WO2025226325A2

WO2025226325A2 - In vitro systems for generation of skin equivalents and uses thereof and methods for diagnosis and treatment of connective tissue disorders

Info

Publication number: WO2025226325A2
Application number: PCT/US2025/013638
Authority: WO
Inventors: Ganna BILOUSOVA; Parker JESBERG; Igor KOGUT
Original assignee: University of Colorado System; University of Colorado Colorado Springs
Current assignee: University of Colorado System; University of Colorado Colorado Springs
Priority date: 2024-01-29
Filing date: 2025-01-29
Publication date: 2025-10-30
Anticipated expiration: 2026-07-29
Also published as: WO2025226325A3

Abstract

Embodiments of the instant disclosure relate to an in vitro system for generating 3D skin equivalent, compositions, and methods for modeling and analyzing potential therapeutic treatments of Ehlers-Danlos Syndrome (EDS), including personalized therapies and regimens. In certain embodiments, the in vitro system can be utilized to facilitate diagnosis, drug discovery, investigation of disease mechanisms, and treatment of EDS and other connective tissue diseases. In certain embodiments, compositions, and methods for making and using matrix metalloproteinase 9 (MMP-9) inhibitors are disclosed to treat EDS. In some embodiments, a subject has hypermobility type EDS (hEDS).

Description

IN VITRO SYSTEMS FOR GENERATION OF SKIN EQUIVALENTS AND USES THEREOF AND METHODS FOR DIAGNOSIS AND TREATMENT OF CONNECTIVE TISSUE DISORDERS

PRIORITY

[0001] This International Application claims the benefit of U.S. Provisional Application No. 63/626,388, filed on January 29, 2024. This provisional application and any applicable appendices are incorporated herein by reference in their entireties for all purposes.

GOVERNMENT FUNDING

[0002] This invention was made with government support under grant number HT9425-23-1- 0019 awarded from the United States Department of Defense. The government has certain rights in the invention.

FIELD

[0003] Embodiments of the instant disclosure relate to in vitro systems for generating three- dimensional (3D) skin equivalents, uses thereof, and compositions and methods for analyzing, diagnosing and treating Ehlers-Danlos Syndrome (EDS) and related connective-tissue disorders thereof. In certain embodiments, the systems for generating 3D skin equivalents can be used to assess therapeutic agents for treatment including personalized treatment of EDS and related connective-tissue disorders thereof in a subject. In other embodiments, compositions, and methods for making and using matrix metalloproteinase 9 (MMP-9) inhibitors are disclosed to treat EDS.

BACKGROUND

[0004] Ehlers-Danlos syndrome (EDS) is a heterogeneous group of skin and connective tissue disorders characterized by hyperextensible skin, joint hypermobility, cutaneous fragility, delayed wound healing, and chronic pain. Mutations in a variety of genes encoding for extracellular matrix (ECM) components, such as tenascin-X, Collagen (Col) III, and V, as well as defects in intracellular processing of ECM have been linked to EDS. However, in many typical cases, genetic defects are not detected in genes typically found associated with EDS. This is especially true for the hypermobility type of EDS (hEDS), which is the most common form of EDS. Because of overlapping symptoms and a poorly defined genetic basis, diagnosing hEDS remains a challenge, and many patients are left undiagnosed. Even in cases when the diagnosis of hEDS is confirmed, there are no treatment options for patients except for symptomatic care and pain management. Further, lack of acceptable EDS models that reliably recapitulates normal skin or the clinical phenotype of the disease further complicates studies of EDS mechanisms and hampers progress toward developing effective therapeutic strategies for variations of this disease. Therefore, a need exists for improved diagnosis and treatment of EDS and hEDS and availability and use of models to study EDS and hEDS and related connective tissue conditions.

SUMMARY

[0005] Embodiments of the instant disclosure relate to in vitro systems for generating 3D skin equivalents for diagnosing connective tissue disorders and assessing therapeutic efficacy for treating Ehlers-Danlos Syndrome (EDS) and other connective-tissue conditions in a subject. In certain embodiments, an in vitro system for generating 3D skin equivalents from a cell sample are disclosed. In some embodiments, an in vitro system for generating 3D skin equivalents from a cell sample having a plurality of wells, a plurality of inserts having a lip extended for supporting and positioning each insert within the plurality of wells where each insert can fit within one well of the plurality of wells, a spacer configured to fit beneath an insert lip and on top of a well to provide spacing from a base of the well and a bottom of an insert, and a gel scaffold positioned within each of the plurality of inserts are disclosed herein. In certain embodiments, gel scaffolds disclosed herein contain cells (e.g., layers of cells) that remain on top of each of a bottom of the plurality of inserts where each insert bottom is permeable to receive a buffer or media. In some embodiments, the spacer can be an adjustable spacer or substitutable spacer for adjusting height of an insert from a bottom of a well.

[0006] In certain embodiments and further to paragraph [0005] above, methods for culturing cells within the in vitro system are disclosed. In accordance with these embodiments, gel scaffolds can be introduced to one or more inserts, culture media can be introduced to one or more of a plurality of wells outside of the one or more inserts and cells can be embedded within and/or on top of each gel scaffold. In some embodiments, in vitro systems can be used to initially culture cells embedded within and/or on top of each gel scaffold within the one or more inserts fully surrounded or submersed in a first media (e.g., a loaded gel). In other embodiments, the one or more inserts can be progressively or systematically transitioned from a first media to a second media or until the one or more inserts containing the gel scaffold and cells are essentially exposed only to a second media (e.g. about 90% or more to the second media); optionally, where when the one or more inserts containing the gel scaffold and cells are exposed only to second media and the gel scaffold can be detached from insert sidewalls to generate 3D skin equivalents and the one or more inserts raised via the spacer. In accordance with these embodiments, the one or more inserts can have increased air exposure and reduced media exposure as the one or more inserts are raised and the gel scaffold detached to generate 3D skin equivalents contacting an aqueous solution or media at a liquid interface (e.g., relaxed gel), for example, through a permeable bottom of the one or more inserts. In certain embodiments, inserts can be linked together as a sheet of attached inserts and progressively lifted and moved from a set of wells to a different set of wells having a first media, a mix of a first media and a second media and then a second media per periods disclosed herein.

