Microphysiological device, system and method

Abstract

Aspects of the present invention relate to a microphysiological device including a first component comprising a frame having a top surface, a bottom surface, a thickness therebetween, a first cavity having an opening at the top surface of the frame, and a second cavity having an opening at the top surface of the frame, the first cavity and second cavity each comprising a first tissue construct and second tissue construct, respectively, a second component comprising a frame having at least a first opening passing through the frame forming an interior region, a holder comprising a frame having at least one opening passing through the frame, and a membrane having a top and bottom surface positioned across the opening, the membrane comprising a first cell culture and second cell culture on the top and bottom surface, respectively, wherein the holder is removably positioned within the interior region of the second component, and the first component and second component are fixedly and removably attached thereby fluidly connecting the first cavity, the second cavity, and the interior region.

Description

[0001] Attorney Docket No.: 204606-0207-00WO

[0002] TITLE

[0003] Microphy si ologi cal Device, System and Method for Modeling a Joint

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 778,842, filed on March 27, 2025, incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under TR003281, AR081025, and AG088071 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND OF THE INVENTION

[0008] Rheumatoid arthritis (RA) is a chronic joint disease with diverse clinical representations and therapy responses. It is driven by immune and stromal cells’ interactions in the synovium leading to painful joint inflammation and destruction.

[0009] Recent findings show synovial cellular and molecular heterogeneity among RA patients, indicating distinct disease subtypes including some (e.g. pauci-immune) that are refractory to existing conventional and biologic disease modifying antirheumatic drugs (DMARDs) (Di Matteo, A., et al., Lancet 402, 2019-2033 (2023), Zhao, J., et al., Front Immunol 12, 790122 (2021)). Animal models, while useful, do not fully capture human synovial complexity due to species differences and artificial disease induction approaches (Wooley, P. H. BestPract Res Clin Rheumatol 18, 47-58 (2004)). In vitro models are good for mechanistic studies but oversimplify the in vivo environment, which limit their clinical predictive value because they lack key cellular and molecular interactions (Mishra, A. P. et al. ACS Pharmacology & Translational Science 7, 2280-2305 (2024), Damerau, A. & Gaber, T. Int J Mol Sci 21 (2020)). There remains a need in the art for in vitro joint models.

[0010] SUMMARY OF THE INVENTIONAttorney Docket No.: 204606-0207-00WO

[0011] In some aspects, the invention relates to a microphysiological device.

[0012] In some embodiments, the microphysiological device comprises a first component comprising a frame having a top surface, a bottom surface, a thickness therebetween, a first cavity having an opening at the top surface of the frame, and a second cavity having an opening at the top surface of the frame, the first cavity and second cavity each comprising a first tissue construct and second tissue construct, respectively, a second component comprising a frame having at least a first opening passing through the frame forming an interior region, a holder comprising a frame having at least one opening passing through the frame, and a membrane having a top and bottom surface positioned across the opening, the membrane comprising a first cell culture and second cell culture on the top and bottom surface, respectively, wherein the holder is removably positioned within the interior region of the second component, and the first component and second component are fixedly and removably attached thereby fluidly connecting the first cavity, the second cavity, and the interior region.

[0013] In some embodiments, the first component of the device further comprises a third cavity having an opening at the top surface of the frame of the first component, the third cavity being fluidly connected to the first cavity, the second cavity, and the interior region.

[0014] In some embodiments, the third cavity of the device further comprises a sensor. In some embodiments, the sensor comprises a photonic ring resonator sensor. In some embodiments, the first tissue construct of the device comprises a bone tissue construct.

[0015] In some embodiments, the second tissue construct of the device comprises a cartilage tissue construct.

[0016] In some embodiments, the first cell culture of the device comprises endothelial cells.

[0017] In some embodiments, the first cell culture of the device comprises a vascular barrier.

[0018] In some embodiments, the second cell culture of the device comprises synovial cells.

[0019] In some embodiments, the second cell culture comprises a hydrogel.Attorney Docket No.: 204606-0207-00WO

[0020] In some embodiments, the device comprises a flow insert removably positioned in the interior region above the holder, the flow insert comprising two ports configured to apply or remove fluid.

[0021] In some embodiments, the first and second tissue cavities of the device each comprise at least two ports configured to apply or remove fluid.

[0022] In some aspects, the invention relates to a method for making a microphysiological system.

[0023] In some embodiments, the method comprises providing a microphysiological device of the invention, culturing one or more cells of a first cell culture on the bottom surface of the membrane of the device, culturing one or more cells of a second cell culture on the top surface of the membrane of the device, culturing one or more cells of a third cell culture in the first cavity of the device, culturing one or more cells of a fourth cell culture in the second cavity of the device, assembling the first and second component of the device and co-culturing all the cells together for a third period of time.

[0024] In some embodiments, the method comprises treating the interior region, first cavity, or second cavity of the device.

[0025] In some embodiments, the treatment comprises administering a disease modifying antirheumatic drug (DMARD).

[0026] In some embodiments, the first cell culture of the method comprises synovial cells.

[0027] In some embodiments, the first cell culture of the method replicates a cell type abundance profile of rheumatoid arthritis.

[0028] In some embodiments, the second cell culture of the method comprises endothelial cells.

[0029] In some embodiments, the third cell culture of the method comprises a bone tissue construct.

[0030] In some embodiments, the fourth cell culture of the method comprises a cartilage tissue construct.

[0031] BRIEF DESCRIPTION OF THE DRAWINGSAttorney Docket No.: 204606-0207-00WO

[0032] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0033] Figure 1, comprising Figures 1A through Figure IB, depicts an overview of the modular pSIM platform. Figure 1A depicts an exemplary pSIM platform with interchangeable pSiM membranes (nanoporous or microporous), a flow channel insert, and sensor integration for diverse tissue culture configurations. Figure IB depicts an exemplary rheumatoid arthritis joint-on-a-chip (RA-JoC) assembly with a multiplayer flow system, dual-scale pSiM membranes, and integrated sensors to monitor the cytokines in the synovial microenvironment in real time, in proximity to wells designed to house cartilage and bone scaffold in the bottom component.

[0034] Figure 2 depicts a schematic of the RA-JoC design and construction. Figure 3, comprising Figures 3A through 3B depicts a schematic of the design of the RA-JoC. Figure 3A depicts an exploded view of the new chip components. Figure 3B depicts a half-cutaway view of the assembled chip showing the media microfluidic connections.

[0035] Figure 4 depicts exemplary RA-JoC components.

[0036] Figure 5 depicts an exemplary dual-scale pSIM membrane.

[0037] Figure 6, comprising Figures 6a through 6c, depicts an exemplary Photonic Ring Resonator Sensor. Figure 6a depicts an exemplary laser confocal microscopy image of an exemplary Photonic Ring Resonator Sensor. Figure 6b depicts exemplary head camera images from the Scienion SX piezoelectric spotter of exemplary Photonic Ring Resonator Sensors. Figure 6c depicts an exemplary calibration of an exemplary functionalized sensor.

[0038] Figure 7 depicts an exemplary assembled RA-JoC.

[0039] Figure 8 depicts an illustrative computer architecture for a computer for practicing the various embodiments of the invention.

[0040] Figure 9 depicts an exploded view of an exemplary microphysiological device.Attorney Docket No.: 204606-0207-00WO

[0041] Figure 10 depicts exemplary synovial cell compositions in rheumatoid arthritis cell type abundance profiles (CTAPs).

[0042] DETAILED DESCRIPTION

[0043] It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity many other elements found in related systems and methods. Those of ordinary skill in the art may recognize that other elements and / or steps are desirable and / or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art.

[0044] Definitions

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, exemplary materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0046] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0047] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.Attorney Docket No.: 204606-0207-00WO

[0048] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate.

[0049] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal amenable to the systems, devices, and methods described herein. The patient, subject or individual may be a mammal, and in some instances, a human.

[0050] As used herein, the term “gel” refers to a three-dimensional polymeric structure that itself is insoluble in a particular liquid but which is capable of absorbing and retaining large quantities of the liquid to form a stable, often soft and pliable, but always to one degree or another shape-retentive, structure. When the liquid is water, the gel is referred to as a hydrogel. Unless expressly stated otherwise, the term “gel” will be used throughout this application to refer both to polymeric structures that have absorbed a liquid other than water and to polymeric structures that have absorbed water, it being readily apparent to those skilled in the art from the context whether the polymeric structure is simply a “gel” or a “hydrogel.”

[0051] As used here, “biocompatible” refers to any material, which, when implanted in a mammal, does not provoke an adverse response in the mammal. A biocompatible material, when introduced into an individual, is not toxic or injurious to that individual, nor does it induce immunological rejection of the material in the mammal.

[0052] As used herein, to “alleviate” a disease, defect, disorder or condition means reducing the severity of one or more symptoms of the disease, defect, disorder or condition.

[0053] As used herein, to “treat” means reducing the frequency with which symptoms of a disease, defect, disorder, or adverse condition, and the like, are experienced by a patient.

[0054] As used herein, a “therapeutically effective amount” is the amount of a composition of the invention sufficient to provide a beneficial effect to the individual to whom the composition is administered.

[0055] Ranges: throughout this disclosure, various aspects of the invention can beAttorney Docket No.: 204606-0207-00WO

[0056] presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0057] Microphysiological Device, System, and Method

[0058] Contemplated herein is a novel device, system, and method that provides a tool for investigating tissue physiology, pathophysiology, and potential treatments thereof. In some aspects, the present invention relates to a microphysiological device comprising a vascular and synovial component removably attached to at least one tissue component. In some embodiments, the tissue component comprises a bone tissue construct and a cartilage tissue construct. The device may provide separate or substantially separate and / or customizable cell culturing conditions per component or per tissue construct. In some aspects, the present invention relates to a system comprising the disclosed microphysiological device and one or more reservoir components for holding and providing culture medium to the vascular and synovial components and each of the at least one tissue components. In some embodiments, the device further comprises a sensor for monitoring a characteristic of the vascular and synovial component, the one or more tissue components, and / or the communication between the vascular and synovial component and the one or more tissue components. The sensor may be in fluid communication with the vascular and synovial component and / or the tissue component. In some embodiments, an assembly jig is further provided with the system. In some aspects, the invention relates to a novel method of replicating an anatomical structure or features of interest of an anatomical structure (e.g., a joint) or disease (e.g., Rheumatoid Arthritis) in vitro. It should be appreciated that aspects of the present invention relate to incorporating subject-specific, or patient derived cells to develop personalized treatmentsAttorney Docket No.: 204606-0207-00WO

[0059] and therapies. It should be appreciated that aspects of the present invention relate to incorporating cell type combinations that relate to cell type combinations found in a subject to develop personalized treatments and therapies.

[0060] Referring now to Figure 9, depicted is an exploded view of an exemplary microphysiological device 100 (e.g., Rheumatoid Arthritis Joint-on-a-Chip (RA-JoC) device) according to aspects of the present invention. In some embodiments, microphysiological device 100 provides a first component 110 (e.g. a tissue component) for culturing one or more tissue constructs and a second component 140 (e.g., a vascular and synovial component) for culturing one or more cellular monolayers and / or 3D hydrogel cultures. In some embodiments, device 100 further provides a sensor 180. In some embodiments, the first component 110 is designed such that it may hold or secure the sensor 180. In some embodiments, the first component 110 comprises a first tissue cavity 112, a second tissue cavity 114, and a sensor cavity 116, in which a first tissue construct, a second tissue construct, and a sensor 180 may be placed or positioned, respectively. In some embodiments, the second component 140 is designed such that it may hold or secure a holder 160. For example, the second component 140 may comprise a second component cavity 142 in which the holder 160 may be placed or positioned. In some embodiments, the device 100 comprises a flow insert 190. The flow insert 190 may be designed such that it fits in the second component cavity 142 above the holder 160. The flow insert 190 may be designed or configured to aid in providing media to the holder 160, the membrane 170, and / or the top of the membrane 170. The holder 160 may comprise a membrane 170 for culturing cells thereon. The membrane 170 may allow for the culture of a vascular tissue and / or a synovial tissue. In some embodiments, cells are cultured on either side of the membrane 170 (e.g., the top and bottom of the membrane 170). In some embodiments, a vascular tissue is cultured on one side of the membrane 170 and a synovial tissue is cultured on the opposite side of the membrane 170. In some embodiments, the microphysiological device 100 provides a media channel component 120. The media channel component 120 may comprise a media channel 122. The media channel component 120 and / or the media channel 122 may be configured to allow fluid communication between the one or more tissue constructs, cellular monolayers, 3D hydrogel cultures, and / or the sensor 180 of the device 100. In some embodiments, theAttorney Docket No.: 204606-0207-00WO

[0061] microphy si ologi cal device 100 provides a spacer component 130. The spacer component 130 may be configured to provide a desired thickness to the device 100. The spacer component 130 may comprise a spacing 132 to allow direct contact between the bottom of the membrane 170 and media within the media channel component 120. The device 100 may also provide a flow insert. It should be appreciated that the first component 110, media channel component 120, spacer component 130, and second component 140 may be stacked to form the device 100.

[0062] Aspects of the present invention relate to a first component 110 for culturing one or more tissue constructs. In some embodiments, first component 110 comprises a top and bottom surface and has a thickness therebetween. The first component may comprise any number of cavities. In some embodiments, the first component 110 comprises any number of cavities configured for the placement of a tissue construct and any number of cavities configured for the placement of a sensor. For example, a cavity configured for the placement of a tissue construct may have an appropriate geometry for the positioning of a tissue construct in the cavity. For example, a cavity configured for the placement of a sensor may have an appropriate geometry for the positioning of a sensor in the cavity. The cavities may be open at the top surface of the first component 110 and may be closed at the bottom surface of the first component 110. In some embodiments, the first component comprises a first tissue cavity 112, a second tissue cavity 114, and a sensor cavity 116. The first tissue cavity 112, second tissue cavity 114, and sensor cavity 116 may be in fluid communication. In some embodiments, the sensor cavity 116 is positioned between first tissue cavity 112 and second tissue cavity 114 such that a sensor positioned in sensor cavity 116 provides for measurement of any aspects of communication between first tissue 112 and second tissue cavity 114. For example, a sensor positioned in sensor cavity 116 may be configured to detect and / or measure any molecule(s) of interest secreted by a tissue construct, cell culture, or cell thereof. In some embodiments, the thickness of the first component is in a range between 0.01 mm and 1 cm. In some embodiments, the bottom surface of the first component 110 and or the entire first component 110 is optically clear or substantially optically clear such that the contents of any cavities of the first component 110 may be imaged optionally in real-time.Attorney Docket No.: 204606-0207-00WO

[0063] Cavities for the placement of a tissue construct may be any geometry and contain any tissue construct. In some embodiments, the opening of a cavity is a circle, ellipse, rectangle, square, or any other geometry. The opening of a cavity may be any surface area. The depth of a cavity may be any depth. For example, the depth of a cavity may be between 0.01 mm and 1 cm. In some embodiments, the depth of the cavity is or is approximately 1 mm. In some embodiments, the opening of the cavity is a circle with a diameter of 3 mm or approximately 3 mm.

