J61899.AK, Thermo Fisher Scientific Inc., USA) for 30 min at room temperature. Following three 5-min PBS washes, cells were permeabilized with 0.3% Triton X-100 (X-100, Sigma-Aldrich, USA) for 15 min at room temperature. After three additional 5-min PBS washes, non-specific binding was blocked with 20% bovine serum albumin (BSA) (A8022, Sigma-Aldrich, USA) for 24 h at 4 °C. Primary antibodies were applied overnight at 4 °C following BSA removal: anti-a-smooth muscle actin [1A4] (ab7817, Abeam, UK) (1 :100), myocilin [F-12] (sc-137233, Santa Cruz Biotechnology, USA) (1 :100), anti-FAK (phospho Y397) [EP2160Y] (ab81298, Abeam, UK) (1 :400), anti-MMP2 (ab97779, Abeam, UK) (1 :100), and anti-collagen type I (COL1A1) (MAB3391 , Sigma-Aldrich, USA) (1 :100). Following three 5-min PBS washes, secondary antibodies were applied for 1 h at room temperature in darkness: phalloidin-TRITC (P1951 , Sigma-Aldrich, USA) (1 :1000), antirabbit Alexa Fluor 488 (ab150077, Abeam, UK) (1 :200), anti-rabbit Alexa Fluor 594 (A-11012, Thermo Fisher Scientific Inc., USA) (1 :1000), and anti-mouse CF 488A (SAB4600388, Sigma- Aldrich, USA) (1 :200). After three 5-min PBS washes, nuclei were counterstained with Hoechst 33258 (H3569, Thermo Fisher Scientific Inc., USA) (1 :3000) for 15 min at room temperature, followed by a final 5-min PBS wash. Samples were imaged using a Nikon Eclipse Ti-E inverted microscope.

[0289] Image Processing and Statistical Analysis

[0290] Image processing was performed using Nikon NIS-Elements AR 5.0 (Nikon Corp., Japan) and Fiji / lmageJ2 (NIH Image, USA) for large tile images, z-stack orthogonal views, and scale bar addition. Statistical analysis and graphing were conducted using OriginPro 2024 (OriginLab Corp., USA). Each data point represents one well or hydrogel (n > 3 per condition). All measurement data are presented as mean ± standard deviation (SD).

[0291] A Hydrogel-Integrated Microfluidic System

[0292] A hydrogel-integrated microfluidic system (HIMS) was successfully designed and developed that enables multiplexed experiments under precisely controlled material and mechanical conditions (FIG. 6A). The system includes six layers: a glass substrate, five PDMS layers with specific functional roles, and integrated GelMA hydrogels. A unique cross-shaped microfluidic channel design allows for the simultaneous integration of four homogeneous GelMA hydrogels, while a U-shaped hydraulic pressure channel enables concurrent inflation of the four GelMA / PDMS composite membranes. This integrated approach addresses an important limitation in current mechanobiology platforms, which typically lack the capability for simultaneous multi-condition testing under controlled mechanical environments.

[0293] Performance validation demonstrated that the fabricated system achieved optimal dimensional specifications with precise tolerances: 4 mm inlet / outlet ports, 0.25 mm well, hydrogel, and hydraulic layers, a 0.1 mm PDMS membrane, and a 1 mm glass substrate. The PDMS-glass interface achieved robust bonding through a 12-hour post-baking process, with bonding strength exceeding ~80 psi — sufficient to withstand hydraulic pressures applied by a 1mL syringe. The 8.5 mm wells successfully accommodated 8 mm cover glasses while providing adequate space for TM cell culture, and precise layer alignment was maintained while minimizing integration errors. These specifications ensure mechanical stability necessary to support 4 mm diameter hydrogels without inducing PDMS membrane bowing, a common failure mode in similar microfluidic systems.

[0294] The HI MS uniquely enables triaxial stretch of the integrated hydrogel through hydraulic pressure applied in the microfluidic channels (FIG. 6B). Unlike previous research utilizing uniaxial or biaxial stretching, this triaxial mechanical actuation provides more physiologically relevant conditioning that better mimics the complex loading conditions experienced by trabecular meshwork (TM) cells in vivo. The hydraulic pressure-driven mechanism ensures uniform strain distribution across the hydrogel surface, which is important for obtaining reproducible cellular responses and avoiding artifacts associated with non-uniform mechanical stimulation.