[0007] In certain embodiments and further to paragraphs [0005]-[0006] above, in vitro systems disclosed herein can be used to screen one or more therapeutic agents for efficacy to treat EDS or related connective tissue conditions disclosed herein. In certain embodiments, in vitro systems disclosed herein can be used to screen a one or more therapeutic agents for efficacy to treat EDS or related connective tissue conditions for personalized or patient-specific medicine based on cells obtained from a patient to be treated or subject (e.g., genetically related) to the patient. In certain embodiments, in vitro systems can include adding culture media to the one or more plurality of wells, providing gel scaffolds to one or more inserts, embedding cells obtained from the patient or subject related to the patient within the gel scaffold of the one or more inserts. In some embodiments, cells (e.g. the same or different cells) can be layered on a surface of the gel scaffold. In accordance with these embodiments, the in vitro system cells and gel scaffold with the plurality of inserts can be fully submerged in first media; then at a predetermined time, the one or more inserts can be progressively transitioned to a second media, until the gel scaffold and cells are exposed to essentially only the second media. In certain embodiments, the gel scaffold can be detached from internal insert walls by mechanical or other suitable methods, to generate a 3D skin equivalent and the one or more inserts having the 3D skin equivalent can be raised to expose 3D skin equivalents to air with essentially the second media via the permeable bottom of the inserts. In certain embodiments, 3D skin equivalents can be exposed to one or more therapeutic agents. In certain embodiments, effect of the one or more therapeutic agents can be tested on the 3D skin equivalent; optionally, to determine whether the one or more therapeutic agents enhance and/or stimulate production of connective tissue or connective tissue restoration. In some embodiments, the one or more therapeutic agents can be assessed for a personalized effect on a patient’s source of cells. In other embodiments, the one or more therapeutic agents can be assessed for a more generalized effect on EDS or other connective tissue disorder. In certain embodiments, the in vitro system can be used to for assessing treatment efficacy or predicted efficacy of one or more therapeutic agents on subjects, by exposing the 3D skin equivalent generated herein to one or more therapeutic agents and measuring the effect of the one or more therapeutic agents on the 3D skin equivalent before, during, and after treating the subject with the one or more therapeutic agents that demonstrated efficacy in repairing or inducing connective tissue in the subject. In certain embodiments, efficacy can be demonstrated by assessing one or more of increased proliferation or expansion of fibroblast cells, increased matrix deposition; and/or fibroblast quiescence, matrix remodeling and/or cell differentiation as the in vitro system progresses from a loaded gel to a relaxed gel system.

[0008] In certain embodiments and further to paragraphs [0005]-[0007] above, the gel scaffold of one or more inserts can include a cross-linkable gel material capable of harboring cells in a cell-expansion permissive matrix. In some embodiments, the gel scaffold can be of a consistency to permit or allow at least one of, cell migration, cell expansion, cell differentiation, and/or the gel scaffold can be biodegradable, optionally, where cells are embedded within or layered upon the gel scaffold In some embodiments, the gel scaffold materials can include, but are not limited to hyaluronan, alginate, collagen, dextran, chitosan, silk fibroin, sericin, and synthetic polyethylene glycol (PEG) polymers, or combinations thereof. In some embodiments, the hyaluronan can be crosslinked. In certain embodiments, the hyaluronan can be crosslinked with polyethylene glycol (PEG) or the like or equivalent agent thereof and the gel scaffold can further include, one or more of: fibrinogen, aprotinin, thrombin, CaCh, cell culture media, serum, or the like, or a combination thereof.

[0009] In some embodiments and further to paragraphs [0005]-[0008] above, kits are provided for making, storing, transporting and using in vitro systems disclosed herein. In certain embodiments, kits can include a multi-well plate; one or more inserts; and one or more of positive and negative controls and at least one container.

[0010] In certain embodiments and further to paragraphs [0005]-[0009] above, compositions, and methods for making and using compositions containing inhibitors of matrix metalloproteinase 9 (MMP-9), also known as matrix metal loprotease 9, to treat EDS or other related connective tissue disorder in a subject are disclosed herein. In some embodiments, EDS in a subject can include newly identified molecular phenotype of EDS. In certain embodiments, compositions and methods disclosed herein concern treating EDS by inhibiting or eliminating MMP-9 activity. In some embodiments, a subject being treated has hypermobility type EDS (hEDS). Other embodiments disclosed herein concern combining inhibition of MMP-9 to treat EDS and/or hEDS in combination with other treatments. In certain embodiments, compositions to treat EDS, newly identified type of EDS, and/or hEDS can be used for short-term or prolonged treatment depending on the subject being treated and severity of the condition. In certain embodiments, inhibition or elimination of EDS can be accomplished by a specific inhibitor of MMP-9 such as a chemical agent, antibody (e.g., polyclonal, or monoclonal antibody) or other suitable agent. In certain embodiments, an animal model can be used for studying hEDS in a skin xenograft model of hEDS where a healing wound associated on the back of an immunodeficient animal can include human keratinocytes and EDS-subject specific fibroblasts for studying or testing treatment efficacy of EDS.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following drawings form part of the present specification and are included to further demonstrate certain embodiments of the present disclosure. Certain embodiments can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0012] FIGS. 1A-1B represent xenografting experimental layout. 1A) illustrates a xenograft schematic. IB) illustrates a grafting process using a grafting chamber to position human cells within an inflicted wound in accordance with some embodiments disclosed herein.

[0013] FIGS. 2A-2B represent exemplary schematics for an in vitro 3D skin equivalent airlift cell culture system. To compliment the in vivo model represented in FIG. 1, this in vitro cellbased model of hEDS was developed by generating 3D skin equivalents using fibroblasts from hEDS patients embedded in thiol ated-hyaluronan (HA-SH) fibrin gel scaffolds (Fig. 2). 2A) illustrates a crosslinked gel scaffold. 2B) illustrates embodiments of in vitro systems disclosed herein where gel scaffolds and cells can be cultured in one or more inserts within a well and liquid-air interface employing a custom-designed airlift spacer that can adjust the height of the one or more inserts in accordance with certain embodiments disclosed herein for a loaded gel or a relaxed gel. 2C) represents an image highlighting some issues of standard approaches lacking a custom airlift spacer.

[0014] FIGS. 3A-3D represents specifications of a custom spacer designed for production of 3D skin equivalents disclosed herein illustrating a top view (3 A), a short side view (3B), a long side view (3C), and isometric view (3D). Each of these illustrations is in accordance with some embodiments disclosed herein.