[0064] In some embodiments, the device provides separate or substantially separate and customizable cell culturing conditions for each tissue cavity. For example, a tissue cavity may be in fluid communication with a media inlet channel and a media outlet channel. For example, first tissue cavity 112 may be in fluid communication with first tissue cavity inlet channel 111 and first tissue cavity outlet channel 113. Similarly, second tissue cavity 114 may be in fluid communication with second tissue cavity inlet channel 115 and second tissue cavity outlet channel 117. The inlet and outlet channels of a tissue cavity may be any depth or width. In some examples, the inlet and outlet channels have a depth that is approximately equal to the depth of the associated tissue cavity.

[0065] The inlet channel and outlet channel of a tissue cavity may be in fluid communication with a media source and a media sink, respectively. The device may be configured to support constant media flow, sporadic media flow, or simple media changes in a tissue cavity. A tissue cavity may be subjected to any desired flow. For example, the flow that a tissue cavity is subjected to may be 1.5 dynes / cm2approximately 1.5 dynes / cm2.

[0066] It should be appreciated that first tissue cavity 112 and second tissue cavity 114 of first component 110 are configured to retain, culture and / or image a tissue construct, and therefore may comprise or be at least partially fluidly filled with any tissue constructs known by one of ordinary level of skill in the art. In some embodiments, the tissue construct comprises any electrospun tissue, hydrogel, and / or any tissue engineered scaffold known in the art. In some embodiments, first tissue cavity 112 and second tissue cavity 114 each comprise at least one tissue construct, wherein the tissue construct comprises one or more polymers (e g., biopolymer, synthetic polymer, or hybridAttorney Docket No.: 204606-0207-00WO

[0067] polymer) known in the art, with a plurality of cells dispersed within. In some embodiments, the polymer comprises any of hydrogel, collagen, type I / III collagen hydrogel, or the like. In some embodiments, the plurality of cells comprise any of mammalian cells, patient derived cells, stem cells, induced pluripotent stem cells (iPSCs), stem cell-derived cells, iPSC-derived cells, bone cells, cartilage cells, chondrocytes, mesenchymal progenitor cells.

[0068] In some embodiments, the plurality of cells comprises cells isolated from bone or cartilage tissue from any patient, subject, or model organism. In some embodiments, the plurality of cells comprises cells isolated from bone or cartilage tissue from any patient, subject, or model organism that has rheumatoid arthritis. In some embodiments, the plurality of cells comprises bone or cartilage cells collected from a joint, bone tissue, or cartilage tissue that may be verified to be bone or cartilage cells by the expression of a bone or cartilage marker. In some embodiments, the plurality of cells comprises bone cells or cartilage cells in an inflammatory state. The inflammatory state of bone or cartilage cells may be assessed by measuring the gene expression profile of the cells. Inflamed bone or cartilage cells may allow for the modeling of an inflamed joint and / or rheumatoid arthritis.

[0069] In some embodiments, one of the first tissue cavity 112 and second tissue cavity 114 comprises a tissue construct configured to mimic bone tissue and one of the first tissue cavity 112 and second tissue cavity 114 comprises a tissue construct configured to mimic cartilage tissue.

[0070] A tissue construct configured to mimic bone tissue may comprise a bone scaffold. For example, a suitable bone scaffold may comprise one or more decellularized trabecular bone discs. In some embodiments, the bone tissue construct comprises mesenchymal progenitor cells (MPCs), monocytes, osteoblasts, osteoclasts, and / or any bone cells. In some embodiments, cells are derived from induced pluripotent stem cells. For example, MPCs may be comprise induced pluripotent stem cell - derived MPCs (iMPCs). In some examples, osteoblasts and / or osteoclasts are derived from or differentiated from induced pluripotent stem cell - derived monocytes. In some examples, differentiated osteoclasts express TRAP, Cathepsin K, and / or NF ATcl.

[0071] Induced pluripotent stem cells may be derived from a subject, a patient, or a modelAttorney Docket No.: 204606-0207-00WO

[0072] organism. In some embodiments, cells are isolated from bone or bone marrow of a subject or model organism. For example, primary osteoblasts and primary osteoclasts may be isolated from the bone or bone marrow of a subject, a patient, or a model organism. In some embodiments, the cells are seeded on one or more trabecular bone discs.

[0073] The media presented to the construct configured to mimic bone tissue may also be customized to promote the viability, maturation, differentiation, culture of, or matrix deposition of mesenchymal progenitor cells, monocytes, osteoblasts, osteoclasts, and / or any bone cells. For example, a suitable media may comprise osteogenic media. An osteogenic media may comprise dexamethasone, ascorbic acid-2-phosphate, and / or P-glycerophosphate. In some examples, osteogenic media comprises or further comprises M-CSF, RANKL, and / or serum. In some embodiments, the bone tissue construct is cultured in osteogenic medium for approximately 21 days to promote the maturation of the bone tissue construct. In some examples, the medium promotes the differentiation of induced pluripotent stem cell (iPSC) - derived monocytes to osteoclasts. In some embodiments, a suitable medium for differentiation of iPSC-derived monocytes to osteoblasts comprises M-CSF, RANKL, and / or serum. In some examples, the osteoblast differentiation comprises M-CSF in a concentration between 25 ng / mL to 50 ng / mL. In some examples, the osteoblast differentiation comprises RANKL in a concentration between 30 ng / mL to 100 ng / mL. In some examples, the cells are cultured in osteoblast differentiation medium for approximately 7 - 14 days. In some embodiments, the bone tissue construct is cultured for any duration before being positioned in the tissue cavity. In some embodiments, the bone tissue construct is positioned in the cavity and then cultured for any duration.

[0074] A tissue construct configured to mimic cartilage tissue may comprise a cartilage scaffold and cartilage cells. In some embodiments, the cartilage scaffold mimics the composition and / or structure of native cartilage. Any biologic hydrogel, synthetic hydrogel, or combination thereof known in the art for the culture of cartilage cells or for recapitulating the composition and / or structure of native cartilage may be used. In some embodiments, the cartilage scaffold comprises a hydrogel. In some embodiments, the cartilage scaffold comprises a photocrosslinkable hydrogel matrix. In some embodiments,Attorney Docket No.: 204606-0207-00WO

[0075] the cartilage scaffold comprises type II collagen and / or hyaluronic acid methacrylate (HAMA). In some embodiments, the cartilage scaffold comprises an ionically crosslinked hydrogel, for example alginate. In some embodiments, the cartilage scaffold comprises a thermally cured hydrogel, for example agarose.

[0076] The cartilage cells of the cartilage tissue construct may comprise chondrocytes. In some embodiments, chondrocytes comprise iPSC-derived chondrocytes and / or chondrocytes isolated from cartilage tissue of a subject, patient, or model organism. In some embodiments, chondrocytes are differentiated from mesenchymal stem cells or iMSCs.

[0077] The media presented to the construct configured to mimic cartilage tissue may also be customized to promote the viability, maturation, differentiation, or culture of cartilage. In some embodiments, a suitable medium comprises a chondrogenic medium. In some examples, chondrogenic medium comprises TGF-P3, ascorbic acid, dexamethasone, ITS supplement, proline, and / or pyruvate.

[0078] In some embodiments, mesenchymal stem cells or MSCs are cultured in the cartilage scaffold for approximately 14 days to 21 days, optionally in a medium comprising chondrogenic medium, to promote the differentiation of MSCs to chondrocytes. The MSCs may be cultured in the cartilage scaffold before or after the cartilage tissue construct is positioned in the device 100.

[0079] Further, it should be appreciated that any hydrogel known by one of ordinary level of skill in the art may be used in a tissue construct and may include any additives or cross-linking agents known in art in order to cross-link or set the hydrogel.

[0080] In some embodiments, the tissue construct of any tissue cavity of the device 100 comprises one or more polymers (e.g., biopolymer, synthetic polymer, or hybrid polymer). Non-limiting examples of suitable polymers include but are not limited to PLGA, PLA, PGA, PCL, PLL, cellulose, poly(ethylene-co-vinyl acetate), polystyrene, polypropylene, dendrimer-based polymers, polyethylene glycol (PEG), branched PEG, polysialic acid (PSA), carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, starch, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polysebacates, poly(glycerolsebacates), poly acryloylmorpholine,Attorney Docket No.: 204606-0207-00WO

[0081] polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anhydride, polystyrene-co- maleic acid anhydride, poly(l-hydroxymethylethylene hydroxymethylformal) (PHF), 2-methacryloyloxy-2'-ethyltrimethylammoniumphosphate (MPC), polyethylene glycol propionaldehyde, copolymers of ethylene glycol / propylene glycol, monomethoxypolyethylene glycol, carboxymethylcellulose, polyacetals, poly- 1,3 -di oxolane, poly-1,3,6- trioxane, ethyl ene / maleic anhydride copolymer, poly (P-amino acids) (either homopolymers or random copolymers), poly(n- vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers (PPG) and other polyakylene oxides, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (POG) (e.g., glycerol) and other polyoxyethylated polyols, polyoxyethylated sorbitol, or polyoxyethylated glucose, colonic acids or other carbohydrate polymers, Ficoll or dextran, biopolymers such as hyaluronans, chitosans, alginates, collagen, dextran, pectin, carrageenan, polylysine, gelatin or agarose, and combinations or mixtures thereof.

[0082] In some embodiments, any tissue construct of the device 100 may comprise one or more hydrogels. Hydrogels can generally absorb a great deal of fluid and, at equilibrium, typically are composed of 60-90% fluid and only 10-30% polymer. In a preferred embodiment, the water content of hydrogel is about 70-80%. Hydrogels are particularly useful due to the inherent biocompatibility of the cross-linked polymeric network (Hill-West, et al. ,1994, Proc. Natl. Acad. Sci. USA 91:5967-5971). Hydrogel biocompatibility may be attributed to hydrophilicity and ability to imbibe large amounts of biological fluids (Brannon-Peppas. Preparation and Characterization of Cross-linked Hydrophilic Networks in Absorbent Polymer Technology, Brannon-Peppas and Harland, Eds. 1990, Elsevier: Amsterdam, pp 45-66; Peppas and Mikos. Preparation Methods and Structure of Hydrogels in Hydrogels in Medicine and Pharmacy, Peppas, Ed. 1986, CRC Press: Boca Raton, Fla., pp 1-27). The hydrogels may be prepared by crosslinking hydrophilic biopolymers or synthetic polymers. Examples of the hydrogels formed from physical or chemical crosslinking of hydrophilic biopolymers, include but are not limited to, hyaluronans, chitosans, alginates, collagen, dextran, pectin, carrageenan, polylysine, gelatin or agarose, (see.: W. E. Hennink and C. F. van Nostrum, 2002, Adv. Drug Del. Rev. 54, 13-36 and A. S. Hoffman, 2002, Adv. Drug Del. Rev. 43, 3-12). These materialsAttorney Docket No.: 204606-0207-00WO

[0083] consist of high-molecular weight backbone chains made of linear or branched polysaccharides or polypeptides. Examples of hydrogels based on chemical or physical crosslinking synthetic polymers include but are not limited to (meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, poly(ethylene glycol) diacrylate (PEGDA), poly(ethylene glycol) (PEO), polypropylene glycol) (PPO), PEO-PPO-PEO copolymers (Pluronics), poly(phosphazene), poly(methacrylates), poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethylene imine), etc. (see A. S Hoffman, 2002, Adv. Drug Del. Rev, 43, 3-12).

[0084] In some embodiments, the hydrogel comprises at least one biopolymer. In some embodiments, the hydrogel further comprises at least two biopolymers. In some embodiments, the hydrogel comprises at least one biopolymer and at least one synthetic polymer. The hydrogel may be cast or deposited into the first tissue cavity 112 or second tissue cavity 114 in a manner that produces any desired hydrogel property. For example, hydrogel fiber alignment and length may be altered to create or mimic any desired cellular environment.

[0085] Hydrogels closely resemble the natural living extracellular matrix (Ratner and Hoffman. Synthetic Hydrogels for Biomedical Applications in Hydrogels for Medical and Related Applications, Andrade, Ed. 1976, American Chemical Society: Washington, D.C., pp 1-36). Hydrogels may also be made degradable in vivo by incorporating PLA, PLGA or PGA polymers. Moreover, hydrogels may be modified with fibronectin, laminin, vitronectin, or, for example, RGD for surface modification, which may promote cell adhesion and proliferation (Heungsoo Shin, 2003, Biomaterials 24:4353-4364; Hwang et al., 2006 Tissue Eng. 12:2695-706). Indeed, altering molecular weights, block structures, degradable linkages, and cross-linking modes may influence strength, elasticity, and degradation properties of the instant hydrogels (Nguyen and West, 2002, Biomaterials 23(22):4307-14; Ifkovits and Burdick, 2007, Tissue Eng.

[0086] 13(10):2369-85).

[0087] Contemplated hydrogels include but are not limited to fibrinogen, collagen, hyaluronic acid, alginate, polyacrylamide, polyethylene glycol, and the like. The hydrogel can be cross-linked based on the type(s) of hydrogel used, such as by photo-cross-linking, thermal-cross-linking, chemical cross-linking, and the like.Attorney Docket No.: 204606-0207-00WO

[0088] Hydrogels may also be modified with functional groups for covalently attaching a variety of proteins or compounds such as therapeutic agents. It is contemplated that linkage of the therapeutic agent to the hydrogel may be via a protease sensitive linker or other biodegradable linkage.