[0295] The ability of HIMS to support multi-condition testing in parallel was confirmed by colored dye loading, which showed clearly separated and independently controlled environments in the four chambers (FIG. 6C). This design enables simultaneous testing of four different pressures with four different hydrogel stiffnesses in duplicate samples within a single system, representing a significant improvement in experimental efficiency compared to traditional single-condition platforms. The versatility of the platform extends to supporting multiple cell culture configurations, allowing cells to be cultured at the bottom of wells, within the hydrogel matrix, or on the hydrogel surface for co-culture applications. This flexibility is particularly valuable for mechanobiology studies where different cellular microenvironments may elicit distinct responses to mechanical stimuli. The ability to simultaneously tests with multiple stiffness and mechanical strain conditions while maintaining precise control over both material properties and mechanical loading conditions addresses a longstanding need in the field for mechanobiology platforms that can accelerate the understanding of cellular mechanotransduction. This capability is directly leveraged in later sections (3.6 and 3.7) to investigate the interplay of stiffness and stretch on TM cell mechanobiology, showcasing the utility of HIMS for complex biomechanical investigations.

[0296] Optimization of GelMA Hydrogel Stability and Mechanical Properties

[0297] To develop reliable GelMA hydrogel substrates for biomechanical studies, it was systematically evaluated how UV crosslinking duration and polymer concentration affect the dimensional stability and stiffness of the hydrogels. The effect of UV exposure time on the surface area of 5% GelMA hydrogels after 24-hour incubation in PBS was first assessed (FIG. 8A-B). A clear inverse relationship was observed between UV duration and hydrogel shrinkage. Hydrogels crosslinked for 60 seconds exhibited the highest shrinkage (8.0 ± 0.2%), which decreased with longer exposures: 90 seconds (3.6 ± 0.1 %), 120 seconds (3.2 ± 0.1 %), and 150 seconds (1.5 ± 0.1 %). Shrinkage plateaued beyond 150 seconds, with no significant differences between 150-, 180-, and 300-second exposures. This trend is attributed to increased crosslinking density with longer UV exposure, reducing unreacted methacrylate groups and forming more stable networks.

[0298] As shown in FIG. 8A, hydrogels exposed to shorter durations showed visible swelling and shrinkage, evidenced by the deviation between the original and post-incubation outlines. In contrast, longer exposures produced hydrogels with uniform morphology and minimal size changes. The 8-fold reduction in shrinkage between 60 and 150 seconds (from 8.0% to 1.5%) significantly enhances substrate reliability for cell seeding, ensuring uniform cell distribution and minimizing experimental variability. Based on these findings, 150 seconds was selected as the optimal UV exposure time for preparing dimensionally stable 5% GelMA hydrogels. The mechanical properties of GelMA hydrogels with increasing polymer concentrations were then characterized. Rheological analysis revealed a concentration-dependent increase in Young’s modulus: 1.23 ± 0.21 kPa (5%), 10.74 ± 4.81 kPa (10%), and 21.47 ± 1.42 kPa (20%) (FIG. 9A- B). These values span physiologically relevant stiffness ranges for the trabecular meshwork (TM), with 5% GelMA positioned near the lower end of the normal TM range (0.5-10 kPa), 10% representing the transition from normal to early glaucomatous conditions, and 20% mimicking early-to-moderate disease progression. While these formulations effectively model the biomechanical environment of early-stage glaucoma, they fall substantially below the mean stiffness of glaucomatous eyes (80.8 kPa; range 0.5-565.3 kPa), indicating that additional crosslinking strategies or higher polymer content may be needed for advanced disease modeling. Notably, the 10% GelMA group showed higher variability in modulus values (±4.81 kPa), possibly due to inconsistencies in crosslinking at this intermediate concentration. The ~8-fold increase in stiffness between the 5% and 10% formulations underscores the sensitivity of mechanical properties to small changes in polymer content, reinforcing the importance of formulation precision. These findings identify 150-second UV exposure as the optimal crosslinking condition for producing dimensionally stable 5% GelMA hydrogels. Furthermore, by tuning GelMA concentration, a panel of hydrogels was established with controllable mechanical properties that replicate key aspects of early glaucoma biomechanics. This dual-optimization strategy provides a robust and reproducible hydrogel platform for investigating how mechanical cues influence TM cell behavior and glaucoma progression and offers a foundation for future therapeutic studies targeting mechanotransduction pathways.