[0015] FIG. 4A-4B illustrates an exemplary timeline schematic of use of an in vitro system for generation of 3D skin equivalents culturing over time (4A) with representative orientations of a well and insert in a Loaded and Relaxed gel state (4B) in accordance with some embodiments disclosed herein.

[0016] FIG. 5. represents exemplary images of morphological analyses of an in vivo (xenograft) model. In these examples, human specific antibody staining confirms presence of human tissue in the engrafted area (left panel); H&E staining of engrafted tissue (center panel); electron microscopy of collagen fibers in engrafted dermis (right panel) in accordance with some embodiments disclosed herein.

[0017] FIG. 6 illustrates in an exemplary 3D skin model system (left panel); a scan illustrating disorganized collagen fibers in 3D skin EDS dermis (middle panel) vs healthy dermis generated using primary patient fibroblasts (right panel) in accordance with some embodiments disclosed herein.

[0018] FIG. 7A-7C represents a schematic illustration of utility of iPSC-derived (organoid- derived) fibroblasts in 3D skin modeling. 7A) illustrates a pathway for developing novel therapeutic strategies using a skin biopsy of use herein; 7B) illustrates H&E staining of primary and iPSC-derived fibroblasts; 7C) illustrates transmission Electron Microscopy (TEM) of primary and iPSC-derived fibroblasts in accordance with some embodiments disclosed herein. [0019] FIG. 8 illustrates morphological analyses of an in vitro generated (3D skin equivalent) model using iPSC-derived (organoid-derived) fibroblasts. In this example, human specific antibody staining(s) verifies formation of human epidermis and dermis, respectively (left panel); H&E staining of 3D skin equivalents is represented (center panel). Electron microscopy of collagen fibers in 3D skin dermis is represented (right panel) in accordance with some embodiments disclosed herein. [0020] FIGS. 9A-9F illustrates effects of an MMP-9 inhibitor on skin samples of control and in an EDS model under electron microscopy analysis. Using TEM for example, restoration of highly organized collagen networks in both primary and organoid-derived EDS models were demonstrated (9C-9F), compared to controls (9 A, 9B) in accordance with some embodiments disclosed herein.

[0021] FIGS. 10A-10D represents exemplary images of effects of an MMP-2 inhibitor

(ARP 100) on collagen network samples on experimental samples (10C, 10D), and controls (10A, 10B) in accordance with some embodiments disclosed herein.

[0022] FIGS. 11A-11B represent images of effects of ARP 100 on collagen network samples in untreated (11 A) and treated (1 IB) organoid-derived EDS models in accordance with some embodiments disclosed herein.

[0023] FIG. 12A-12C illustrates exemplary images depicting similar effects of MMP-1 (FIG.12B) and MMP-9 (FIG. 12C) inhibitors on the collagen network of the sole sample that responded to MMP-1 inhibition as compared to control (FIG 12A), in accordance with certain embodiments disclosed herein.

[0024] FIG. 13 represents exemplary images of effects of an exemplary MMP inhibitor (Funalenone; an MMP-1 inhibitor) on collagen network of a majority of hEDS samples, demonstrating a lack of response in this example.

DEFINITIONS

[0025] Terms, unless defined herein, have meanings as commonly understood by a person of ordinary skill in the art relevant to certain embodiments disclosed herein or as applicable.

[0026] Unless otherwise indicated, all numbers expressing quantities of agents and/or compounds, properties such as molecular weights, reaction conditions, and as disclosed herein are contemplated as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters in the specification and claims are approximations that can vary from about 10% to about 15% plus and/or minus depending upon the desired properties sought as disclosed herein. Numerical values as represented herein inherently contain standard deviations that necessarily result from the errors found in the numerical value's testing measurements. [0027] As used herein, “individual”, “subject”, “host”, and “patient” can be used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, prophylaxis, or therapy is desired, for example, humans, pets, livestock, horses or other animals. [0028] As used herein, “treat,” “treating” or “treatment” can refer to treating, reversing, ameliorating, or inhibiting onset or inhibiting progression of a health condition or disease or a symptom of the health condition or disease.

[0029] As used herein, “MMP” refers to matrix metalloproteinase, which is also known, matrix metalloprotease. The terms are interchangeable as used herein. Multiple isoforms of MMPs are known, including MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP- 14, MMP-15, MMP-16, MMP-17, MMP-18, MMP-19, MMP-20, MMP-21, MMP -23 A, MMP-23B, MMP-24, MMP-25, MMP-26, MMP-27, and MMP-28. MMP inhibitors may inhibit one or more MMP isoforms.

DETAILED DESCRIPTION OF THE INVENTION

[0030] In the following sections, certain exemplary compositions and methods are described in order to detail certain embodiments of the invention. It will be obvious to one skilled in the art that practicing the certain embodiments does not require the employment of all or even some of the specific details outlined herein, but rather that concentrations, times, and other specific details can be modified through routine experimentation. In some cases, well known methods, or components have not been included in the description.

[0031] Embodiments of the instant disclosure relate to Ehlers-Danlos Syndrome (EDS) and other connective tissue related conditions and systems for diagnosing and assessing efficacy of therapeutic agents in the treatment and/or progression of EDS and other connective tissue related conditions in general and/or personalized to a patient. EDS is a group of genetic connective tissue disorders characterized by hyperextensible skin, joint hypermobility, and cutaneous fragility. EDS is primarily caused by mutations in genes encoding collagens or proteins involved in collagen biogenesis. Lack of reliable in vivo and in vitro models precludes ability to fully characterize an EDS phenotype, especially in the most common form of EDS, hypermobility -type (hEDS). Genetic causes of hEDS are poorly understood. Therefore, there is a need for recapitulating an EDS phenotype in a reliable model. Therefore, a 3D model as disclosed herein opens doors to diagnosis, phenotype characterization, causative mutation validation, quantitative diagnostic modalities, and therapeutic testing to identify treatments of these elusive conditions. To date, there is no treatment for the condition of EDS, only treatment of side effects of these conditions.