[0089] In certain embodiments, one or more multifunctional cross-linking agents may be utilized as reactive moieties that covalently link biopolymers or synthetic polymers. Such bifunctional cross-linking agents may include glutaraldehyde, genipin, epoxides (e.g., bis-oxiranes), oxidized dextran, p-azidobenzoyl hydrazide, N-[a.-maleimidoacetoxy]succinimide ester, p-azidophenyl glyoxal monohydrate, bis-[(3-(4-azidosalicylamido)ethyl]disulfide, bis[sulfosuccinimidyl]suberate, dithiobis[succinimidyl proprionate, disuccinimidyl suberate, l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and other bifunctional cross-linking reagents known to those skilled in the art. It should be appreciated by those skilled in the art that the mechanical properties of the hydrogel are greatly influenced by the crosslinking time and the amount of cross-linking agents.

[0090] In some embodiments, utilizing a cross-linking agent, polyacrylated materials, such as ethoxylated (20) trimethylpropane triacrylate, may be used as a nonspecific photo-activated cross-linking agent. Components of an exemplary reaction mixture would include a thermoreversible hydrogel held at 39°C, polyacrylate monomers, such as ethoxylated (20) trimethylpropane triacrylate, a photo-initiator, such as eosin Y, catalytic agents, such as l-vinyl-2-pyrrolidinone, and triethanolamine.

[0091] Continuous exposure of this reactive mixture to long- wavelength light (>498 nm) would produce a cross-linked hydrogel network.

[0092] In some embodiments, the hydrogel comprises a UV sensitive curing agent which initiates hydrogel polymerization. For example, In some embodiments, a hydrogel comprises the photoinitiator 4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone. In some embodiments, polymerization is induced by 4-(2-hydroxy ethoxy )phenyl-(2-hydroxy-2-propyl)ketone upon application of UV light. Other examples of UV sensitive curing agents include 2-hydroxy-2-methyl-l-phenylpropan-2-one, 4-(2-hydroxyethoxy)phenyl (2-hydroxy-2-phenyl-2-hydroxy-2-propyl)ketone, 2,2-dimethoxy-2-phenyl-acetophenone 1 -[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-Attorney Docket No.: 204606-0207-00WO

[0093] methyl-1 -propane-1 -one, 1 -hydroxycyclohexylphenyl ketone, trimethyl benzoyl diphenyl phosphine oxide and mixtures thereof.

[0094] The stabilized cross-linked hydrogel of the present invention may be further stabilized and enhanced through the addition of one or more enhancing agents. By “enhancing agent” or “stabilizing agent” is intended any compound added to the hydrogel, in addition to the high molecular weight components, that enhances the hydrogel by providing further stability or functional advantages. Suitable enhancing agents, which are admixed with the high molecular weight components and dispersed within the hydrogel, include many of the additives described earlier in connection with the hydrogel discussed above. The enhancing agent may include any compound, especially polar compounds, that, when incorporated into the cross-linked hydrogel, enhance the hydrogel by providing further stability or functional advantages.

[0095] Exemplary enhancing agents for use with the stabilized cross-linked hydrogels include polar amino acids, amino acid analogues, amino acid derivatives, intact collagen, and divalent cation chelators, such as ethylenediaminetetraacetic acid (EDTA) or salts thereof. Polar amino acids are intended to include tyrosine, cysteine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, arginine, lysine, and histidine. The preferred polar amino acids are L-cysteine, L-glutamic acid, L-lysine, and L-arginine. Suitable concentrations of each particular preferred enhancing agent are the same as noted above in connection with the hydrogel. Polar amino acids, EDTA, and mixtures thereof, are preferred enhancing agents. The enhancing agents may be added to the matrix composition before or during the crosslinking of the high molecular weight components.

[0096] Cavities for the placement of a sensor may be any geometry and contain any sensor. In some embodiments, the opening of a cavity is a circle, ellipse, rectangle, square, or any other geometry. The opening of a cavity may be any surface area. The depth of a cavity may be any depth. For example, the depth of a cavity may be between 0.01 mm and 1 cm. The sensor cavity may be in fluid communication with any tissue constructs and / or vascular and synovial constructs of the device. The sensor may be any sensor. The sensor may measure or detect media components or any secreted factors from any tissue constructs and / or vascular and synovial constructs of the device. For example,Attorney Docket No.: 204606-0207-00WO

[0097] sensor cavity 116 may be designed such that a Photonic Ring Resonator Sensor may fit within the cavity. Exemplary Photonic Ring Resonators for use with the device are described in U.S. Patent Application No. 17 / 585,914, U.S. Patent Application No.

[0098] 18 / 865,135, and Bucukovski, J. & Miller, B. L. Everything's under Control: Maximizing Biosensor Performance through Negative Control Probe Selection. Anal Chem 97, 3525-3535 (2025), each of which is incorporated by reference herein in its entirety.

[0099] In some embodiments, device 100 comprises sensor 180. In some embodiments, sensor 180 is positioned in sensor cavity 116. Sensor 180 may be any suitable sensor such as any sensor described herein. Sensor 180 may measure or detect any media characteristic(s). For example, sensor 180 may detect and / or measure any molecule(s) of interest secreted by a tissue construct, cell culture, or cell thereof. Sensor 180 may measure or detect media components or any secreted factors from any tissue constructs and / or vascular and synovial constructs of device 100. For example, sensor 180 may comprise a Photonic Ring Resonator Sensor. Exemplary Photonic Ring Resonators are described in U.S. Patent Application No. 17 / 585,914, U.S. Patent Application No. 18 / 865,135, and Bucukovski, J. & Miller, B. L. Everything's under Control: Maximizing Biosensor Performance through Negative Control Probe Selection. Anal Chem 97, 3525-3535 (2025), each of which is incorporated by reference herein in its entirety.

[0100] In some embodiments, any sensors of device 100 including sensor 180 is communicatively and electronically connected to a computer (e.g., computer 1000). In some embodiments, the one or more sensors of device 100 comprise any of a biosensor, a real-time sensor, or the like. In some embodiments, the one or more sensors monitor secretory profiles and provide valuable information on disease progression and therapeutic efficacy. In some embodiments, the one or more sensors may be configured or selected to sense or analyze one or more analytes, selected from any of TNF-a, IFN-y, IL- let, IL- 1 , IL-6, IL- 17, IL-21, CXCL-13, C-reactive protein (CRP). In some embodiments, the one or more sensors may be configured or selected to sense or analyze cartilage degradation products, bone degradation products, synovial tissue degradation products, products indicative of osteoclast activity, and / or products indicative of cartilage extracellular matrix (ECM) loss. Cartilage degradation products may comprise C2MAttorney Docket No.: 204606-0207-00WO

[0101] and / or CTX-II. Bone degradation products may be indicative of bone resorption. In some embodiments, bone degradation products comprise CTX-I and / or ICTP. Synovial tissue degradation products may comprise C3M. Products indicative of osteoclast activity may comprise Tartrate-resistant acid phosphatase (TRAP). Products indicative of cartilage ECM loss may comprise glycosaminoglycans (GAGs). Although exemplary analytes are provided, it should be appreciated that other analytes such as pro-inflammatory protein factors, and other protein factors known by one of ordinary level of skill in the art may be sensed or analyzed as well.

[0102] In some embodiments, device 100 further comprises one or more embedded or functionalized reagents for detecting a target in a sample. In some embodiments, the one or more embedded or functionalized agents are positioned within any interior regions, channels, or interior volumes of the device. Exemplary reagents include, but are not limited to, oligonucleotide probes, antibodies, antibody fragments, peptides, proteins, guideRNAs, CRISPR-Cas polypeptides, and the like. In some embodiments, device 100 may comprise one or more biosensing elements positioned within the interior volumes, including but not limited to, nanowires, ring resonators, and the like.

[0103] Aspects of the present invention relate to a media channel component. The media channel component may be stacked on the first component. The media channel component may be stacked between the first component and the second component. The media channel component may provide a media channel that fluidly connects any of the tissue constructs of the device, a vascular and synovial construct of the device, and / or any number of sensors of the device. For example, as in Figure 9, media channel component 120 may comprise a frame 124 and media channel 122. Media channel 122 may comprise an opening in the frame. The opening of media channel 122 may span the thickness of the frame 124 and may have any surface area. Media channel 122 may be designed with a geometry and surface area such that any tissue cavities, and sensor cavities of the first component 110 are directly underneath the media channel 122 and the membrane 170 of the device 100 is directly above the media channel 122 in the assembled device 100. For example, the media channel 122 may fluidly connect the first tissue cavity 112, the second tissue cavity 114, the sensor cavity 116, and the membrane 170 of the device 100.Attorney Docket No.: 204606-0207-00WO

[0104] The media channel 122 may be any thickness. The thickness may be designed such that the media channel 122 contains a desired volume of media when filled.

[0105] As depicted in Figure 9, the first tissue cavity 112, second tissue cavity 114, and membrane 170 may have substantially separate and customizable culturing conditions. For example, the tissue cavity 112, second tissue cavity 114, and membrane 170 may each comprise a separate media inlet and media outlet that is proximal to the first tissue cavity 112, second tissue cavity 114, and membrane 170, respectively.

[0106] However, the media channel 122 may fluidly connect each of these components and the sensor cavity 116. Therefore, the media channel 122 may comprise a gradient of any of the media supplied to the first tissue cavity 112, second tissue cavity 114, and membrane 170. Additionally, the sensor cavity 116 may be placed such that the sensor 180 is positioned at an approximate midpoint among the first tissue cavity 112, second tissue cavity 114, and membrane 170 such that the sensor 180 may be in contact with some mixture of the provided media. Additionally, the sensor 180 may be positioned such that it may be contacted by and may detect any secreted factors of interest derived from any tissue constructs and / or vascular and synovial constructs of the device 100.

[0107] Aspects of the present invention relate to a spacer component. As in Figure 9, the spacer component 130 may be stacked under the second component 140. The spacer component 130 may comprise a spacing 132 such that the membrane 170 may be in fluid communication with the media channel 122. The spacer 132 may have any surface area or geometry. For example, the geometry of the spacer 132 may match the geometry of the bottom surface of the holder 160 or the membrane 170.

[0108] Aspects of the present invention relate to a second component 140 comprising a frame and an interior region. In some embodiments, second component 140 comprises a frame 144 having top and bottom surfaces and a thickness therebetween, and at least one opening in the frame passing through from the top to the bottom surface and forming the interior region 142. In some embodiments, the perimeter of the interior region 142 is at least partially formed in the shape of a circle, polygon, rectangle, clover, cruciform, rounded cruciform, decagon, hexagon, cluster shaped, rosette shaped, daisy shaped, flower shaped, globular shaped. It should be appreciated that the shape of the perimeter of interior region 142 is designed and sized to allow the placement of holderAttorney Docket No.: 204606-0207-00WO

[0109] 160 without damaging holder 160 and / or membrane 170.

[0110] In some embodiments, a holder 160 is fixedly and removably positioned in the interior region 142 of the second component 140. In some embodiments, holder 160 is positioned between the interior region 142 of the second component 140 and spacing 132 and at least partially separates the two regions. In some embodiments, holder 160 is positioned between the interior region 142 of the second component 140 and media channel 122 and at least partially separates the two regions.

[0111] In some embodiments, holder 160 at least partially spans an opening of the interior region 142, or at least partially fills the area within an opening of the interior region 142. In some embodiments, holder 160 and interior region 142 are sized and shaped to allow holder 160 to fit within interior region 142 with a compression and / or friction fit. In some embodiments, holder 160 is sized and shaped to allow fluid from interior region 142 to flow around holder 160 into spacing 132, and / or media channel 132. In some embodiments, holder 160 is sized and shaped such that no fluid or no substantial amount of fluid flows around the holder from interior region 142 to flow around holder 160 into spacing 132, and / or media channel 132. In this embodiment, fluid may flow through the membrane 170 to flow from interior region 142 into spacing 132, and / or media channel 132.

[0112] In some embodiments, holder 160 comprises at least one membrane 170 having a top and bottom surface spanning at least a portion of the holder 160 and spanning the thickness of holder 160. In some embodiments, the top surface and bottom surface of membrane 170 each comprise a cell culture including a cell monolayer, a tissue construct. In some embodiments, the top surface and / or bottom surface of membrane 170 comprises a scaffold comprising a cell culture. In some embodiments, the bottom surface of membrane 170, including any cell culture is a short distance from the top surface of a sensor 180 when the device 100 is assembled.

[0113] In some embodiments, the length, width, and height of the holder 160 each range between 0.1 mm and 2 cm.

[0114] In some embodiments, membrane 170 comprises any of an ultra-thin membrane, a dual-scale membrane, or a porous membrane. In some embodiments, membrane 170 is manufactured or composed of any membrane material in any thicknessAttorney Docket No.: 204606-0207-00WO

[0115] known by one of ordinary level of skill in the art. Tn some embodiments, membrane 170 has a height or thickness ranging between 0.001 mm and 1 mm.

[0116] In some embodiments, membrane 170 supports the formation and culturing of a cell culture against the top and / or bottom surface of the membrane 170 while also enabling migration of cells through the membrane 170 and / or the passage of cell secreted molecules through the membrane 170 (e.g. signaling molecules or specifically paracrine signaling molecules). For example, membrane 170 may contain any number of pores with an area large enough to support cell transmigration and / or may contain any number of pores with an area large enough to allow the passage of cell secreted molecules but small enough such that cell migration through the pores is not enabled.

[0117] For example, membrane 170 may support the culturing of an endothelial cell monolayer on the top surface of membrane 170 while also allowing for endothelial cell migration through the membrane 170 and / or allowing for paracrine signaling between cells on either side of the membrane 170. For example, the pores in membrane 170 may allow for the migration of any immune cells including leukocytes and / or monocytes. In some examples, cells cultured on the top surface of membrane 170 may migrate through the membrane 170 and enter spacing 132 and / or media channel 122. In some examples, cells cultured on the top surface of membrane 170 may migrate through the membrane and enter a hydrogel or scaffold that is adhered or otherwise contacting the bottom surface of the membrane 170. In some embodiments, membrane 170 comprises a Dual-Scale (DS) silicon nitride porous membrane with 5 pm pores dispersed over a nano-porous (<100 nm pores) background. In some embodiments, membrane 170 comprises a Dual-Scale (DS) silicon nitride porous membrane with 3 pm pores or approximately 3 pm pores dispersed over a nano-porous (<100 nm pores) background.