[0299] Strain Analysis and Bursting Strength

[0300] Mechanical testing revealed distinct strain-volume relationships across membrane formulations, with PDMS exhibiting the highest deformation capacity, followed by 5%, 10%, and 20% GelMA hydrogels (FIG. 10A-B). This trend corresponds with increased stiffness due to higher GelMA concentrations, where greater crosslinking density limits polymer chain mobility. Notably, an excellent linear correlation (R2= 0.99) was observed between input volume and membrane deformation, demonstrating predictable and reproducible mechanical behavior. PDMS and 5% GelMA membranes achieved up to -50% linear strain, while 10% and 20% GelMA membranes reached -40% before nearing their elastic limits. Bursting pressure analysis confirmed membrane integrity up to 95 pL of input volume, with no leakage or delamination observed through 90 L (FIG. 10C), highlighting the robustness of the HIMS under high-pressure actuation. These strain capacities significantly exceed the -20% physiological strain experienced by trabecular meshwork (TM) cells, providing a broad operational window for studying both normal and pathological loading conditions.

[0301] Among the hydrogel options, 5% GelMA demonstrated an optimal balance between mechanical flexibility and biological compatibility, offering high strain capacity similar to PDMS while providing a more biomimetic substrate stiffness for TM cell culture. Reliable mechanical coupling between the GelMA hydrogel and TMSPPA-modified PDMS membrane was essential for preserving integrity under large deformation. The chemical bonding strategy effectively prevented delamination, ensuring consistent strain transmission to cells — an important factor for precise mechanobiological investigations. The HIMS platform supports large, reproducible, and tunable mechanical strain profiles across physiologically relevant stiffness ranges. This enables the study of TM mechanobiology under dynamic loading conditions while preserving membrane integrity and culture quality, establishing a strong foundation for advanced glaucoma research.

[0302] Finite Element Analysis of HIMS

[0303] The finite element models demonstrated exceptional predictive accuracy with coefficient of determination values exceeding 0.998 for all formulations (FIG. 11). Maximum membrane deflections from simulation and experiment were nearly identical: PDMS (1.738 mm vs. 1.731 mm), 5% GelMA / PDMS (1.927 mm vs. 1.927 mm), and 20% GelMA / PDMS (1.704 mm vs. 1.703 mm), validating the accuracy of the Mooney-Rivlin material model and the reliability of the computational framework. Gel MA concentration inversely affected deflection behavior: the 5% GelMA / PDMS composite exhibited 13.1 % greater maximum deflection than the 20% formulation, consistent with expectations from polymer network theory. Increased GelMA concentration raises crosslink density, limiting polymer chain mobility and increasing stiffness, thus reducing membrane deformation under hydraulic loading (FIG. 11 B and 12A). Stress analysis yielded important insights. Although the 5% GelMA / PDMS composite showed the highest deflection, it also experienced 14.2% higher maximum internal stress (243.5 kPa) compared to the 20% formulation (213.2 kPa), due to the greater deformation of the softer material. Stress patterns also differed: the 5% formulation displayed broader, more uniform stress distributions, while the 20% composite exhibited more localized stress concentrations (FIG. 12B), which may influence fatigue resistance and cell response. Surface stress profiles measured from center to edge further highlighted these trends. Pure PDMS exhibited the highest surface stress gradient (57.9 to 14.9 kPa), followed by 20% GelMA / PDMS (23.7 to 4.6 kPa) and 5% GelMA / PDMS (16.3 to 3.3 kPa) (FIG. 11C). Despite differences in magnitude, the GelMA composites maintained similar gradient ratios. Meridional and hoop stress ratios were also consistent across formulations (FIG. 11 D), indicating that overall stress distribution behavior is preserved, with PDMS providing the dominant mechanical contribution.