[0032] In certain embodiments and further to paragraph [0031] above, in vitro cell-based systems can be generated using 3D skin equivalents including cells (e.g. fibroblast cells) embedded in gel scaffolds within inserts situated in wells (e.g., transwell). In further embodiments, inserts harboring embedded cells can be separated from wells with a spacer for maintaining or increasing separation of the insert from the well. In certain embodiments, a spacer can be made of polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), or polyethylene terephthalate glycol (PETG), or any material suitable for three-dimensional (3D) printing. In some embodiments, the spacer can be placed under a lip of the insert top to support its position in the well where the spacer is placed on top of the well and under the lip of the insert establishing increasing clearance of the insert from a base of the well (See for example FIGS. 2A-2C).

[0033] In some embodiments and further to paragraphs [0031 ]-[0032] above, gel scaffolds disclosed herein can include any gel capable of permitting cell expansion and growth while maintaining cells within the matrix of the gel scaffold. In certain embodiments, cells of use to generate 3D skin equivalent modeling disclosed herein can include, but are not limited to, fibroblasts, chondrocytes, keratinocytes, melanocytes, Langerhans cells, Merkel cells, induced pluripotent stem cells (iPSCs), reprogrammed or rejuvenated cells obtained from a biopsy, established cell lines thereof, or combinations thereof. In accordance with these embodiments, the cells can originate from a skin biopsy, including a skin biopsy from a subject suffering from EDS or a subject genetically related to a patient having EDS. In accordance with these embodiments, the cells can be of human origin, or other mammalian origin, (e.g., including pets (e.g., dog, cat, rabbit, hamster etc.), livestock, horses, wild animals and other mammals) or other origin. In some embodiments, the cells can be from an infant, a child, a juvenile, an adolescent, a young adult, an adult or an elder adult. In some embodiments, the gel scaffold can include hyaluronan, gelatin, alginate, collagen, dextran, chitosan, silk fibroin, sericin, synthetic PEG polymers, or similar agent, or combinations thereof, optionally crosslinked with another agent. In certain embodiments, the hyaluronan can be thiolated-hyaluronan (HA-SH). In other embodiments, the hyaluronan can be thiolated-hyaluronan (HA-SH)-fibrin gel scaffolds. In some embodiments and as disclosed herein, skin equivalents can be derived from primary EDS fibroblasts and/or organoid-derived EDS fibroblasts differentiated from patient iPSCs due in part to being morphologically similar to a xenograft model.

[0034] In certain embodiments and further to paragraphs [0031 ]-[0033] above, gel scaffolds of in vitro systems disclosed herein can be placed within one or more inserts having permeable bottoms and gel scaffold filled inserts can be placed within wells. In certain embodiments, wells and inserts can be separated with adjustable spacers as described above that can progressively lift the insert away from a base of a well removing an gel scaffold within the inserts progressively out of culture media within wells to shift the skin equivalents from fibroblast proliferation and matrix deposition (e.g., a Loaded Gel scaffold) to fibroblast quiescence, matrix remodeling, and/or human epithelial keratin differentiation to produce 3D skin equivalents for further use. In certain embodiments, a 3D skin equivalent is mechanically moved away from side walls of the insert to permit further analysis and testing.

[0035] In certain embodiments and further to paragraphs [0031]-[0034] above, primary fibroblasts from a subject having EDS can be exposed to rejuvenation or conditional immortalization techniques to preserve cellular functionality during scale-up processes. In accordance with these embodiments, these rejuvenation methods can provide a renewable and consistent source of patient-derived cells suitable for modeling EDS and evaluating a broad spectrum of therapeutic agents within a 3D skin modeling system disclosed herein.

Rejuvenation through iPSCs (e.g., as cell source of use herein) of a subject are known in the art and all compositions and methods of use to rejuvenate a population of iPSCs is contemplated herein. For example, cellular rejuvenation can be achieved through reprogramming via iPSCs or alternative methodologies, resulting in enhanced collagen production. It is understood that increases in collagen synthesis resulting from cellular rejuvenation can be advantageous for development and use of 3D models for studying collagen-related diseases. In certain embodiments, treatment of mature 3D skin equivalents of in vitro assays disclosed herein with targeted agents can assess efficacy of the targeted agents to affect a connective tissue disorder disclosed herein. In some embodiments, a target MMP-9 inhibitor can be used to assess efficacy of the target MMP-9 inhibitor to trigger and/or enhance reorganization of collagen network using an in vitro 3D cell model disclosed herein for assessing efficacy and resemble those of healthy collagen network of control samples. [0036] In certain embodiments and further to paragraphs [0031 ]-[0035] above, a skin xenograft model of hEDS can be developed herein using healing wounds inflicted on the backs of an immunodeficient animal model (e.g., mice) using human keratinocytes and EDS patient fibroblasts. In accordance with these embodiments, the developed EDS skin xenografts can be used to demonstrate disorganized collagen fibrils in the extracellular matrix, resembling those observed in skin biopsies of hEDS patients. In other embodiments, to complement this in vivo model, an in vitro skin model was developed as disclosed herein.

SYSTEMS AND METHODS FOR GENERATING 3D SKIN EQUIVALENTS

[0037] Embodiments of the instant disclosure and further to paragraphs [0031 ]-[0036] above relate to in vitro systems for preparing 3D skin equivalents and methods for culturing cells with the in vitro system. In accordance with these embodiments, these in vitro systems can be used for diagnosing EDS, a specific type of EDS, hEDS, or other connective-tissue related disorder in a subject. In other embodiments, these in vitro systems can be used for screening one or more therapeutic agents for effects on skin, connective tissue and/or for generalized or personalized treatment of EDS or other connective tissue disorder in a subject or patient. In certain embodiments, devices disclosed herein can include, but are not limited to, an in vitro system for generating 3D skin equivalents from a cell sample having a plurality of wells, a plurality of inserts having a lip extended for supporting and positioning each insert within the plurality of wells where each insert can fit within one well of the plurality of wells, a spacer configured to fit beneath an insert lip and on top of a well to provide spacing from a base of the well and a bottom of an insert, and a gel scaffold positioned within each of the plurality of inserts are disclosed herein. In certain embodiments, the gel scaffold can contain cells (e.g., layers of cells) that remain on top of each of a bottom of the plurality of inserts where each insert bottom is permeable to receive a buffer or media. An in vitro system for generating 3D skin equivalents from a cell sample having a plurality of wells, a plurality of inserts having a lip extended for supporting and positioning each insert within the plurality of wells where each insert can fit within one well of the plurality of wells, a spacer configured to fit beneath an insert lip and on top of a well to provide spacing from a base of the well and a bottom of an insert, and a gel scaffold positioned within each of the plurality of inserts are disclosed herein. In certain embodiments, the gel scaffold contains cellular layers (e.g., that remain on top of each of a bottom of the plurality of inserts where each insert bottom is permeable to receive a buffer or media.