[0118] In some embodiments, a cellular monolayer is cultured on the top surface of membrane 170. In some embodiments, the top surface of membrane 170 comprises a cellular monolayer comprising one or more cells. In some embodiments, the top surface of membrane 170 is seeded with the one or more cells. In some embodiments, the one or more cells comprise any of mammalian cells, human cells, tissue cells, endothelial cells (EC), epithelial cells, immune cells, monocytes, macrophages, red blood cells, peripheralAttorney Docket No.: 204606-0207-00WO

[0119] blood mononuclear cells (PBMC) In some embodiments, EC cells are cultured on membrane 170 to form a cohesive vascular barrier with developed junction proteins. In some embodiments, membrane 170 may comprise supporting cells such as stellate cells, pericyte cells, fibroblast cells, mesenchymal cells, or other supporting cells known by one of ordinary level of skill in the art. In some embodiments, the one or more cells are derived from induced pluripotent stem cells. In some embodiments, the induced pluripotent stem cells are derived from a patient.

[0120] In some embodiments, synovial cells are cultured on the bottom surface of membrane 170. In some embodiments, a hydrogel comprising synovial cells is attached, adhered, or otherwise positioned on the bottom surface of membrane 170. Synovial cells may comprise NK cells, T cells, Myeloid cells, Stromal cells, fibroblasts, fibroblast / stromal cells, B / plasma cells, endothelial cells, or any subcategory thereof. Exemplary subcategories of synovial cell types are depicted in Figure 10. Synovial cell types may be sorted by the presence of surface markers or any other molecular markers. Exemplary surface and molecular markers that define subsets of synovial cell types are described in Zhang, F. et al. (Zhang F. et al., Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes. Nature 623, 616-624 (2023)) which is hereby incorporated by reference in its entirety.

[0121] In some embodiments, the synovial hydrogel comprises primary patient-derived synovial cells. In some embodiments, the synovial hydrogel comprise synovial cells derived from induced pluripotent cells. In some embodiments, the synovial cells included in the hydrogel comprise a combination of synovial cell types that reflects the combination of synovial cell types in a subject, in a model organism, in a subject having rheumatoid arthritis, and / or in a subject having any pathotype of rheumatoid arthritis. In some embodiments, the synovial cells include a combination of cell types known to be indicative of any cell type abundance profile (CTAP) of rheumatoid arthritis. Exemplary cell type abundance profiles of rheumatoid arthritis include CTAP-M, CTAP-TM, CTAP-TB, CTAP-TF, CTAP-F, and CTAP-EFM. Exemplary distributions of cells that relate to CTAP-TB, CTAP-M, and CTAP-F are depicted in Figure 10. Synovial cell distributions indicative of CTAP-TB may be enriched in T cells, B / plasma cells, and fibroblasts.

[0122] Synovial cell distributions indicative of CTAP-M may be enriched in myeloid cells andAttorney Docket No.: 204606-0207-00WO

[0123] stromal cells. Synovial cell distributions indicative of CTAP-F may be enriched in fibroblasts and endothelial cell subsets with minimal immune infiltration. In some embodiments, patient-derived synovial cells seeded in the hydrogel preserve the CTAP composition of the patient. For example, synovial cells may be dissociated from and / or isolated from a synovial biopsy of a patient, subject, or model organism. In some embodiments, synovial cells including any synovial cell type or subtype are derived from iPSCs. Exemplary cell type abundance profiles (CTAPs) of rheumatoid arthritis are described in Zhang, F. et al. (Zhang F. et al., Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes. Nature 623, 616-624 (2023)) which is hereby incorporated by reference in its entirety.

[0124] The synovial hydrogel may comprise any hydrogel. The synovial hydrogel may comprise any physiologically relevant substrate for the adhesion, migration, and / or signaling of stromal cells and / or immune cells. In some embodiments, the hydrogel comprises any collagen subtype or combinations thereof including but not limited to type I collagen and / or type III collagen, gelatin mathacryloyl (GelMA), fibrin, fibrin-based hydrogels. In some embodiments, the synovial hydrogel may mimic or model an inflamed synovium. For example, the inclusion of fibrin or a fibrin-based hydrogel in the synovial hydrogel may aid in the modeling of an inflamed synovium.

[0125] The synovial hydrogel may have any geometry including any thickness. In some embodiments, the synovial hydrogel has a thickness of approximately 300 pm.

[0126] Aspects of the present invention relate to a flow insert for the device. The flow insert may be configured to such that it fits within the interior region 142 of the second component 140. As depicted in Figure 9, device 100 may comprise flow insert 190 positioned in interior region 142. In some embodiments the bottom of flow insert 190 rests against the holder 160. In some embodiments, there is a spacing between the bottom of flow insert 190 and the holder 160. A spacing between the bottom of flow insert 190 and the holder 160 may be designed such that a desired volume of media may fit in the spacing. In some embodiments, the flow insert 190 comprises an inlet opening and an outlet opening. In some embodiments, media is flowed from the inlet opening to the outlet opening such that the cell culture is exposed to a flow. In some embodiments, the flow rate is or is approximately 1.5 dyn / cm2.Attorney Docket No.: 204606-0207-00WO

[0127] In some embodiments, an inlet opening is fluidly connected to an inlet channel of a tissue cavity and an outlet opening is fluidly connected to an outlet channel of a tissue cavity. In some embodiments, as in Figure 9, there are openings in the media channel component 120, spacer component 130, and second component 140 such that when the device 100 is assembled a lumen forms from the bottom to the top of the device 100 which fluidly connects each tissue cavity inlet channel to a media source and each tissue cavity outlet channel to a media sink. In some embodiments, a separate media source and media sink is connected to each tissue cavity. In some embodiments, media is continuously circulated in each cavity as well as interior region 142.

[0128] Any fluid including medium may be introduced to any tissue cavity or interior region 142. In some embodiments, the media introduced to any tissue cavity, interior region 142 or any other medium used with the device may comprise cells. In some embodiments, any medium used with the device may comprise blood cells or peripheral blood mononuclear cells. In some embodiments, the cells are derived from a patient. In some embodiments the cells are derived from induced pluripotent stem cells. In some embodiments, the fluid may comprise cells, media, growth factors, therapeutic agents (e.g., small molecules, peptides, nucleic acid molecules, antibodies) or the like. In some embodiments, the media comprises any drugs for the treatment of Rheumatoid Arthritis or inflammation. In some embodiments, the media comprises conventional and / or biologic disease modifying antirheumatic drugs (DMARDs). Exemplary drugs include but are not limited to Methotrexate, Etanercept, Tocilizumab, Abatacept, Rituximab, CCX354, EC359, DAPT, RO4929097, Anti-NRR3, CB-103, CCR1 antagonists, LIFR inhibitors, Gamma-secretase inhibitors, Tofacitinib, JAK1 / 3 inhibitors, baricitinib, JAK1 / 2 inhibitors, Upadacitinib, filgotinib, JAK1 inhibitors, selective JAK1 inhibitors, and NOTCH inhibitors.

[0129] In some embodiments, any components of the device 100 may be aligned with the aid of an assembly jig.

[0130] Device 100 may comprise components that are removably attached, and therefore surfaces of the components may comprise one or more adhesives or adhesive layers. The tops and bottom surfaces of first component 110, media channel component 120, spacer component 130, and / or second component 140 may comprise one or moreAttorney Docket No.: 204606-0207-00WO

[0131] adhesives or adhesive layers. In some embodiments, first component 110, media channel component 120, spacer component 130, and / or second component 140 may comprise one or more coatings on their exterior surfaces. Exemplary coatings include antimicrobial coatings, anti -reflective coatings, waterproof coatings, or the like.

[0132] Aspects of the present invention relate to exemplary methods for assembly and use of a microphysiological system and device.

[0133] In some embodiments, the method comprises the steps of providing the device 100, positioning a synovial hydrogel on the bottom of a membrane of the device, positioning endothelial cells on the top of the membrane, positioning a bone tissue construct in a first tissue cavity of the device, positioning a cartilage tissue construct in a second tissue cavity of the device, assembling the device, and flowing a first medium, second medium, and third medium in the interior region, first tissue cavity, and second tissue cavity of the device, respectively. In some embodiments, the method further comprises applying a treatment to one or more of the mediums of the method. In some embodiments, the method further comprises analyzing one or more of the synovial hydrogel, endothelial cells, bone tissue construct, or cartilage tissue construct.

[0134] In some embodiments, positioning a synovial hydrogel on the bottom membrane of the device comprises thawing synovial cells. In some embodiments, positioning a synovial hydrogel on the bottom membrane of the device comprises suspending synovial cells in a hydrogel comprising type I collagen and seeding the hydrogel seeded with synovial cells onto the underside of a membrane of the device. In some embodiments, the membrane is a pSiM membrane. In some embodiments, the synovial hydrogel is cultured on the bottom of the membrane of the device for some period of time before the device is assembled. In some examples, the period of time is in the range of three days to ten days. In some examples, the synovial hydrogel is cultured in a 1:1:1 mix of synovial cell media, leukocyte media, and endothelial cell media. In some examples, the composition of the hydrogel comprises components that mimic any characteristic of the synovial environment. In some examples, the synovial cells comprises a combination of cells that recapitulates a combination of cells of any Rheumatoid Arthritis Cell Type Abundance Profile (CTAP).

[0135] In some embodiments, endothelial cells are positioned on the top of theAttorney Docket No.: 204606-0207-00WO

[0136] membrane of the device after the synovial hydrogel is positioned on the bottom of the device. In some embodiments, the synovial hydrogel is cultured on the bottom of the membrane of the device for some period of time before endothelial cells are positioned on the top of the membrane. In some examples, the synovial hydrogel is cultured for approximately seven days before endothelial cells are seeded on the membrane. In some embodiments, endothelial cells are cultured for 72 hours or approximately 3 days after being positioned or seeded on the membrane before the device is assembled. In some embodiments, the membrane comprising seeded endothelial cells is cultured in conditions that promote the formation of a vascular barrier. In some examples, the membrane comprising seeded endothelial cells are cultured in the presence of shear stress, for example 1.5 dynes / cm2.

[0137] In some embodiments, a bone tissue construct is positioned in a tissue cavity of the device. In some embodiments, bone tissue constructs are first produced and matured before being positioned in a tissue cavity of the device. In some embodiments, bone tissue constructs are produced and matured within a tissue cavity of the device 100 before the device is assembled. In some embodiments, bone tissue constructs are produced by seeding decellularized trabecular bone discs with mesenchymal progenitor cells. In some embodiments, the mesenchymal progenitor cells are derived from induced pluripotent stem cells. In some embodiments, the decellularized trabecular bone discs are cultured for approximately 21 days to allow for maturation of the bone tissue construct. In some embodiments, the bone tissue construct is matured before being placed in the device. In some embodiments, the bone tissue construct is cultured or exposed to an osteogenic medium. In some embodiments, the bone tissue construct is allowed to mature after being placed in the device.

[0138] The media presented to the construct configured to mimic bone tissue may also be customized to promote the viability, maturation, differentiation, culture of, or matrix deposition of mesenchymal progenitor cells, monocytes, osteoblasts, osteoclasts, and / or any bone cells. For example, a suitable media may comprise osteogenic media. An osteogenic media may comprise dexamethasone, ascorbic acid-2-phosphate, and / or 0-glycerophosphate. In some examples, osteogenic media may comprise or further comprise M-CSF, RANKL, and / or serum. In some embodiments, the bone tissueAttorney Docket No.: 204606-0207-00WO

[0139] construct is cultured in osteogenic medium for approximately 21 days to promote the maturation of the bone tissue construct. In some examples, the medium promotes the differentiation of induced pluripotent stem cell (iPSC) - derived monocytes to osteoclasts. In some embodiments, a suitable medium for differentiation of iPSC-derived monocytes to osteoblasts comprises M-CSF, RANKL, and / or serum. In some examples, the osteoblast differentiation comprises M-CSF in a concentration between 25 ng / mL and 50 ng / mL. In some examples, the osteoblast differentiation comprises RANKL in a concentration between 30 ng / mL and 100 ng / mL. In some examples, the cells are cultured in osteoblast differentiation medium for a duration in the range of approximately 7 days to 14 days. In some embodiments, the bone tissue construct is cultured for some time before positioning in the tissue cavity. In some embodiments, the bone tissue construct is positioned in the cavity and then cultured for any amount of time.

[0140] Exemplary bone tissue constructs for use in the device are described in Lin, Z. et al. (Lin, Z. et al., Osteochondral Tissue Chip Derived From iPSCs: Modeling OA Pathologies and Testing Drugs. Front Bioeng Biotechnol 7, 411 (2019)) which is incorporated by reference herein in its entirety.

[0141] In some embodiments, a cartilage tissue construct is positioned in a tissue cavity of the device. In some embodiments, cartilage tissue constructs are first produced and matured before being positioned in a tissue cavity of the device. In some embodiments, cartilage tissue constructs are produced and matured within a tissue cavity of the device 100 before the device is assembled. In some embodiments, the cartilage tissue construct comprises a cartilage disc. In some embodiments, the cartilage tissue construct is formed by photocuring chondrocytes in a hydrogel. In some embodiments, the hydrogel comprises type II collagen and / or Methacrylated hyaluronic acid (HAMA). In some embodiments, the cartilage tissue construct is cultured in a chondrogenic medium. In some embodiments, the cartilage tissue construct is cultured in a medium that promotes maturation.

[0142] The media presented to the cartilage tissue construct may also be customized to promote the viability, maturation, differentiation, or culture of cartilage. In some embodiments, a suitable medium comprises a chondrogenic medium. In some examples, chondrogenic medium comprises TGF-P3, ascorbic acid, dexamethasone, ITSAttorney Docket No.: 204606-0207-00WO

[0143] supplement, proline, and / or pyruvate.

[0144] In some embodiments, mesenchymal stem cells or MSCs are cultured in the cartilage scaffold for approximately 14 to 21 days, optionally in a medium comprising chondrogenic medium, to promote the differentiation of MSCs to chondrocytes. The MSCs may be cultured in the cartilage scaffold before or after the cartilage tissue construct is positioned in the device 100.