[0304] These results demonstrate that GelMA concentration can be used to precisely tune mechanical responses in the HIMS platform. Softer composites like 5% GelMA support greater deformation and broader stress distribution, favoring uniform mechanical stimulation for cell culture. In contrast, stiffer formulations like 20% GelMA offer localized stress patterns that may elicit more targeted mechanobiological effects. The validated simulation framework provides a robust tool for predictive design and optimization, enabling rapid prototyping of membrane mechanics for diverse biomedical applications.

[0305] TM Cell Morphology

[0306] CellTracker™ staining revealed elongated and partially aligned TM cells on 10% GelMA hydrogels, confined to the hydrogel regions (FIG. 13). FIG. 14A-C show cell morphology including cell aspect ratio and size under various stiffness and location of the curvature. As presented in FIG. 14A, lower stiffness (5% GelMA) allowed for greater cellular elongation in response to regional cues, a trend absent at higher stiffness (20% GelMA), where cells maintained a more uniformly spread shape. The elongated morphology on 20% GelMA is consistent with mechanobiological principles where stiffer substrates promote cell spread by active cytoskeletal formation. This confirms that the HIMS was able to capture stiffness-dependent cell behaviors. These findings highlight the important role of substrate stiffness in TM cell morphology and the potential impact of genetic alterations on cellular mechanosensing. Combining with stretch conditions, TM cells on 5% GelMA showed increasing aspect ratio from “No Stretch” to “Edge,” while on 20% GelMA, the aspect ratio remained consistently around 1.0. gTM cells exhibited similar trends, but with less elongation on 5% GelMA at the “Edge” and a notable decrease in aspect ratio at the “Top” region for both 5% and 20% GelMA. The attenuated elongation of gTM and their unique reduction in aspect ratio at the “Top” region suggest altered mechanosensing or cytoskeletal remodeling due to phenotypic alterations.

[0307] Stiffness and stretch also impact on cell spreading area changes relative to the unstretched 5% GelMA condition. FIG. 14C shows the distribution of cell spreading area. For the TM cells, the 5% GelMA under stretch case shows stretch-induced area reduction at the Top region (0.25-fold of baseline) that progressively recovered toward the Edge (0.9-fold). This coordinated result shows that cells on compliant substrates can efficiently redistribute their cytoplasmic volume to achieve directional elongation. Cells on 20% GelMA maintained area ratios closer to unstretched levels (0.5-0.75-fold) despite constrained elongation, indicating that substrate stiffness prevents normal mechanosensitive remodeling by restricting cytoskeletal reorganization necessary for coordinated morphological adaptation. However, gTM shows decrease of cell spreading area with minimal reduction at the Top region (0.85-fold), suggest that the glaucomatous phenotype specifically disrupts cellular mechanotransduction machinery. These findings demonstrate that pathological tissue stiffening creates a mechanical environment that limits normal cellular mechanosensing while exacerbating intrinsic mechanotransduction defects in glaucomatous cells, potentially contributing to progressive trabecular meshwork dysfunction.