[0038] In certain embodiments and further to paragraphs [0031 ]-[0037] above, methods for culturing cells within the in vitro system are disclosed. In accordance with these embodiments, gel scaffolds can be introduced to one or more inserts, culture media can be introduced to one or more of a plurality of wells outside of the one or more inserts, and cells can be embedded within each gel scaffold. In some embodiments, the in vitro system can be used to initially culture the cells and gel scaffold within the one or more inserts fully surrounded or submersed in a first media (e.g., a Loaded Gel as described herein). In other embodiments, the first media can be progressively transitioned to second media by for example dilution or replacement or gradual replacement. In some embodiments, the first media is transition to a second media over a period by replacing a percentage of the first media within the wells with the second media. In other embodiments, the one or more inserts can be progressively raised, optionally via an adjustable spacer, until the one or more inserts containing the gel scaffold and cells have reduced contact to culture media and increased exposure to air; optionally, where the one or more inserts containing the gel scaffold and cells have increased air exposure and reduced media exposure as the insert is raised to have only the base having a liquid-air interface (e.g., Relaxed Gel as described herein). In some embodiments, the plurality of inserts can be raised or lowered within the plurality of wells. In other embodiments, the bottom of each of the plurality of inserts can be permeable to an aqueous solution (e.g., buffer or media), permitting gel scaffold access to the aqueous solution and capable of absorbing the aqueous solution. In some embodiments, the aqueous solution can be a media and further contain one or more agents for promoting cell growth and/or differentiation and/or modeling (e.g., fibroblast differentiation, matrix remodeling etc.) In some embodiments, the permeable membrane or component of the one or more inserts can be a mesh, a filter, or other permeable material, including, but not limited to, polycarbonate, high pore density PET membranes, and the like. In certain embodiments, the in vitro system can be configured such that inserts of the in vitro system do not contact internal sidewalls of the well, permitting an aqueous solution to envelope the external surface of an insert, if desired. In accordance with these embodiments, level of the aqueous solution can be controlled during a process from a Loaded Gel to a Relaxed Gel, for example. In certain embodiments, inserts can be linked together as a sheet of attached inserts (e.g. 3D generated sheet of inserts configured to sit in a complimentary set of wells) and progressively lifted and moved from a set of wells to a different set of wells having a first media, a mix of a first media and a second media and then a second media per periods disclosed herein. In certain embodiments, the mix of the first and the second medias can be for example a 10: 1, 5:1, 3: 1, 2:1, 1 : 1, 1 :2, 1 :3, 1 :4 1 :5, 1 : 10 or any ratio in between of the first media to the second media as a block of inserts is moved from one block of wells to a second block of wells with increasing second media to first media ratios to 100% second media prior to detachment of the gel scaffold disclosed herein.

[0039] In certain embodiments, and further to paragraphs [0031 ]-[0038] above, wells and inserts can be made with any material suitable for use in cell culture. In certain embodiments, wells (also known as culture wells) can be made from polystyrene, polyethylene terephthalate (PET), high- and low-density polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polycarbonate, borosilicate glass, or similar material. In certain embodiments, insert side walls (e.g. transwell inserts) can be prepared from polystyrene, polyethylene terephthalate (PET), high- and low-density polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polycarbonate, borosilicate glass, or similar material. In other embodiments, permeable surfaces of a base of an insert can be made of any suitable permeable membrane or surface, for example, polycarbonate, high pore density PET membranes, or similar material. In certain embodiments, spacers or adjustable spacers can be made of polylactic acid (PLA), polylactic acid (PLA), acrylonitrile butadiene styrene (ABS), or polyethylene terephthalate glycol (PETG), or any material suitable for making a spacer by manufacture or by 3D printing. It is contemplated herein that any system or method for raising and lowering an insert within a well can be used with in vitro systems and methods disclosed in the instant application.

[0040] In certain embodiments, and further to paragraphs [0031 ]-[0039] above, gel scaffolds or matrices can include crosslinked gel materials capable of supporting cell growth and differentiation. In some embodiments, gel scaffolds and crosslinked gel scaffolds disclosed herein can be configured or of a material to allow or permit at least one of cell adhesion, cell migration, cell expansion, cell differentiation and/or be biodegradable. In some embodiments, gel scaffold materials can include, but are not limited to, hyaluronan, gelatin, alginate, collagen, dextran, chitosan, silk fibroin, sericin, synthetic PEG polymers, or combinations thereof or other cross-linkable material. In some embodiments, gel scaffolds or matrices can include crosslinked hyaluronan. In accordance with these embodiments, hyaluronan of use herein is not limited. In other embodiments, hyaluronan of use herein can be thiolated hyaluronan (HA-SH), high molecular weight hyaluronan (e.g., hyaluronan having a molecular weight of greater than 500 kDa), low molecular weight hyaluronan (e.g., hyaluronan with a molecular weight of 500 kDa or less), or the like. In some embodiments, hyaluronan of use in gel scaffolds disclosed herein can be crosslinked with one or more of acrylate, diacrylate, methacrylate, maleimide, thiol, hexadecylamide, carbodiimide, succinimide, triazole, diimidazole, tyramine, formaldehyde, divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) or other suitable cross-linking agent. In certain embodiments, hyaluronan of use in gel scaffolds disclosed herein can be crosslinked with polyethylene glycol diacrylate (PEGDA). In certain embodiments, the molar ratio between the gel material and crosslinker can be about 0.1 : 1 to about 10: 1; about 0.25: 1 to about 5: 1; about 0.25: 1 to about 2.5: 1; or about 0.5: 1 to about 2: 1. In certain embodiments, the molar ratio can between the gel material and crosslinker can be about 0.1 : 1, about 0.25: 1; about 0.5: 1; about 1 : 1; about 2: 1, about 2.5: 1; about 5: 1, or about 10: 1. In certain embodiments, the molar ratio between hyaluronan and crosslinker can be about 0.5: 1; about 1 : 1; or about 2: 1, or any ratio therebetween. In certain embodiments, the gel scaffolds or gel matrices can further include at least one additional agent to supplement expansion and/or differentiation of the cells in the in vitro system. In accordance with these embodiments, the at least one additional agent can include, but is not limited to, fibrinogen, aprotinin, thrombin, CaCE, culture media, serum, a non-human serum for human cell-containing gel scaffolds, or a combination thereof.