[0145] In some embodiments, the device is assembled after the synovial hydrogel is matured on the membrane 170, after the endothelial cells form a vascular barrier on the membrane 170, after the bone tissue construct is matured, and / or after the cartilage tissue construct is matured. In some embodiments, the device is assembled after the synovial hydrogel is positioned on the membrane 170, after the endothelial cells are positioned on the membrane 170, after the bone tissue construct is positioned in a tissue cavity of the device, and / or after the cartilage construct is positioned in a tissue cavity of the device. In some embodiments, the device is assembled with the aid of pressure-sensitive adhesives. In some embodiments, the device is assembled with the aid of a jig. In some embodiments, the jig is a custom jig.

[0146] In some embodiments, the method comprises flowing a first medium, second medium, and third medium in the interior region, first tissue cavity, and second tissue cavity of the device, respectively. The media may have any flow rate. In some embodiments, the first medium comprises a medium configured for the maintenance of endothelial cells and / or synovium. In some embodiments, the second medium comprises a medium configured for the maintenance or maturation of a bone tissue construct. In some embodiments, the third medium comprises a medium configured for the maintenance or maturation of a cartilage tissue construct. In some embodiments, the medium comprises cells. In some embodiments, the cells are patient-derived cells. In some embodiments, the cells are PBMCs. In some embodiments, the flow rate is configured such that a desired gradient among different media types is achieved in the media channel of the device. In some embodiments, the flow rate is configured such that medium that is proximal to the interior region or tissue cavities of the device is substantially that medium.

[0147] In some embodiments, the method further comprises a step of collectingAttorney Docket No.: 204606-0207-00WO

[0148] cells from a subject and forming a tissue construct with the patient cells (e.g., collecting patient-derived cells). Patient cells such as isolated blood cells, endothelial cells, synovial cells, bone cells, or cartilage cells may be collected from any patient sample or tissue, including, but not limited to, blood samples, oral swabs, and tissue biopsies (e.g., synovial tissue, scar tissue, retinal tissue, skin tissue, organ tissue, cancerous tissue, etc.). In some embodiments, cells may be sorted and / or isolated based on gene expression profde, the expression of any marker genes, and / or the expression of any surface proteins to acquire the cells of interest. For example, cells may be sorted based on any synovial cell type markers, bone cell type markers, cartilage cell type marker, blood cell type markers, immune cell type markers, or markers of inflammatory state. In some embodiments, patient cells may be treated with an agent prior to forming a tissue construct. Examples of agents include, but are not limited to, small molecules, nucleic acids, peptides, siRNAs, shRNAs, miRNAs, ribozymes, antisense nucleic acids, antagonists, inhibitors, agonists, partial agonists, inverse agonists, aptamers, peptidomimetics, viruses, bacteria, cells, or any combination thereof. In some embodiments, the one or more agents are cellular differentiation-inducing agents, agents for preventing cellular differentiation, agents for reversing cellular differentiation, agents for preventing reversal of cellular differentiation, agents for inducing pluripotency, anti-fibrotic agents, anti-inflammatory agents, anti-cancer therapies, anti-cancer drugs, antiviral drugs, anti-microbial drugs, DMARDs, and / or anti-arthritic drugs.

[0149] In some embodiments, the tissue constructs may be cultured in the assembled device for any period of time before tissue constructs are analyzed. In some examples, the tissue constructs may be cultured in the assembled device in the range of approximately 7 to 14 days.

[0150] In some embodiments, the method further comprises a step of treating the interior region 142, first tissue cavity 112, and / or second tissue cavity 114. In some embodiments, treating a region comprises administering an agent. Examples of agents include, but are not limited to, small molecules, nucleic acids, peptides, siRNAs, shRNAs, miRNAs, ribozymes, antisense nucleic acids, antagonists, inhibitors, agonists, partial agonists, inverse agonists, aptamers, peptidomimetics, viruses, bacteria, cells, or any combination thereof. In some embodiments, the one or more agents are anti-fibroticAttorney Docket No.: 204606-0207-00WO

[0151] agents, anti-inflammatory agents, anti-cancer therapies, anti-cancer drugs, anti-viral drugs, anti-microbial drugs, DMARDs, and / or anti-arthritic drugs. In some embodiments, treating a region comprises changing the conditions of the region. Examples of condition changes include, but are not limited altering the pH of the region, altering the temperature of the region, altering the atmosphere of the region (e.g., increasing or decreasing the amount of CO2), altering the humidity of the region, altering the stress or strain applied to the region, altering the orientation of the region, exposing or halting exposure of the region to electromagnetic radiation (e.g., UV therapy, IR therapy, ionizing radiation treatment), exposing or halting exposure of the region to a magnetic field, exposing or halting exposure of the region to sound (e.g., sonotherapy), or any combination thereof.

[0152] In general, cells, fluids, cell components, and cell supernatants may be isolated from either interior region 142, membrane 170, first tissue cavity 112, and / or second tissue cavity 114 in order to perform any desired assays. Exemplary assays include, but are not limited to, any DNA / RNA sequencing, RT-PCR, RT-qPCR, proteomics, transcriptomics, genomics, metabolomics, metabolic profiling, mass spectrometry (MS), nuclear magnetic resonance (NMR) analysis, Western blotting, Southern blotting, Northern blotting, ELISA assays, cell-sorting, scratch assays, scrape loading / dye transfer assays, beat synchronization assays, signal conductivity assays, luciferase assays, optical assays, enzyme activity assays, protein binding assays, cell proliferation assays, cell viability assays, oxidation assays, reduction assays, reactive oxygen species (ROS) assays, and immunoassays known by one of ordinary level of skill in the art. In some embodiments, the assay comprises any assay for cellular and molecular markers of a cell type abundance profile of synovium. In some embodiments, the assay comprises single-cell RNA-sequencing, immunofluorescence microscopy either on live or fixed cells, microscopy imaging of synovial hyperplasia, endothelial activation and leukocyte transendothelial migration. In some embodiments, longitudinal PhRR sensor-based cytokine release profiles are analyzed. In some embodiments, the assay comprises an assessment of bone and cartilage degradation. In some embodiments, the degradation assessment is based on tartrate-resistant acid phosphatase (TRAP), glycosaminoglycan (GAG) release, the presence of collagen degradation products including C2M and CTX-II (specific to cartilage degradation), C3M (synovial tissueAttorney Docket No.: 204606-0207-00WO

[0153] degradation), and / or CTX-I and ICTP (bone resorption). In some embodiments, TRAP or GAG release and the presence of collagen degradation products is measured by ELISA. In some embodiments, the assay comprises the assessment of secreted inflammatory cytokines. Exemplary secreted inflammatory cytokines comprise TNF-a, IFN-y, IL-10, IL-6, etc. and C-reactive protein (CRP). Assays for the assessment of analytes or cytokines including secreted inflammatory cytokines may comprise ELISA or any Luminex type assays.

[0154] In some embodiments, the method further comprises the step of collecting one or more cell supernatants, or fluids, from the interior region 142, first tissue cavity 112, second tissue cavity 114, and / or media channel 122. In some embodiments, the method further comprises the step of imaging the membrane 170, first tissue cavity 112, and / or second tissue cavity 114.

[0155] In some embodiments, the method may further comprise the step of measuring one or more morphological changes in the cells of the bone tissue construct, cartilage tissue construct, endothelial cell layer, and / or the synovial hydrogel. In some embodiments, the method may further comprise the step of detecting one or more analytes from the cells of the bone tissue construct, cartilage tissue construct, endothelial cell layer, and / or the synovial hydrogel.

[0156] The method may further comprise conducting or performing one or more assays or analysis before, during, or after any disclosed steps, selected from: secrotome analysis, cytokine analysis, cytotoxicity assay, supernatant assays, functional Assays, Luminex Multiplex Immunoassay, Human Luminex Discovery Assays, endpoint assays, immunostaining, Immunohistochemistry (IHC), fluorescence Microscopy, Bulk Construct Analysis Techniques, Collagen Contraction Quantification, a-SMA Quantification, Live-Stain Imaging, Quantification of Macrophage Transmigration, yH2A.X Quantification, Cellular alignment, cell signaling assay, RNA quantification, or the like. In some embodiments, the method may further comprise performing a reactome pathway enrichment analysis, analyzing the upregulation or downregulation of proteins (e.g., CD31, VE-cadherin, vWF, ICAM-1, VCAM-1, Collagen Type II, Aggrecan, GAG, ALP, Runx2, TRAP, cathepsin K, SOX2, OCT4, SSEA4, TRA-1-60, NFATcl, LIFR, NOTCH3, CCL14, CCR1, C2M, CTX-II, C3M, CTX-I, ICTP).Attorney Docket No.: 204606-0207-00WO

[0157] In some embodiments, the method further comprises the quantification of selected human cytokines and chemokines in cell supernatants collected from device 100. The selected human cytokines and chemokines may comprise TNF-a, IFN-y, IL-1 a, IL-10, IL-6, IL- 17, IL-21, CXCL-13.

[0158] In some embodiments, the method may further include steps for drug screening or drug development. For example, the administered agent may be any number of potential therapeutics and any desired assay may be performed at any time after the administration of potential therapeutics. The fibrotic state, inflammatory state, or any disease state of the tissue may be ascertained using any relevant assay.

[0159] In some embodiments, any analysis, quantification, and / or characterization is performed after any period of time after the microphysiological device is assembled and is used for culture, or any period of time after a treatment is administered to any portion of the device or cell culture of the device. For example any analysis, quantification, and / or characterization may be performed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks 3 weeks, or 4 weeks after the device is assembled or after a treatment is administered.

[0160] Computing Device

[0161] In some aspects of the present invention, software executing the instructions provided herein may be stored on a non-transitory computer-readable medium, wherein the software performs some or all of the steps of the present invention when executed on a processor.

[0162] Aspects of the invention relate to algorithms executed in computer software. Though certain embodiments may be described as written in particular programming languages, or executed on particular operating systems or computing platforms, it is understood that the system and method of the present invention is not limited to any particular computing language, platform, or combination thereof. Software executing the algorithms described herein may be written in any programming language known in the art, compiled, or interpreted, including but not limited to C, C++, C#, Objective-C, Java, JavaScript, MATLAB, Python, PHP, Perl, Ruby, or Visual Basic. It is further understood that elements of the present invention may be executed on anyAttorney Docket No.: 204606-0207-00WO

[0163] acceptable computing platform, including but not limited to a server, a cloud instance, a workstation, a thin client, a mobile device, an embedded microcontroller, a television, or any other suitable computing device known in the art.

[0164] Parts of this invention are described as software running on a computing device. Though software described herein may be disclosed as operating on one particular computing device (e.g. a dedicated server or a workstation), it is understood in the art that software is intrinsically portable and that most software running on a dedicated server may also be run, for the purposes of the present invention, on any of a wide range of devices including desktop or mobile devices, laptops, tablets, smartphones, watches, wearable electronics or other wireless digital / cellular phones, televisions, cloud instances, embedded microcontrollers, thin client devices, or any other suitable computing device known in the art.

[0165] Similarly, parts of this invention are described as communicating over a variety of wireless or wired computer networks. For the purposes of this invention, the words “network”, “networked”, and “networking” are understood to encompass wired Ethernet, fiber optic connections, wireless connections including any of the various 802.11 standards, cellular WAN infrastructures such as 3G, 4G / LTE, or 5G networks, Bluetooth®, Bluetooth® Low Energy (BLE) or Zigbee® communication links, or any other method by which one electronic device is capable of communicating with another. In some embodiments, elements of the networked portion of the invention may be implemented over a Virtual Private Network (VPN).

[0166] Fig. 8 and the following discussion are intended to provide a brief, general description of a suitable computing environment in which the invention may be implemented. While the invention is described above in the general context of program modules that execute in conjunction with an application program that runs on an operating system on a computer, those skilled in the art will recognize that the invention may also be implemented in combination with other program modules.

[0167] Generally, program modules include routines, programs, components, data structures, and other types of structures that perform particular tasks or implement particular abstract data types. Moreover, those skilled in the art will appreciate that the invention may be practiced with other computer system configurations, including hand-Attorney Docket No.: 204606-0207-00WO

[0168] held devices, multiprocessor systems, microprocessor-based or programmable consumer electronics, minicomputers, mainframe computers, and the like. The invention may also be practiced in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules may be located in both local and remote memory storage devices.

[0169] Fig. 8 depicts an illustrative computer architecture for a computer 1000 for practicing the various embodiments of the invention. The computer architecture shown in Fig. 8 illustrates a conventional personal computer, including a central processing unit 1050 (“CPU”), a system memory 1005, including a random access memory 1010 (“RAM”) and a read-only memory (“ROM”) 1015, and a system bus 1035 that couples the system memory 1005 to the CPU 1050. A basic input / output system containing the basic routines that help to transfer information between elements within the computer, such as during startup, is stored in the ROM 1015. The computer 1000 further includes a storage device 1020 for storing an operating system 1025, application / program 1030, and data.

[0170] The storage device 1020 is connected to the CPU 1050 through a storage controller (not shown) connected to the bus 1035. The storage device 1020 and its associated computer-readable media provide non-volatile storage for the computer 1000. Although the description of computer-readable media contained herein refers to a storage device, such as a hard disk or CD-ROM drive, it should be appreciated by those skilled in the art that computer-readable media can be any available media that can be accessed by the computer 1000.

[0171] By way of example, and not to be limiting, computer-readable media may comprise computer storage media. Computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer-readable instructions, data structures, program modules or other data. Computer storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used toAttorney Docket No.: 204606-0207-00WO

[0172] store the desired information and which can be accessed by the computer.

[0173] According to various embodiments of the invention, the computer 1000 may operate in a networked environment using logical connections to remote computers through a network 1040, such as TCP / IP network such as the Internet or an intranet. The computer 1000 may connect to the network 1040 through a network interface unit 1045 connected to the bus 1035. It should be appreciated that the network interface unit 1045 may also be utilized to connect to other types of networks and remote computer systems.