[0308] Effect of Stiffness on TM Cell Mechanobiology

[0309] Substrate stiffness significantly modulated mechanotransduction signaling in both normal trabecular meshwork (TM) and glaucomatous trabecular meshwork (gTM) cells. Phosphorylated focal adhesion kinase (pFAK), the primary mechanosensor, exhibited strong stiffness-dependent activation in both TM and gTM. As presented in FIG. 17A, high stiffness (20% GelMA) resulted in a 2.3-fold increase in pFAK in TM cells and a 2.4-fold increase in gTM cells compared to low stiffness controls (5% GelMA). These findings indicate that the fundamental ability to detect mechanical cues is preserved in glaucomatous cells. However, the pathological implications lie downstream, where the same mechanosensing machinery may drive exaggerated or maladaptive cellular responses in the diseased state. Stiffness also triggered changes in extracellular matrix (ECM) remodeling proteins. Matrix metalloproteinase-2 (MMP2) showed modest upregulation (1.4-fold in TM, 1.2-fold in gTM) under high stiffness, reflecting a mild proteolytic response. In contrast, collagen type I (Coll) expression was highly stiffness-sensitive. TM cells demonstrated a 2.1 -fold increase, while gTM cells exhibited a 2.7-fold increase, suggesting a stronger fibrotic response in the diseased phenotype (FIG. 17A). This enhanced collagen production in gTM under mechanical stress could exacerbate ECM accumulation, leading to increased outflow resistance — a hallmark of glaucomatous pathology. High stiffness conditions also elevated a- smooth muscle actin (a-SMA) levels, marking a shift toward a contractile, myofibroblast-like phenotype. TM cells exhibited a 1.6-fold increase and gTM cells a 1.9-fold increase in a-SMA expression (FIG. 17B). This contractile phenotype, particularly pronounced in gTM cells, may further restrict aqueous humor outflow by increasing cellular tension and trabecular resistance. Additionally, myocilin (MYOC), a glaucoma-associated protein involved in outflow regulation and disease progression, was modestly upregulated under high stiffness (1.4-fold in TM, 1.6-fold in gTM) as presented in FIG. 17B. Although modest in magnitude, the upregulation of MYOC under mechanical stress provides a mechanistic link between ECM stiffness and molecular hallmarks of glaucoma. Taken together, these findings suggest that while both TM and gTM are mechanically responsive, gTM cells display exaggerated profibrotic, contractile, and glaucomaspecific protein expression in response to increased substrate stiffness. This increased mechanosensitivity likely contributes to a self-reinforcing cycle of dysfunction in the glaucomatous outflow pathway, where elevated tissue stiffness promotes pathological remodeling that further worsens fluid drainage and intraocular pressure regulation.

[0310] Coupled Effects of Stiffness and Stretch

[0311] To further explore the mechanical microenvironment of TM, the combined effects of substrate stiffness and static mechanical stretch (10%) on TM and gTM cells cultured on low (5% GelMA) and high (20% GelMA) stiffness substrates were examined. pFAK expression revealed distinct response patterns. Under stiffness-only conditions, both TM and gTM cells showed robust activation (2.3-fold and 2.4-fold increases, respectively), similar to earlier observations. When stretch was applied to soft substrates, pFAK activation was minimal (1.0-fold in TM, 0.8-fold in gTM), suggesting that stretch alone is insufficient to trigger mechanotransduction. However, combining high stiffness with stretch maintained a 1.6-fold pFAK increase in both cell types (FIG. 17A), indicating that pathological stiffness supports sustained mechanosensitivity even under dynamic loading. The interaction of stiffness and stretch amplified ECM remodeling in a conditionspecific manner. MMP2 expression increased slightly under stiffness alone (1.4-fold TM, 1.2-fold gTM) and reached peak levels with the combined stiffness-stretch condition (1.6-fold in both cell types). This synergy suggests that gTM cells remain proteolytically active under dynamic mechanical stimuli. Coll expression, a more pronounced fibrotic marker, revealed contrasting effects. While high stiffness alone elevated Coll levels (2.1-fold TM, 2.7-fold gTM), the addition of stretch on soft substrates suppressed Coll in TM cells (0.6-fold) and maintained baseline levels in gTM cells. Under the combined stiffness-stretch condition, TM cells again exhibited reduced Coll (0.6-fold), whereas gTM cells maintained elevated levels (1.6-fold) (FIG. 17A). This differential response suggests that while stretch may mitigate fibrotic remodeling in healthy cells, gTM cells are resistant to such regulatory effects, maintaining a pathogenic synthetic program even under fluctuating mechanical environments. a-Smooth muscle actin (a-SMA) expression demonstrated that substrate stiffness was the primary driver of contractile phenotype acquisition. Stiffness alone strongly induced a-SMA (1.6- fold TM, 1.9-fold gTM). Stretch on soft substrates reduced a-SMA in TM (0.5-fold) but maintained expression in gTM (1.3-fold). When stretch was added to high-stiffness substrates, TM cells partially attenuated a-SMA levels (0.8-fold), but gTM cells preserved elevated expression (1.4- fold) (FIG. 17B). This indicates that while stretch may offer some protective effects against contractile phenotype acquisition in normal cells, these effects are largely lost in gTM cells. Myocilin (MYOC) expression provided the most interesting example of synergistic stiffness-stretch interactions (FIG. 17B). High stiffness alone modestly increased MYOC levels (1.4-fold in TM cells, 1 .6-fold in gTM cells). However, the combination of high stiffness (20% GelMA) and stretch, TM cells exhibited the highest MYOC expression observed (1.9-fold), while gTM cells displayed only modest changes (0.9-fold). This suggests that normal TM cells remain responsive to pathological mechanical cues and may undergo disease-associated molecular changes when exposed to the glaucomatous mechanical environment. In contrast, gTM cells may already be saturated in their mechanosensitive pathways or less plastic in MYOC regulation.