[0041] In certain embodiments, and further to paragraphs [0031]-[0040] above, gel scaffolds can be prepared by mixing or otherwise combining components of gel scaffolds. In certain embodiments, cells can be included within the gel scaffold mixture and, as gel scaffolds polymerize, the cells can become embedded within the gel scaffold. In certain embodiments, gel scaffolds can include a mixture of thiolated hyaluronan, aprotinin, fibrinogen, thrombin, CaCk, PEGDA, culture media, serum, non-human serum, and, optionally, cells. In certain embodiments, culture medium within the gel scaffold can include minimum essential media, such as Dulbecco’s Minimum Essential Medium (DMEM) or other similar media. In certain embodiments, serum can include, but is not limited to, bovine serum, or fetal bovine serum or other non-human serum or synthetic version thereof. In certain embodiments, cells of use in systems disclosed herein can include, but are not limited to, fibroblasts, keratinocytes, melanocytes, Langerhans cells, Merkel cells, induced pluripotent stem cells (iPSCs), other suitable cells, or combinations thereof. In some embodiments, cells of use in systems disclosed herein, can include, but are not limited to cells from a skin biopsy (e.g. from a subject having a connective tissue disorder). In accordance with these embodiments, a connective tissue disorder can include but is not limited to EDS and hEDS.

[0042] In certain embodiments, and further to paragraphs [0031 ]-[0041 ], a first set of one or more cells can be embedded within or introduced to gel scaffolds of in vitro systems disclosed herein to generate 3D skin equivalent. In accordance with these embodiments, cells can be primary cells from the patient or a related subject or donor subject, or from a cell line. In certain embodiments, cells can be obtained from a biopsy, such as a skin biopsy from a patient or other subject. In further embodiments, skin biopsy samples can be obtained from a subject having EDS or other connective tissue related conditions; optionally, where the subject has hEDS. In certain embodiments, a second set of one or more cells can be layered on gel scaffolds of in vitro systems with a first set of one or more cells embedded within the gel scaffolds described herein. In certain embodiments, cells obtained from a patient or other subject (optionally, a subject genetically related to the patient) include, but are not limited to, fibroblasts, chondrocytes, keratinocytes, melanocytes, Langerhans cells, Merkel cells, induced pluripotent stem cells (iPSCs), or combination thereof. iPSCs can be prepared from cells obtained from a subject by any rejuvenating or cell revival technique known in the art. In certain embodiments, iPSCs can be rejuvenated using compositions and methods disclosed in PCT/US2016/063258 or applications related thereto where all compositions and methods disclosed are incorporated by reference in their entireties for all purposes. In certain embodiments, iPSCs can be prepared according to compositions and methods disclosed in PCT/2020/050665 or applications related thereto, where all compositions and methods disclosed are incorporated by reference for all purposes. In certain embodiments, at least one of fibroblasts, chondrocytes, keratinocytes, melanocytes, Langerhans cells, Merkel cells, or a combination thereof can be used in systems and methods disclosed herein. In certain embodiments, a first set of one or more cells can include fibroblasts from a subject or iPSCs derived from a subject’s fibroblast cells can be embedded within gel scaffolds (e.g., as the gel forms or firms up) and a second set of one or more cells can include keratinocytes layered on gel scaffolds. In certain embodiments, the second set of one or more cells layered on gel scaffolds can be from the same or different source as the first cells. [0043] In certain embodiments, and further to paragraphs [0031 ]-[0042] above, in vitro systems of 3D skin equivalents can be used to culture cells, for example, to prepare 3D skin equivalents. In accordance with these embodiments, methods for culturing and analyzing cells can include adding culture media to one or more of a plurality of wells of the in vitro system. The wells can be free standing or part of a plate having multiple wells. Size of wells disclosed herein are not limited but can be of size suitable to minimize expenditures and appropriate for sufficient testing (e.g., minimize need for large numbers of cells, conserve therapeutic agent concentration for testing, while providing reliable return on analysis) and can be appropriate for the type and size of gel scaffolds and cells utilized. In certain embodiments, multiple inserts (e.g., connected to one another) can be inserted into a single well. In other embodiments, a single insert can be inserted into each well. In further embodiments, gel scaffolds or matrices can be provided to the one or more of the plurality of inserts. In certain embodiments, one or more cells can be embedded on, intermixed with, or introduced to existing gel scaffolds residing within inserts. In certain embodiments, inserts can be lowered in the culture media such that the gel scaffold and cells are completely submerged (e.g., Loaded Gel configuration). In some embodiments, adjustable spacers can be included and adjusted, continuously or in increments (e.g., predetermined intervals), to progressively raise the insert containing the gel scaffold and cells over a period to increase surface area of the gel scaffold and cells exposed to ambient air. In some embodiments, the temperature of the ambient air is about 25° C to about 40° C, about 30° C to about 40° C, or about 37° C. In certain embodiments, the gel scaffold and cells can be incubated under standard conditions appropriate for cells embedded within the gel scaffold, including up to about 10% CO2 and about 5% to about 30% O2. In certain embodiments, normoxic conditions can include about 1.0 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% CO2 or any percentage in between. In certain embodiments, normoxic conditions can include about 10.0 % to about 25.0% O2, about 15% and about 20% O2, or about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%, O2 or any percentage in between. In certain embodiments, gel scaffolds can be incubated at about 75% relative humidity (RH) or above, about 80% RH or above, about 85% RH or above, about 90% RH or above, about 95% RH or above, or up to 100% RH. In certain embodiments, normoxic condition can include about 37° C, about 5% CO2, about 20% O2, and about 95% RH. In certain embodiments, the gel scaffold and cells can be fully raised, and the gel scaffold and cells can be in contact with the culture media principally at the bottom of the inserts and have a liquid-air interface and detached from insert side walls (e.g. Relaxed Gel configuration). In accordance with these embodiments, a liquid-air interface can occur when culture media contacts the gel scaffold and cells only through a permeable bottom or permeable layer of the insert. In certain embodiments, when the gel scaffold and cells are submerged or partially submerged, the structure can be considered a “Loaded Gel,” having increased fibroblast proliferation and increased matrix deposition. In other embodiments, when the gel scaffold and cells emerge from the culture media and are in contact with the culture media essentially or principally through an inserts permeable bottom and the gel scaffold and cells are detached from insert sidewalls, the structure can be considered a “Relaxed Gel,” having increased fibroblast quiescence, matrix remodeling and/or epithelial keratinocyte differentiation. (See for example FIG. 2B.)