[0174] The computer 1000 may also include an input / output controller 1055 for receiving and processing input from a number of input / output devices 1060, including a keyboard, a mouse, a touchscreen, a camera, a microphone, a controller, a joystick, or other type of input device. Similarly, the input / output controller 1055 may provide output to a display screen, a printer, a speaker, or other type of output device. The computer 1000 can connect to the input / output device 1060 via a wired connection including, but not limited to, fiber optic, Ethernet, or copper wire or wireless means including, but not limited to, Wi-Fi, Bluetooth, Near-Field Communication (NFC), infrared, or other suitable wired or wireless connections.

[0175] As mentioned briefly above, a number of program modules and data files may be stored in the storage device 1020 and / or RAM 1010 of the computer 1000, including an operating system 1025 suitable for controlling the operation of a networked computer. The storage device 1020 and RAM 1010 may also store one or more applications / programs 1030. In particular, the storage device 1020 and RAM 1010 may store an application / program 1030 for providing a variety of functionalities to a user. For instance, the application / program 1030 may comprise many types of programs such as a word processing application, a spreadsheet application, a desktop publishing application, a database application, a gaming application, internet browsing application, electronic mail application, messaging application, and the like. According to an embodiment of the present invention, the application / program 1030 comprises a multiple functionality software application for providing word processing functionality, slide presentation functionality, spreadsheet functionality, database functionality and the like.

[0176] The computer 1000 in some embodiments can include a variety of sensors 1065 for monitoring the environment surrounding and the environment internal to theAttorney Docket No.: 204606-0207-00WO

[0177] computer 1000. These sensors 1065 can include a Global Positioning System (GPS) sensor, a photosensitive sensor, a gyroscope, a magnetometer, thermometer, a proximity sensor, an accelerometer, a microphone, biometric sensor, barometer, humidity sensor, radiation sensor, or any other suitable sensor.

[0178] EXPERIMENTAL EXAMPLES

[0179] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0180] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0181] e 1: Modeling Synovial Heterogeneity in Rheumatoid Arthritis in a Joint-on-a-

[0182] Modeling synovial heterogeneity in new alternative methods (NAMs) is essential for accurate representation of RA and discovery of new DMARDs for refractory subtypes.

[0183] While there are well accepted clinical RA classification criteria, synovial pathotyping is gaining acceptance as a clinically relevant method to stratify synovial pathologies based on distinct molecular and cellular features that define clinical RA subtypes which are linked with different clinical outcomes and responses to treatments (Aletaha, D. et al. Arthritis Rheum 62, 2569-2581 (2010), Arnett, F. C. et al. Arthritis Rheum 31, 315-324 (1988), Aletaha, D. et al. Ann Rheum Dis 69, 1580-1588 (2010), Steiner, G., et al., RMD Open 10, e003851 (2024), Humphreys, J. H. et al. Ann Rheum Dis 72, 1315-1320 (2013), Lewis, M. J. et al. Cell Rep 28, 2455-2470.e2455 (2019),Attorney Docket No.: 204606-0207-00WO

[0184] Pitzalis, C., et al., Current Opinion in Rheumatology 25, 334-344 (2013), Rivellese, F. et al. Nat Med 28, 1256-1268 (2022).). In RA patients, regardless of disease stage, activity, imaging, or other clinical parameters, three synovial histologic pathotypes, lympho-myeloid, diffuse myeloid, and pauci-immune, have been identified, each showing distinct relationships with disease progressionjoint damage, and response to treatment (Asif Amin, M., et al., Semin Immunopathol 39, 385-393 (2017), Humby, F. et al. Annals of the Rheumatic Diseases 78, 761-772 (2019), Kemble, S. & Croft, A. P. Front Immunol 12, 715894 (2021)).

[0185] A comprehensive analysis of inflamed synovial tissues from 79 RA patients using single-cell cellular indexing of transcriptomes and epitomes (CITE-Seq) histological methods has been conducted. Based on these analyses, the innovative concept of Cell-Type Abundance Phenotypes (CTAPs) has been introduced (Zhang, F. et al. Nature 623, 616-624 (2023)). CTAPs offer a more nuanced molecular framework for understanding the diversity of synovial tissues in RA compare to histology pathotyping. This framework categorizes inflamed synovial tissues into six distinct pathotypes, with unique molecular signatures, including key cytokines, chemokines, and surface markers, which notably are not at all predictable from the clinical profile of the patients.

[0186] Evidence has suggested synovial CTAPs significantly influence therapeutic outcomes and can be altered by therapy. For instance, CTAP-F, a pauci-immune subtype, poorly responded to biologic therapies like rituximab and tocilizumab, while CTAP-TB and CTAP-TF, enriched in lymphocytes and fibroblasts, responded better. These predictions have been confirmed in RAI 1. Large synovial biopsy-driven clinical trials have demonstrated the power of synovial molecular analytics for precision medicine treatment approaches (Humby, F. et al. Lancet 397, 305-317 (2021).

[0187] There is an unmet drug development need to accurately represent synovial heterogeneity in drug development tools offering a scientifically compelling rationale for NAMs, which can address the pharmaceutical industry’s insatiable demand for superior cost-efficiencies in drug discovery and validation.

[0188] While the synovial histologic pathotyping paradigm has been reported and is increasingly utilized in research settings, its full integration into routine clinical practice is still evolving. The emerging molecular and cellular subtyping, which provides a higherAttorney Docket No.: 204606-0207-00WO

[0189] resolution classification of RA synovium based on 6 distinct CTAPs, offers rich datasets that could help identify alternative therapeutic pathways for refractory subtypes and advance precision medicine in RA management. It was posited in this example that representing the synovial CTAPs in combinational and synergistic NAMs, such as the RA Joint-on-Chip (RA-JoC), offers a transformative framework to leverage the patient’s cellular and molecular data to revolutionize precision medicine in arthritis, aligning treatment strategies with the underlying biology of disease subtypes.

[0190] RA-JoC: An in vitro model of Rheumatoid Arthritis

[0191] The RA-JoC uses the pSiM platform which incorporates significant technological innovations (Fig. 1A). It utilizes ultra-thin, transparent, and permeable silicon nanomembranes (-100 nm thick) with adjustable pore sizes from -60 nm to 10 pm for constructing endothelialized synovial membranes, enabling detailed studies of paracrine signaling and immune cell migration under static or flow conditions (Ajalik, R. E. et al. Adv Healthc Mater 14, e2403116 (2025), Masters, E. A. et al. Nanomedicine 21, 102039 (2019), McCloskey, M. C. et al. Adv Healthc Mater 11, e2200804 (2022), Mossu, A. et al. J Cereb Blood Flow Metab. 39, 395-410 (2019)). Additionally, the platform features a modular design that supports both live and endpoint imaging, multiplex protein detection, and single-cell assays, including scRNA-Seq and flow cytometry. It also integrates Photonic Ring Resonator (PhRR) sensors for real-time, label-free detection of proteins, providing high specificity and sensitivity for monitoring inflammatory cytokines and ECM degradation products, essential for tracking disease progression. This NAM is highly innovative in that it uses a new design of a versatile and modular joint-on-a-chip to experimentally model the crosstalk between immune cells and a vascularized synovial lining of the joint, engineered with the patient’s distinct CTAP, with engineered cartilage and bone constructs (Fig. IB).

[0192] The human Tendon-on-a-Chip (hToC) has been developed as a model of the myofibroblast microenvironment in peritendinous adhesions (Ajalik, R. E. et al. Adv Healthc Mater 14, e2403116 (2025)). Key fibrotic processes, including vascular inflammation, myofibroblast activation, and cytokine secretion, were modeled with TGF-pi treatment, which led to increased mTOR activity, consistent with in vivo observations. More importantly, treatment with the mTOR inhibitor rapamycin reduced TGF-01-Attorney Docket No.: 204606-0207-00WO

[0193] induced fibrosis and inflammation, including markers such as a-SMA and Ki67 and M1 / M2 macrophage ratio. The feasibility and importance of incorporating physiological fluid flow into the hToC was demonstrated. Key responses of the activated endothelium under flow, which affected the inflammatory cellular infiltration into the myofibroblast microenvironment hydrogel was demonstrated. Furthermore, advances in biosensor performance, including optimized sensitivity and specificity highlight the potential for integrating state-of-the-art, label-free PhRR sensors in platforms like the hToC for dynamic disease state monitoring, which are translated seamlessly to the RA-JoC (Bucukovski, J. & Miller, B. L. Anal Chem 97, 3525-3535 (2025)).

[0194] Modeling RA’s CTAP heterogeneity in a vascularized synovial joint on a chip Two component tissue chips are customized by redesigning its bottom module while retaining the standard top component, with a 100 pl well that accommodates pSiM membrane chips and a flow insert, to create a vascularized synovium. The bottom component incorporates fluidic channels to house cartilage and bone constructs on either side of a PhRR sensor. This modular design enables separate culture of tissues optimized for their cell types, which are later assembled after achieving phenotype stability under fluidic conditions. The device integrates patient’s PBMCs flowing in a blood compartment over iPSC-derived endothelial cells on the top (blood) side of the pSiM membrane. The bottom (tissue) side of the membrane is lined with a synovial hydrogel seeded with primary patient-derived synovial cells, preserving the CTAP composition of the donor. Three synovial subtypes are modeled, fibroid (F), lymphoid (T+B), and myeloid (M), to simulate RA’s synovial heterogeneity and distinct responses to treatments. iPSC-derived chondrocytes (iCH) and mesenchymal progenitor cells (iMPC) are used to create a cartilage hydrogel and bone scaffold, respectively. To achieve a synovial joint configuration, a novel bottom component with fluidic channels that hold the cartilage and bone constructs close to a PHRR sensor was designed (Fig. 2).

[0195] The isogenic device integrates patient’s PBMCs flowing in a blood compartment lined with iPSC-derived endothelial cells on the top side of the pSiM membrane and interfacing with a synovial hydrogel seeded with primary patient-derived synovial cells (Figure 2, zoomed inset). In the bottom component, iPSC-derived osteoblasts and chondrocytes cultured within a bone scaffold and a cartilage hydrogel in proximity toAttorney Docket No.: 204606-0207-00WO

[0196] PhRR sensor for real-time cytokine monitoring. This system recapitulates the synovial joint microenvironment and facilitates CTAP-based synovial representation, offering a platform to evaluate responses to DMARDs and test novel drug candidates.

[0197] Bone constructs were produced by seeding decellularized trabecular bone discs with iMPCs and culturing in osteogenic media for 21 days, while cartilage discs were formed by photocuring iCH in a type II collagen / HAMA hydrogel, maintained in chondrogenic media for 21 days as described by Lin et al (Lin, Z. et al. Front Bioeng Biotechnol 7, 411 (2019)). Ten days prior to assembly, synovial cells were thawed, suspended in type I collagen hydrogel, and seeded onto the underside of the pSiM membrane to form a 300 pm thick synovial lining, cultured in a 1:1:1 mix of media optimized for synovial cells, leukocytes, and endothelial cells for 72 hours. Three days before assembly, iECs were plated on the top side of the pSiM and matured under 1.5 dynes / cm2shear stress to establish a vascular barrier (Linares, I. et al. Scientific Reports 15, 3227 (2025)). Once all components mature, bone and cartilage constructs were positioned in the bottom module, and the top and bottom components were assembled using pressure-sensitive adhesives and custom jigs. Patient PBMCs were then introduced under flow both in the blood and bone compartments, allowing 7-14 days of monitoring inflammatory responses and DMARD effects.

[0198] Key cellular and molecular markers characteristic of each cell subtype are assessed using flow cytometry and single-cell RNA sequencing (scRNA-seq) to demonstrate CTAP stability in vitro. Live and end point immunofluorescence microscopy imaging of synovial hyperplasia, endothelial activation, and leukocyte transendothelial migration, as well as longitudinal PhRR sensor-based cytokine release profiles are used to characterized outcomes. Bone and cartilage degradation is assessed based on tartrateresistant acid phosphatase (TRAP) and glycosaminoglycan (GAG) release using ELISA. Bone and cartilage degradation is also assessed based on collagen degradation products including C2M and CTX-II (specific to cartilage degradation) C3M (synovial tissue degradation), and CTX-I and ICTP (bone resorption) using ELISA.

[0199] Greater than or about 90% cell viability and density within 10% of in vivo levels are maintained. Cell viability and density are verified with live / dead assays and microscopy. CTAP heterogeneity is modeled and demonstrated in the RA-JoC byAttorney Docket No.: 204606-0207-00WO

[0200] replicating the distinct cellular compositions of each CTAP subtype (F, TB, and M) using flow cytometry as observed in patient synovial biopsies (±10%) and depicted in Figure 4. This is further validated by using single-cell CITE-Seq to ensure molecular pathways activated in the RA-JoC align with those observed in clinical RA samples stratified by CTAP subtypes, achieving a Pearson and Spearman Correlation Coefficient of 0.8 or higher. Additionally, a significant overlap in expressed genes with a Jaccard Index of 0.5 or higher is confirmed. At least 80% of key genes like cytokines and chemokines may align with known patient RA synovial profiles, assessed via scRNA-seq and qPCR. Cytokine levels may deviate less than 15% from patient synovial fluid, measured using ELISA and multiplex sensor assays.

[0201] Modeling CTAP-specific responses in the RA-JoC to conventional and biologic DMARDs

[0202] To establish the translational relevance of the RA-JoC, the ability to predict conventional and biologic DMARDs therapeutic responses across distinct CTAP subtypes is evaluated. Tested drugs include conventional, synthetic, and biologic DMARDs (Table 1, below). To mirror clinical conditions as closely as possible, these drugs are administered in the blood side (top component well) of the RA-JoC at concentrations reflecting their known safe therapeutic serum levels in humans and their effects are evaluated over up to 7 days post treatment.

[0203] Table 1.