[0312] These findings demonstrate the importance of mechanical context in glaucoma pathophysiology. While stiffness appears to be the primary driver of ECM synthesis and contractile transformation, dynamic stretch modulates these effects in a cell-type-dependent manner. Normal TM cells exhibit adaptive or even protective responses under stretch, while gTM cells retain an exaggerated, maladaptive phenotype regardless of mechanical context. The observation that MYOC is most strongly induced in normal cells under combined stress conditions raises the possibility that mechanical strain in a stiffened microenvironment may serve as a trigger for disease onset in otherwise healthy tissues. This supports the hypothesis of a biomechanical “tipping point” where progressive tissue stiffening, and mechanical stress jointly initiate and perpetuate glaucomatous remodeling. These insights contribute to a growing understanding that glaucoma is not merely a result of static elevation in intraocular pressure, but a dynamic mechanical disease. Therapeutic strategies that target ECM stiffness or mechanotransduction signaling — particularly in early stages — may hold promise in disrupting this self-reinforcing pathological loop.

[0313] This hydrogel-integrated microfluidic system successfully addresses important limitations in trabecular meshwork mechanobiology research while advancing lab-on-a-chip technology. The PDMS-GelMA system achieves unprecedented control over cellular mechanical microenvironments by simultaneously modulating substrate stiffness and applying physiologically relevant triaxial tensile stress — a combination previously unattainable with conventional approaches. The integration of tunable GelMA hydrogels (5%, 10%, and 20% concentrations) successfully recapitulated the stiffness range observed across glaucoma disease progression (1.23 to 21.47 kPa), while the optimized surface modification protocol ensured robust hydrogel- PDMS integration capable of withstanding up to 40% linear strain without mechanical failure. The exceptional predictive accuracy of these finite element models (r2> 0.998) validates the system’s reliability and establishes a framework for rational design of future mechanobiological platforms.

[0314] These biological discoveries reveal that trabecular meshwork cells exhibit dramatically different mechanotransduction responses when exposed to coupled mechanical stimuli that occur physiologically. Significantly, it was demonstrated that disease-associated substrate stiffness creates a mechanically permissive environment that amplifies cellular responses to stretch stimuli, establishing a pathological feedback mechanism where tissue stiffening promotes behaviors that further increase stiffness through enhanced ECM deposition and myofibroblast transformation. The pronounced mechanosensitivity of glaucomatous cells, evidenced by enhanced pFAK activation, a-SMA upregulation, and MYOC expression, provides new insights into how elevated intraocular pressure perpetuates disease progression through mechanotransduction pathways. This coordinated upregulation of ECM remodeling proteins (MMP2, Coll), contractile markers, and disease-specific proteins under disease-stiffness conditions establishes clear mechanistic targets for therapeutic development and suggests that interventions targeting tissue stiffness could interrupt disease progression.