[0044] In certain embodiments, and further to paragraphs [0031]-[0043] above, methods of using in vitro systems disclosed herein can include obtaining one or more cells from a skin biopsy from a subject or patient to be treated or subject genetically related the patient. In accordance with these embodiments, cells of use herein can be obtained from the patient, or subject genetically related to the patient, include, but are not limited to, fibroblasts, chondrocytes, keratinocytes, melanocytes, Langerhans cells, Merkel cells, induced pluripotent stem cell (iPSCs), or combination thereof. In some embodiments, cells from a subject can include a human subject or patient. In other embodiments, cells from a subject can include a non-human subject. In certain embodiments, cells can include a non-human primate, mammal, reptile, bird, or amphibian. Mammals contemplated herein include pets (e.g., dog, cat, rabbit, hamster etc.), livestock, horses, wild animals and other mammals. In some embodiments, the subject is an infant, a child, a juvenile, an adolescent, a young adult, an adult or an elder adult. [0045] In certain embodiments, and further to paragraphs [0031]-[0044] above, in vitro systems disclosed herein containing gel scaffold and embedded cells can be incubated for at least one or multiple days for various stages during disclosed methods. In one embodiment, gel scaffolds with embedded cells can be provided to inserts and stored for later use such as in a kit or other storage system. In other embodiments, gel scaffolds with embedded cells can be provided to inserts about a day to about a week prior to layering a second set of cells on the gel scaffold. In another embodiment, inserts harboring a gel scaffold including cells can be fully (e g., 100%) submerged within culture media for about one to about three days, or about two days. As used herein, fully submerged can mean that gel scaffolds, empty or harboring cells, are entirely covered by an aqueous solution (e.g., culture media). In one embodiment, aqueous compositions (e.g., media) within wells having inserts, the inserts having gel scaffolds harboring cells can be progressively and/or incrementally transitioned from a first media to a second media. In certain embodiments, the gel scaffold with embedded first set of cells (and optionally layered with second set of cells) can be incubated in a first media. Incubation in a first media can be for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. Over time and after incubation in a first media, a portion of the first media within wells can removed and an equal volume of a second media can be added to wells and the system can be incubated for a period. Removal of media from wells and replacement with second media can be repeated until first media is completely or substantially replaced with the second media. In other embodiments, a third or fourth media can be used in these transitions or later in the process of generating these in vitro systems. In certain embodiments, intervals for transitioning media within wells from first media to second media can occur in at least 2, at least 3, at least 4, at least 5, or at least 6 intervals or over enough intervals to transition media within wells from the first media to the second media. The period for incubation at each interval can be hours to about 0.5 to about 4 days, including about 0.5 days (e.g., 12 hours), about 1 day, about 2 days, about 3 days, or about 4 days. In some embodiments, a permeable membrane at a base of an insert remains in contact with media during incubation times (e.g., temporary or intermittent removal of an insert from a well is contemplated for example, when changing media, adjusting a system, introducing one or more therapeutic agent to be tested, etc.). In certain embodiments, after being fully transitioned to second media, gel scaffolds and cells within inserts can be incubated in the transition media (a second media) for another period. The period of time for culture of gel scaffolds and cells in second media can be for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. The number of intervals, amount of 3D skin equivalent exposed to air, and duration of incubation at a given interval can be adjusted as necessary depending upon cells, gel scaffold, and culture media used. [0046] In certain embodiments, and further to paragraphs [0031 ]-[0045] above, gel scaffolds (e.g., in a Loaded Gel state) can be mechanically or manually detached from the walls of the insert to generate a Relaxed Gel state and 3D skin equivalents contemplated herein. Detachment can occur via physical means where an instrument can be inserted between the gel scaffold and insert interior walls to detach gel scaffold from the insert walls or shaken loose (e.g., gently) or pried away from the insert interior walls, each permitting space between an outer gel scaffold and the insert interior walls. Without being bound by theory, detachment from insert sidewalls can provide mechanical stability to the system. Prior to detachment (e.g., in a Loaded Gel state), encapsulated fibroblasts can be under uniaxial tension and can exert contractile force on the scaffold, which can tear apart the scaffold and collapse the gel. Upon detachment, the system can reach a point of equilibrium where cells are not under uniaxial tension and stop contraction. The fibroblast-mediated contraction can also induce high levels of collagen deposition, which can lead to a densely packed extracellular matrix due to macromolecular crowding. In some embodiments, bottom of the gel scaffold remains in contact with the permeable bottom of the insert and in contact with an aqueous solution which can be the same or different solution when generating the 3D skin equivalent. Prior to detachment, the gel scaffold with cells can be considered a Loaded Gel. After detachment, the gel scaffold can also be considered a Relaxed Gel. 3D skin equivalents can be raised to expose all, or a portion of each 3D skin equivalents to air. In certain embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or up to 100% of each 3D skin equivalent can be exposed to air. In certain embodiments, 3D skin equivalents exposed to air can be cultured at the liquid-air interface for a period of time, including at least about 1 day or at least about 24 hours, at least about 2 days, at least about 3 days, at least about 4 days , at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, or up to about 21 days. 3D skin equivalents can be further cultured for a period in presence or absence of an agent such as one or more therapeutic agents. In certain embodiments, the period of time can be at least about half a day (e.g., about 12 hours), at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, or at least about 21 In certain embodiments, the 3D skin equivalent can be exposed to one or more therapeutic agents and effects of the one or more therapeutic agents on the 3D skin equivalent (e.g., the gel scaffold, cells embedded within the gel scaffold, or both) can be measured. In certain embodiments, remodeling such as collagen remodeling can be compared to a healthy control as a positive control or a negative control from a donor subject having a connective tissue disorder.