[0204] Drug CTAP-TB CTAP-M CTAP-F Methotrexate Moderate Moderate Poor Etanercept High High Poor Tocilizumab Moderate-High High Poor Abatacept High Moderate Poor Rituximab High Moderate Poor

[0205]

[0206] Several assays are employed to characterize CTAP-specific treatment responses by incorporating clinical markers of RA pathology. Outcomes are assessed based onAttorney Docket No.: 204606-0207-00WO

[0207] advanced biosensing technologies and conventional assessment of secreted inflammatory cytokines (TNF-a, IFN-y, IL-ip, IL-6, etc. and C-reactive protein (CRP), employing longitudinal PhRR sensors and endpoint sampling using ELISA, respectively. Real-time and endpoint microscopy techniques are used to assess synovial hyperplasia (synovial fibroblast proliferation and invasiveness, endothelial activation, and immune cell infiltration into the synovial hydrogel). Bone and cartilage degradation, based on TRAP and GAG release, as well as collagen degradation products including C2M, CTX-II, C3M, CTX-I, and ICTP are assessed using ELISA. Flow cytometry and scRNA-seq are employed to analyze CTAP changes, comparing pre- and post-treatment for each DMARD. Successful DMARD responses in the RA-IoC were defined as 20% reductions a measured outcome, including disease activity and inflammation markers. Successful treatment induces: 1) >20% reduction in synovial hyperplasia, endothelial activation, leukocyte transendothelial migration, and pro-inflammatory cytokines and CRP levels, 2) >20% reduction in cartilage and bone destruction, replicating expected drug responses based on CTAP-speciftc clinical data (Table 1, above).

[0208] Utilizing the RA-JoC for refractory pauci-immune RA drug discovery Based on clinical evidence, it was expected that CTAP-F be refractory to DMARD treatment in the RA-JoC, which offers an opportunity to pursue a drug discovery approach for novel drug candidates that target pathways enriched in this pauci-immune CTAP.

[0209] Molecular profiling of CTAP-F revealed a fibroblast-dominated microenvironment with enriched signaling pathways. Lining fibroblast-like synoviocytes (FLS) subsets (F-0 and F-l) and CD34+ sub-lining fibroblasts (F-2) were expanded, along with increased LIFR+ and ICAM1+ venular endothelial cells. These endothelial cells expressed high CCL14 levels, while adjacent MERTK+ macrophage neighborhoods exhibited elevated CCR1 expression. This suggested the CCL14 / CCR1 signaling axis and LIFR+ endothelial cells may be therapeutic targets. NOTCH signaling was implicated in CTAP-F pathogenesis. Single-cell transcriptomic and surface protein analyses revealed a distinct fibroblast population expressing high NOTCH3 levels and mural markers enriched in CTAP-F compared to other CTAPs. Cell-cell neighborhoodAttorney Docket No.: 204606-0207-00WO

[0210] analyses indicated these fibroblasts associate with a pro-inflammatory and tissue remodeling microenvironment. Differential expression of NOTCH pathway components suggested aberrant NOTCH signaling activates these fibroblasts, making it a potential therapeutic target (Zhang, F. et al. Nature 623, 616-624 (2023)).

[0211] In this Example, a drug discovery approach is pursued, and novel drug-care candidates are tested for treating the pauci-immune CTAP-F RA-JoC. Specifically, CTAP-F RA-JoC devices are treated with drug candidates (small molecules and antibodies) that target these pathways, and combinational strategies are explored such as dual targeting of NOTCH3+ fibroblasts and CCR1+ macrophage neighborhoods (CCX354+Anti-NRR3) and simultaneous blockade of LIFR and NOTCH3 (EC359+Anti-NRR3) pathways (Table 2, below).

[0212] Table 2.

[0213] Drug Candidate Target Pathway Dosing Reference CCX354 CCR1 antagonist 0.1 - 1 pM Tak, P. P. et al.

[0214] Ann Rheum Dis 72, 337-344 (2013). EC359 LIFR inhibitor 0.1 - 100 nM Ebrahimi, B. et al.

[0215] npj Precision Oncology 8, 118 (2024).

[0216] DAPT Gamma-secretase 10 -20 pM Zhao, F. et al.

[0217] inhibitor (NOTCH) Front Immunol 14,

[0218] 1272133 (2023). RO4929097 Gamma-secretase 1 - 10 pM Zhao, F. et al.

[0219] inhibitor (NOTCH) Front Immunol 14,

[0220] 1272133 (2023). Anti-NRR3 NOTCH3 receptor ~10 pg / mL Wei, K. et al.,

[0221] monoclonal Nature 582, 259- antibody 264 (2020).

[0222]

[0223] Attorney Docket No.: 204606-0207-00WO

[0224] CB-103 NOTCH Zhao, F. et al.

[0225] transcriptional Front Immunol 14, complex inhibitor 1272133 (2023).

[0226]

[0227] The experimental data are analyzed using t-tests, ANOVA, regression, and correlation analyses to investigate relationships and differences within and between CTAP subtypes (CTAP-F, CTAP-TB, and CTAPM), as well as before and after DMARD treatment, each initially sampled from n=5 patients per CTAP. To ensure robust statistical power (80%) and achieve a 5% significance level with medium effect sizes (Cohen’s d = 0.5), sample size calculations suggested approximately n=15 RA-JoC devices per CTAP group for single-cell analyses, totaling 225 devices.

[0228] Technical Characterization of the In Vitro RA-JoC

[0229] Context of Use of the RA-JoC

[0230] The in vitro RA-JoC is a preclinical drug discovery and testing tool designed to recapitulate the synovial microenvironment in rheumatoid arthritis (RA). THe RA-JoC integrates patient-derived synovial cells, preserving their cell-type abundance phenotypes (CTAPs), with isogenic iPSC-derived vascular and chondro-osseous components. It employs advanced imaging to capture spatial cellular interactions and biosensors to monitor inflammatory cytokines in the synovial microenvironment. It is intended to generate human-relevant data for drug discovery, and for evaluating efficacy and safety of candidate therapeutics, informing investigational new drug (IND) submissions and regulatory decision making for RA treatments.

[0231] Quality Control for RA-JoC Components and Devices

[0232] Table 3 outlines quality control (QC) acceptance criteria for components and full devices (Table 3, below). Now referring to Figure 4, depicted are the following exemplary components of the RA-JoC: 1. Top Component, 2. Middle Spacer, 3. Media Channel, 4. Bottom Component, 5. Flow Insert, 6. Dual-Scale pSiM membrane, and 7. PhRR Sensor.Attorney Docket No.: 204606-0207-00WO

[0233] Table 3.

[0234] Component Acceptance Criteria

[0235] Top Component, Middle Spacer, Media • No debris visible by eye, clean feature Channel, Bottom Component lines, part is correctly sized, key features present and positioned correctly

[0236] • Debris free PSA by light microscopy • Ports and well are within ± 0.2 mm of design specifications

[0237] • P200 pipette tip seals in port opening • PSA backing layer removes without stripping COP imaging layer

[0238] Flow Insert • No debris visible by eye, clean feature lines, part is correctly sized, key features present and positioned correctly

[0239] • PSA backing layer removes without stripping channel layer

[0240] • Fluid seal against glass with pipette injection

[0241] Dual -Scale • No debris visible by eye, straight lines (3 pm, 60-100 nm) pSiM for membrane windows and chip edges, membrane features appear positioned correctly • Low debris on membrane area under optical microscope

[0242] • Pore sizes are within spec by STEM • Burst pressure > 5 PSI

[0243] • N2 permeance 1.2 ± 0.2 SLPM

[0244] • Nanopore sizes <100 nm with micropores ~ 3 pm ± 0.5 pm (Figure 5) PhRR Sensor • Full-wafer assessment of the refractive index (netr) of silicon nitride used for photonic waveguides is conducted by

[0245]

[0246] Attorney Docket No.: 204606-0207-00WO

[0247] analyzing interference patterns produced by unbalanced Mach-Zehnder interferometers. This allows culling of sensors with neff outside of design tolerances from reticles (successfully applied to analysis of > 25,000 sensors) • A subset (1%) of sensors from each reticle passing step 1 is imaged via laser confocal microscopy to confirm fidelity to the sensor design (Figure 6a)

[0248] • Antibody nanoprint process - Head camera images from the Scienion SX piezoelectric spotter acquired on 100% of chips to confirm proper placement of antibody and control droplets (Figure 6b) • Calibrations using antigen cocktails are performed on a subset (5%) of functionalized sensors to ensure reproducibility of analytical performance (Figure 6c)

[0249] Assembled RA-JoC • Left-to-right alignment of ports, channel and membrane

[0250] • Membrane centered in channel without contact with insert PSA

[0251] • No evidence of bubbles or debris in contact area

[0252] • Peel-and-seal functionality of flow insert and proper fitting to P200 pipette tips and flow fittings

[0253] • No evidence of leakage during flowruns

[0254]

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[0256] An exemplary Dual-Scale (3 pm, 60-100 nm) pSiM membrane is depicted in Figure 5. An exemplary PhRR sensor is depicted in Figure 6. An exemplary assembled RA-JoC is depicted in Figure 7.

[0257] Quality Control for Cellular Components of RA-JoC

[0258] To ensure the highest quality of cellular inputs for the RA-JoC, a rigorous quality control system encompassing comprehensive viability, sterility, and identity (phenotypic stability) assessments is implemented.

[0259] Primary synovial cells are isolated from synovial biopsies stratified into fibroid, myeloid, and lymphoid CTAPs using flow cytometry and verified using scRNA-Seq. These remaining cells are cryopreserved for subsequent use in creating the synovium hydrogel of the RA-JoC. Patient-matched peripheral blood mononuclear cells (PBMCs) are also cryopreserved. Induced pluripotent stem cell (iPSC) clones generated from these patient cells are thawed and undergo quality control, including verification of >90% positive expression of pluripotency markers (SOX2, OCT4, SSEA4, TRA-1-60), absence of chromosomal abnormalities by karyotyping, and negative mycoplasma testing. From these iPSC lines, endothelial cells (iECs), chondrocytes (iCHs), osteoblasts (iOBs), and osteoclasts (iOCs) derived from iPSC-derived Monocytes are generated. Each derivative is validated for expression of specific markers and then cryopreserved (Table 4, below). Upon thawing for use in the RA-JoC, cells (both primary and iPSC-derived) are only used if they meet the viability threshold of >90%.

[0260] Table 4.

[0261] Cell Type Assay Acceptance Criteria iEC Phenotypic Marker >90% of cells must be Expression positive for CD31, VE- (immunostaining for cadherin, and vWF CD31, VE-cadherin, vWF)

[0262] iEC Adhesion Molecule >85% of cells should Activation exhibit upregulation of adhesion molecules

[0263]

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[0265] (immunostaining for (ICAM-1, VCAM-1) ICAM-1, VCAM-1 after following a standardized inflammatory stimulation) pro-inflammatory stimulus iCH Phenotypic Marker >85% of cells should Expression express collagen type II (i mmunostai ni ng / RT - and aggrecan at levels qPCR for collagen type II within ±20% of a primary and aggrecan) chondrocyte standard iCH Cartilage ECM Production DMMB assay must yield (Alcian Blue staining, >15 pg GAG per pg DNA DMMB assay for GAG (or meets an equivalent quantification) optical density threshold for primary chondrocytes) iOB Phenotypic Marker >85% of cells should Expression express osteoblast markers (immunostaining / RT- with levels within ±20% of qPCR for ALP, Runx2, a primary osteoblast osteocalcin) standard

[0266] iOB Mineralization Alizarin Red optical (Alizarin Red staining) density should be >0.5 at 550 nm after dye extraction (or meets an equivalent optical density threshold for primary osteoblasts) iOC Phenotypic Marker >85% of cells should Expression express osteoclast markers (immunostaining, TRAP, with levels within ±20% of cathepsin K, NFATcl) a primary osteoclast

[0267] standard

[0268]

[0269] Attorney Docket No.: 204606-0207-00WO

[0270] Functionality of NAMs, CTAP Accuracy and Human Relevance:

[0271] The RA-JoC’s ability to recapitulate the complex heterogeneity of the human Synovium is rigorously characterized. This is achieved by benchmarking RA-JoC synovial hydrogel against patient-derived synovial biopsy datasets. Quantitative analysis of cellular composition and cytokine profiles serve to validate the in vitro reproduction of CTAP heterogeneity. In addition, key inflammatory cytokines (TNF-a, IFN-y, IL-la, 1L-ip, IL-6, IL-17, IL-21, CXCL-13 and other disease biomarkers (e.g. C-reactive protein (CRP)) are assessed via immunostaining and ELISA. Real-time functionality is corroborated by photonic ring resonator (PhRR) sensor outputs, which may demonstrate a correlation coefficient of >0.85 with traditional endpoint assays. Acceptance criteria include obtaining a correlation coefficient of >0.8 for cellular and cytokine outputs and maintaining biomarker levels within ±15% of clinical reference values, thereby establishing both CTAP accuracy and human relevance.

[0272] DMARD Predictive Capabilities

[0273] Dose-response experiments are performed with a panel of reference DMARDs, including conventional synthetic DMARDs (Methotrexate), biologic DMARDs targeting inflammatory pathways such as TNF-a (Etanercept), IL-6 receptor (Tocilizumab), costimulatory ligands CD80 and CD86 (Abatacept), and B-cell depletion (Rituximab), as well as targeted small molecule DMARDs such as JAK inhibitors (Tofacitinib) to validate the predictive capabilities to the RA-JoC. These drugs are administered in the blood side (top component well) of the RA-JoC at concentrations reflecting their known safe therapeutic serum levels in humans to mirror clinical conditions. Methotrexate is administered at concentrations ranging from 0.01 to 1 pM, reflecting common safe plasma levels achieved in treated patients (Wallace, C. A., et al., Arthritis & Rheumatism 32, 677-681 (1989)). Etanercept is used at approximately 2 pg / ml, consistent with the steady-state concentrations observed in patients (Gehin, J. E. et al. RMD Open 7 (2021).). Tocilizumab is administered at 10 pg / ml, correlating with its median serum levels (Arad, U. & Elkayam, O., J Rheumatol 46, 1577-1581 (2019).). Similar dosing strategies are employed for Abatacept (> Cmin~10 pg / ml), Rituximab (< Cmax~300 pg / ml based on two IV doses of 1,000 mg)35, and Tofacitinib (1 ng / ml < JoC dose <100 ng / ml, corresponding to Cmin and Cmax of 10 mg dose twice daily) (D'Agostino, M. A. et al.Attorney Docket No.: 204606-0207-00WO

[0274] Clin Rheumatol 36, 2655-2665 (2017), Drugbank. https: / / go(dot)drugbank(dot)com / drugs / DB00073., Pfizer. https: / / www(dot)pfizermedicalinformation(dot)com / xeljanz / clinical-pharmacology). These experiments employ multi-modal assessments, including flow cytometry, ELISA, and high-resolution imaging, to capture changes in key inflammatory biomarkers and cellular activation states.