[0315] From a technological perspective, this platform addresses an important need in the lab- on-a-chip community for systems capable of applying complex, physiologically relevant mechanical stimuli while maintaining precise experimental control. The successful integration of multiple mechanical stimulation modalities within a single, optically transparent device demonstrates potential for investigating mechanobiology in other mechanically active tissues, including cardiovascular, musculoskeletal, and respiratory systems. The validated approach for combining tunable substrate mechanics with dynamic stress application provides a template for next-generation microfluidic platforms designed for drug discovery, disease modeling, and personalized medicine applications. Future directions include investigations of cyclic mechanical loading, integration of flow-based shear stress, and expansion to three-dimensional culture systems. This hydrogel-integrated microfluidic system represents a significant advancement that bridges the gap between simplified in vitro models and complex in vivo mechanical environments, providing both technological tools and biological insights that advance understanding of mechanobiology while supporting the development of targeted therapeutic strategies for glaucoma and other mechanically mediated diseases.

Claims

CLAIMSWhat is claimed:

1. A biomimetic microfluidic device, comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

2. The device of claim 1 , wherein the substrate base layer comprises silanized glass.

3. The device of claim 1, wherein each of the first microfluidic channel layer, second microfluidic channel layer, and membrane layer comprises a polymeric material of polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof.

4. The device of claim 1 , wherein the membrane layer is porous and comprises an electrospun collagen nanofiber membrane.

5. The device of claim 1 , wherein the membrane layer is porous and comprises a porosity of about 70% to about 80%.

6. The device of claim 1 , wherein the membrane layer is porous and comprises an average pore size of about 1 pm to about 5 pm.

7. The device of claim 1 , wherein the membrane layer comprises a flat, concave, or convex shaped curvature upon adjusting the pressure in the first microfluidic channel layer to stretch the population of cells ranging from 0% to 50% linear strain.

8. The device of claim 1 , wherein the membrane layer comprises a uniform thickness across its surface of about 25 pm to about 250 pm.

9. The device of claim 1 , wherein the second microfluidic channel layer comprises Hele- Shaw geometrical dimensions comprising a width of about 2 mm to about 15 mm, a length of about 25 mm to about 30 mm, and a height of about 0.05 mm to about 0.1 mm.

10. The device of claim 1 , wherein the second microfluidic channel layer is configured to modulate shear stress on the population of cells from about 0.01 dyn / cm2to about 30 dyn / cm2.

11. The device of claim 1 , wherein the second microfluidic channel layer is configured for fluid flow rates of about 1 pL / min to about 750 pL / min to modulate shear stress on the population of cells.

12. The device of claim 1 , wherein the hydrogel layer comprises gelatin, collagen, elastin, alginate, or combinations thereof.

13. The device of claim 1 , wherein the hydrogel layer comprises a photo-crosslinkable material comprising gelatin methacrylate (GelMA), polyethylene glycol diacrylate (PEGDA), or combinations thereof.

14. The device of claim 1 , wherein the hydrogel layer comprises gelatin methacrylate (GelMA) at a concentration of about 1 % w / v to about 20% w / v.

15. The device of claim 1 , wherein the hydrogel layer comprises a Young’s modulus of about 1 kPa to about 25 kPa.

16. The device of claim 1 , wherein the concentration and Young’s modulus of the hydrogel layer are associated with a stiffness of the population of cells.

17. The device of claim 1 , wherein the hydrogel layer has a uniform thickness of about 0.05 mm to about 0.5 mm.

18. The device of claim 1 , further comprising one or more of a plurality of receiving channels, outlets, flow sensors, and pressure transducers to measure fluid flow rates of outflow from the population of cells.

19. The device of claim 1 , wherein the population of cells is cultured on the first surface of the membrane layer, the second surface of the membrane layer, or a combination thereof.

20. The device of claim 1 , wherein the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof.

21. The device of claim 1 , wherein the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof.

22. The device of claim 21 , wherein the TM cells comprise glaucomatous TM cells.

23. The device of claim 21 , wherein TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer.

24. The device of claim 1 , wherein the membrane layer mimics a juxtacanalicular tissue structure.

25. The device of claim 1 , wherein the device is a glaucoma eye organ-on-a-chip microfluidic device.

26. The device of claim 1 , wherein the device is capable of simultaneously and independently modulating stiffness, stretch, and shear stress conditions for the population of cells.

27. The device of claim 1 , wherein the membrane layer is optically transparent for real-time cell imaging.

28. The device of claim 1 , wherein the device is optically transparent for real-time cell imaging.

29. A method of culturing and monitoring one or more cell types, the method comprising: inserting a population of cells into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; modulating the hydraulic pressure in the first microfluidic channel layer to alter the shape of the membrane layer and stretch the population of cells; and analyzing the population of cells in the device.

30. The method of claim 29, further comprising applying a fluid flow through the second microfluidic channel layer to modulate the shear stress on the population of cells prior to analyzing the population of cells in the device, wherein the shear stress and stretch are applied simultaneously to the population of cells.

31. The method of claim 29, wherein the population of cells comprises trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof.

32. The method of claim 31 , wherein TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer.

33. The method of claim 31 , wherein the TM cells are derived from a subject having glaucoma.

34. The method of claim 31 , wherein the TM cells are isolated from juxtacanalicular and / or corneoscleral meshwork eye regions of a subject.

35. The method of claim 29, wherein the population of cells is cultured on the hydrogel layer, within the hydrogel layer, or a combination thereof.

36. The method of claim 29, wherein the membrane layer of the biomimetic microfluidic device is surface treated with one or more extracellular matrix proteins comprising collagen type I, fibronectin, or a combination thereof prior to inserting the population of cells into the device.

37. The method of claim 29, wherein the population of cells is analyzed in the biomimetic microfluidic device using one or more imaging techniques.

38. The method of claim 29, further comprising performing one or more biochemical assays on the population of cells.

39. The method of claim 29, further comprising administering one or more anti-glaucoma therapeutic agents into the device.

40. A method of screening one or more anti-glaucoma therapeutic agents, the method comprising: inserting a population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof into a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing the population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer; administering one or more anti-glaucoma therapeutic agents into the device; and analyzing the population of cells in the device.

41. The method of claim 40, wherein TM cells are cultured on the second surface of the membrane layer and SC cells are cultured on the first surface of the membrane layer.

42. The method of claim 40, wherein the membrane layer is porous and comprises an electrospun collagen nanofiber membrane.

43. The method of claim 40, wherein the anti-glaucoma therapeutic agent comprises one or more ion channel agonists or antagonists for Piezol , TRPV4, TREK-1 , or combinations thereof.

44. The method of claim 40, wherein the anti-glaucoma therapeutic agent comprises an actin depolymerization agent, a Rho kinase inhibitor, or a combination thereof.

45. The method of claim 40, further comprising modulating one or more of stiffness, stretch, and shear stress conditions for the population of cells.

46. The method of claim 40, further comprising measuring fluid flow rates of outflow from the population of cells.

47. A kit for culturing and monitoring one or more cell types, the kit comprising: a biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer;optionally, cell culture buffers, reagents, and receptacles; and optionally, one or more of packaging or instruction for use.

48. Use of a biomimetic microfluidic device for culturing and monitoring one or more cell types in response to modulation of stiffness, stretch, and / or shear stress conditions, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow; a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

49. Use of a biomimetic microfluidic device for screening one or more anti-glaucoma therapeutic agents, the biomimetic microfluidic device comprising: a substrate base layer; a first microfluidic channel layer positioned on the substrate base layer and configured for hydraulic pressure modulation, the first microfluidic channel layer being fluidly connected to a hydraulic actuation system comprising a source of one or more pressurized fluids and configured to selectively adjust the pressure in the first microfluidic channel layer; a second microfluidic channel layer positioned adjacent to the first microfluidic channel layer and configured for cellular shear stress modulation by fluid flow;a membrane layer positioned between the first microfluidic channel layer and the second microfluidic channel layer, the membrane layer comprising a first surface in fluid communication with the first microfluidic channel layer and a second surface in fluid communication with the second microfluidic channel layer, each surface of the membrane layer being configured for culturing a population of cells, the population of cells comprising trabecular meshwork (TM) cells, Schlemm’s canal (SC) cells, or a combination thereof; a hydrogel layer disposed on the second surface of the membrane layer; and a plurality of inlets and outlets fluidly connected to one or more of the first microfluidic channel layer, second microfluidic channel layer, membrane layer, or hydrogel layer.

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