[0275] Treatment with effective reference DMARDs in CTAPs TB and M may induce a minimum of a 20% reduction in these markers relative to baseline, and that predictive models derived from these experiments achieve a correlation of at least 85% when validated against independent clinical observations (Table 1, above).

[0276] For testing novel drug candidates for treating CTAP-F pauci-immune RA (Aim C3), aberrant NOTCH, LIFR, and CCR1 signaling is targeted with the drug candidate described in Table 2. These agents are evaluated in the RA-JoC system for potency, specificity, and safety. Potency is quantified using a Z-factor which represents a normalized response between positive (p) and negative (n) controls, accounting for both the response magnitude and variance and is calculated as:

[0277] Z

[0278]

[0279] 1 3(<7p + Un I

[0280] (Zhang, J.-H., et al., Journal of Biomolecular Screening 4, 67-73 (1999), Bar, H. & Zweifach, A. SLAS Discovery 25, 1000-1008 (2020)).

[0281] The positive and negative controls are determined from a dose-response curve for the drug. Typically, Z > 0.5 is considered a hit, but smaller values may be acceptable if disease markers are reduced by » 20%, and the study is appropriately powered (Bar, H. & Zweifach, A. SLAS Discovery 25, 1000-1008 (2020)).

[0282] Defining intra- and inter-laboratory reliability and reproducibility

[0283] To demonstrate the robustness and reproducibility of the RA-JoC, comprehensive intra- and inter-laboratory evaluations are conducted. Intra-lab oratory repeatability is assessed by performing multiple assay runs using identical batches of reagents and cellAttorney Docket No.: 204606-0207-00WO

[0284] sources, with a target coefficient of variation (CV) of less than 10% for critical endpoints such as cytokine concentrations and cellular compositions. For inter-laboratory reproducibility, the complete RA-JoC protocol, including device assembly, cell handling procedures, and data analysis pipelines, are transferred to an independent external laboratory. The evaluation focuses on primary endpoints, expecting inter-laboratory correlation coefficients of at least 0.85. Additionally, endpoint variability is rigorously controlled to within a 10-15% margin. To establish intra-laboratory concordance, the Bland- Altman test is used to statistically assess the agreement between two independent quantitative measurements of the same assay from different labs. This graphical method compares the differences between two measurements against their mean value, and quantifies the limits of agreement (LOA), typically set at two standard deviations above and below the mean difference.

[0285] Design of the RA-JoC

[0286] Figure 3 depicts a novel design of the RA-JoC. Figure 3a depicts an exploded view of the chip components. Figure 3b depicts a half-cutaway view of the assembled chips showing the media microfluidic components.

[0287] Synovial Cell subtypes in CTAPs

[0288] Figure 10 depicts exemplary distributions of synovial cell types in CTAP-TB, CTAP-M, CTAP-F.

[0289] REFERENCES

[0290] The following publications are incorporated herein by reference in their entirety.

[0291] 1. Di Matteo, A., Bathon, J. M. & Emery, P. Rheumatoid arthritis. Lancet 402, 2019 2033 (2023).

[0292] 2. Zhao, J., Guo, S., Schrodi, S. J. & He, D. Molecular and Cellular Heterogeneity in Rheumatoid Arthritis: Mechanisms and Clinical Implications. ront Immunol 12, 790122 (2021).

[0293] 3. Wooley, P. H. The usefulness and the limitations of animal models in identifying targets for therapy in arthritis. Best Pract Res Clin Rheumatol 18, 47-58 (2004).Attorney Docket No.: 204606-0207-00WO

[0294] 4. Mishra, A. P. etal. Emerging Landscape of Tn Vitro Models for Assessing Rheumatoid Arthritis Management. ACS Pharmacology & Translational Science 7, 2280-2305 (2024).

[0295] 5. Damerau, A. & Gaber, T. Modeling Rheumatoid Arthritis In Vitro: From Experimental Feasibility to Physiological Proximity. IntJMolSci 21 (2020). 6. Aletaha, D. etal. 2010 Rheumatoid arthritis classification criteria: an American College of Rheumatology / European League Against Rheumatism collaborative initiative. Arthritis Rheum 62, 2569-2581 (2010).

[0296] 7. Arnett, F. C. et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31, 315-324 (1988).

[0297] 8. Aletaha, D. etal. 2010 rheumatoid arthritis classification criteria: an American College of Rheumatology / European League Against Rheumatism collaborative initiative. Ann Rheum Dis 69, 1580-1588 (2010).

[0298] 9. Steiner, G., Verschueren, P., Van Hoovels, L., Studenic, P. & Bossuyt, X.

[0299] Classification of rheumatoid arthritis: is it time to revise the criteria? RMD Open 10, e003851 (2024).

[0300] 10. Humphreys, J. H. et al. The incidence of rheumatoid arthritis in the UK:

[0301] comparisons using the 2010 ACR / EULAR classification criteria and the 1987 ACR classification criteria. Results from the Norfolk Arthritis Register. Ann Rheum Dis 'll, 1315-1320 (2013).\

[0302] 11. Lewis, M. J. et al. Molecular Portraits of Early Rheumatoid Arthritis Identify Clinical and Treatment Response Phenotypes. Cell Rep 28, 2455-2470. e2455 (2019).

[0303] 12. Pitzalis, C., Kelly, S. & Humby, F. New learnings on the pathophysiology of RA from synovial biopsies. Current Opinion in Rheumatology 25, 334-344 (2013).

[0304] 13. Rivellese, F. etal. Rituximab versus tocilizumab in rheumatoid arthritis: synovial biopsy-based biomarker analysis of the phase 4 R4RA randomized trial. Nat Medl , 1256-1268 (2022).

[0305] 14. Asif Amin, M., Fox, D. A. & Ruth, J. H. Synovial cellular and molecular markers in rheumatoid arthritis. Semin Immunopathol 39, 385-393 (2017).Attorney Docket No.: 204606-0207-00WO

[0306] 1 . Humby, F. et al. Synovial cellular and molecular signatures stratify clinical response to csDMARD therapy and predict radiographic progression in early rheumatoid arthritis patients. Annals of the Rheumatic Diseases 78, 761-772 (2019).

[0307] 16. Kemble, S. & Croft, A. P. Critical Role of Synovial Tissue-Resident Macrophage and Fibroblast Subsets in the Persistence of Joint Inflammation. Front Immunol 12, 715894 (2021).

[0308] 17. Zhang, F. et al. Deconstruction of rheumatoid arthritis synovium defines inflammatory subtypes. Nature 623, 616-624 (2023).

[0309] 18. Humby, F. et al. Rituximab versus tocilizumab in anti-TNF inadequate responder patients with rheumatoid arthritis (R4RA): 16-week outcomes of a stratified, biopsy driven, multicentre, open-label, phase 4 randomised controlled trial.

[0310] Lancet 397, 305-317 (2021).

[0311] 19. Ajalik, R. E. etal. Human Tendon-on-a-Chip for Modeling the Myofibroblast Microenvironment in Peritendinous Fibrosis. Adv Healthc Mater 14, e2403116 (2025).

[0312] 20. Masters, E. A. et al. An in vitro platform for elucidating the molecular genetics of S. aureus invasion of the osteocyte lacuno-canalicular network during chronic osteomyelitis. Nanomedicine 21, 102039 (2019).

[0313] 21. McCloskey, M. C. et al. The Modular microSiM: A Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro. Adv Healthc Mater 11, e2200804 (2022).

[0314] 22. Mossu, A. et al. A silicon nanomembrane platform for the visualization of immune cell trafficking across the human blood-brain barrier under flow. J Cereb Blood Flow Metab. 39, 395-410 (2019).

[0315] 23. Linares, I. et al. Fluid flow impacts endothelial -monocyte interactions in a model of vascular inflammatory fibrosis. Scientific Reports 15, 3227 (2025).

[0316] 24. Bucukovski, J. & Miller, B. L. Everything's under Control: Maximizing Biosensor Performance through Negative Control Probe Selection. Anal Chem 97 , 3525- 3535 (2025).Attorney Docket No.: 204606-0207-00WO

[0317] 25. Lin, Z. et al. Osteochondral Tissue Chip Derived From iPSCs: Modeling OA Pathologies and Testing Drugs. Front Bioeng Biotechnol 7, 411 (2019).

[0318] 26. Felson, D. T. & LaValley, M. P. The ACR20 and defining a threshold for response in rheumatic diseases: too much of a good thing. Arthritis Res Ther 16, 101 (2014).

[0319] 27. Tak, P. P. et al. Chemokine receptor CCR1 antagonist CCX354-C treatment for rheumatoid arthritis: CARAT-2, a randomised, placebo controlled clinical trial. Ann Rheum Dis 72, 337-344 (2013).

[0320] 28. Ebrahimi, B. etal. Pharmacological inhibition of the LIF / LIFR autocrine loop reveals vulnerability of ovarian cancer cells to ferroptosis. npj Precision Oncology , 118 (2024).

[0321] 29. Zhao, F. etal. The Notch signaling-regulated angiogenesis in rheumatoid arthritis:

[0322] pathogenic mechanisms and therapeutic potentials. Front Immunol 14, 1272133 (2023).

[0323] 30. Wei, K. et al. Notch signalling drives synovial fibroblast identity and arthritis pathology. Nature 582, 259-264 (2020).

[0324] 31. Wallace, C. A., Bleyer, W. A., Sherry, D. D., Salmonson, K. L. & Wedgwood, R.J. Toxicity and serum levels of methotrexate in children with juvenile rheumatoid arthritis. Arthritis & Rheumatism 32, 677-681 (1989).

[0325] 32. Gehin, J. E. etal. Serum etanercept concentrations in relation to disease activity and treatment response assessed by ultrasound, biomarkers and clinical disease activity scores: results from a prospective observational study of patients with rheumatoid arthritis. RMD Open 7 (2021).

[0326] 33. Arad, U. & Elkayam, O. Association of Serum Tocilizumab Trough Concentrations with Clinical Disease Activity Index Scores in Adult Patients with Rheumatoid Arthritis. J Rheumatol 46, 1577-1581 (2019).

[0327] 34. D'Agostino, M. A. et al. Body mass index and clinical response to intravenous or subcutaneous abatacept in patients with rheumatoid arthritis. Clin Rheumatol 36, 2655-2665 (2017).

[0328] 35. Drugbank. https: / / go.drugbank.com / drugs / DB00073.

[0329] 36. Pfizer, https: / / www.pfizermedicalinformation.com / xeljanz / clinical-pharmacology.Attorney Docket No.: 204606-0207-00WO

[0330] 37. Zhang, J.-H., Chung, T. D. Y. & Oldenburg, K. R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. Journal of Biomolecular Screening 4, 67-73 (1999).

[0331] 38. Bar, H. & Zweifach, A. Z’ Does Not Need to Be > 0.5. SLAS Discovery 25, 1000- 1008 (2020).

[0332] The disclosures of each and every patent, patent application, and publication cited herein are hereby each incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

Attorney Docket No.: 204606-0207-00WOCLAIMSWhat is claimed is:

1. A microphysiological device comprising:a first component comprising a frame having a top surface, a bottom surface, a thickness therebetween, a first cavity having an opening at the top surface of the frame, and a second cavity having an opening at the top surface of the frame, the first cavity and second cavity each comprising a first tissue construct and second tissue construct, respectively;a second component comprising a frame having at least a first opening passing through the frame forming an interior region;a holder comprising a frame having at least one opening passing through the frame, and a membrane having a top and bottom surface positioned across the opening, the membrane comprising a first cell culture and second cell culture on the top and bottom surface, respectively;wherein the holder is removably positioned within the interior region of the second component, and the first component and second component are fixedly and removably attached thereby fluidly connecting the first cavity, the second cavity, and the interior region.

2. The device of claim 1, wherein the first component further comprises a third cavity having an opening at the top surface of the frame of the first component, the third cavity being fluidly connected to the first cavity, the second cavity, and the interior region.

3. The device of claim 2, wherein the third cavity comprises a sensor.

4. The device of claim 3, wherein the sensor comprises a photonic ring resonator sensor.Attorney Docket No.: 204606-0207-P1US5. The device of any one of claims 1-4, wherein the first tissue construct comprises a bone tissue construct.

6. The device of any one of claims 1-5, wherein the second tissue construct comprises a cartilage tissue construct.

7. The device of any one of claims 1-6, wherein the first cell culture comprises endothelial cells.

8. The device of claim 7, wherein the first cell culture comprises a vascular barrier.

9. The device of any one of claims 1-8, wherein the second cell culture comprises synovial cells.

10. The device of any one of claims 1-9, wherein the second cell culture comprises a hydrogel.

11. The device of any one of claims 1-10, further comprising a flow insert removably positioned in the interior region above the holder, the flow insert comprising two ports configured to apply or remove fluid.

12. The device of any one of claims 1-11, wherein the first and second tissue cavities each comprise at least two ports configured to apply or remove fluid.

13. A method for making a microphy si ologi cal system comprising: providing the device of claim 1;culturing one or more cells of a first cell culture on the bottom surface of the membrane;culturing one or more cells of a second cell culture on the top surface of the membrane;Attorney Docket No.: 204606-0207-P1USculturing one or more cells of a third cell culture in the first cavity; culturing one or more cells of a fourth cell culture in the second cavity; assembling the first and second component and co-culturing all the cells together for a third period of time.

14. The method of claim 13, further comprising treating the interior region, first cavity, or second cavity.

15. The method of claim 14, wherein the treating the interior region, first cavity, or second cavity comprises administering at least one agent.

16. The method of claim 1 , wherein the agent comprises a disease modifying antirheumatic drug (DMARD).

17. The method of any one of claims 13-16, wherein the first cell culture comprises synovial cells.

18. The method of claim any one of claims 13-17, wherein the first cell culture replicates a cell type abundance profile of rheumatoid arthritis.

19. The method of any one of claims 13-18, wherein the second cell culture comprises endothelial cells.

20. The method of any one of claims 13-19, wherein the third cell culture comprises a bone tissue construct.

21. The method of any one of claims 13-20, wherein the fourth cell culture comprises a cartilage tissue construct.

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