[0047] In certain embodiments, and further to paragraphs [0031 ]-[0046] above, systems and methods disclosed herein can be utilized to screen potential therapeutic agents for use to treat a subject having EDS or other connective tissue disorder. In some embodiments, systems and methods disclosed herein can be utilized to screen potential therapeutic agents for use to treat a patient as a personalized medicine approach to that patient and/or to assess progress of treatment of the patient. In one embodiment, systems described above can include providing culture media to one or more of a plurality of wells and providing a gel scaffold to one or more inserts placed within the plurality of wells. Cells can be harvested from a subject or patient such as a skin biopsy or other appropriate sample and cultured according to the method described herein. In some embodiments, culturing methods can include embedding harvested cells within the gel scaffold of the inserts and progressively and incrementally transitioning the gel scaffold and cells from first media to second media using any method, detaching the cell harboring gel scaffold as described above to generate 3D skin equivalents for a subject or patient, and exposing 3D skin equivalents to air as described above. Following withdrawal of the gel scaffold and cells from the second media and detachment as noted above (e.g., only a base of the insert remains in contact with the aqueous solution), the 3D skin equivalents can be exposed to one or more therapeutic agents (e.g., MMP inhibitors or other suitable treatment agent) and effect of the one or more therapeutic agent on the 3D skin equivalent can be assessed or measured (e.g., matrix remodeling, collagen remodeling). In certain embodiments, one or more therapeutic agents can be added directly to media in wells with 3D skin equivalents. In certain embodiments, the subject or patient can be treated with one or more of the one or more therapeutic agents that demonstrate an advantageous or beneficial effect on the 3D skin equivalent (e.g., promotion of connective tissue remodeling). In certain embodiments, the therapeutic agent identified as clinically relevant (e.g., it demonstrates an advantageous or beneficial effect on the 3D skin equivalent) and can be different from one subject or another or from a target patient compared to another with the same or similar condition that present with similar or identical symptoms.

EDS

[0048] Ehlers-Danlos syndrome is a group of inherited disorders that affect connective tissues — primarily skin, joints, and blood vessel walls. Connective tissue is a complex mixture of proteins and other substances that provide strength and elasticity to the underlying structures in the body. Ehlers-Danlos syndrome can be manifested in a subject having overly flexible joints and stretchy, fragile skin. For example, this can become a problem if a patient has a wound that requires stitches, because the skin often isn't strong enough to hold them. A more severe form of the disorder, called vascular Ehlers-Danlos syndrome, can cause the walls of blood vessels, intestines, or uterus to rupture. Vascular Ehlers-Danlos syndrome can also have serious potential complications in pregnancy. There are several different types of Ehlers-Danlos syndrome, but the most common signs and symptoms include: overly flexible joints. Because the connective tissue that holds joints together is looser, joints can move far past the normal range of motion. Joint pain and dislocations are common. Another side effect of EDS is stretchy skin. Weakened connective tissue allows skin to stretch much more than usual. Skin might also feel exceptionally soft and velvety. Another side effect of EDS is fragile skin. Damaged skin often doesn't heal well or at all. For example, stitches used to close a wound often will tear out and leave a gaping scar. Symptom severity can vary and depends on the specific type of EDS. The current most common type is called hypermobile Ehlers-Danlos syndrome.

[0049] In other embodiments, and further to paragraphs [0031]- [0048] above, a subject can have vascular EDS. In accordance with these embodiments, side effects of vascular EDS often display distinctive facial features of a thin nose, thin upper lip, small earlobes, and prominent eyes. A subject can have thin, translucent skin that bruises very easily. In fair-skinned people, the underlying blood vessels are very visible through the skin. Vascular EDS can weaken the heart's largest artery (aorta), as well as the arteries to other regions of your body. A rupture of any of these larger blood vessels can be fatal. The vascular type can also weaken the walls of the uterus or large intestines, which can also rupture. Different types of Ehlers-Danlos syndrome are associated with a variety of genetic causes, some of which are inherited and passed on from parent to child. If you have the most common form, hEDS, there's a 50% chance that you'll pass on the gene to each of your children. Complications depend on the types of signs and symptoms you have. For example, overly flexible joints can result in joint dislocations and early-onset arthritis. Fragile skin may develop prominent scarring. People who have vascular EDS are at risk of often fatal ruptures of major blood vessels. Some organs, such as the uterus and intestines may also rupture. Pregnancy can increase the risk of a rupture in the uterus.

[0050] Because of overlapping symptoms and a poorly defined genetic basis, diagnosing EDS remains a challenge, and many patients are left undiagnosed. Even in cases when the diagnosis of EDS is confirmed, there are no treatment options for patients except for symptomatic care and pain management. As disclosed herein, embodiments concern the development of a 3D ex vivo EDS model by generating 3D skin equivalents using hEDS human fibroblasts embedded in thiolated-hyaluronan (HA-SH) fibrin gel scaffolds. This model emulates EDS phenotypes by demonstrating disorganized collagen fibrils in the extracellular matrix, resembling those seen in skin biopsies of hEDS patients. Therefore, this model is suitable for studying EDS, validating EDS causative mutations and drug screening. Using this model, a monoclonal antibody that acts as a selective matrix metalloproteinase (MMP)-9 inhibitor (e.g., Andecaliximab (ADX)), triggered a drastic restoration of collagen networks in EDS samples, resembling those of healthy controls. The use of other MMP inhibitors, such ARP 100 (MMP-2 inhibitor), did not promote the restoration of collagen network in EDS bioengineered tissues. Surprisingly, inhibition of MMP-1, whose overactivation has been previously implicated as a partial cause of EDS symptoms, demonstrated an inconsistent effect on collagen networks analyzed, demonstrating improved collagen organization in only a single patient amongst a cohort tested. In contrast, inhibition of MMP-9, which has not been previously associated with EDS symptoms, demonstrated efficacy across multiple patients. These findings highlight the heterogeneity of hEDS and suggest that it is likely driven by a variety of overlapping mechanisms, where MMP- 9 activity plays an important role.

[0051] Consequently, and further to paragraphs [0048]-[0050] above, personalized therapeutic agent screenings may be necessary for individual patients beyond MMP-9 inhibition, further highlighting the utility and value of the in vitro 3D skin model system as described in paragraphs [003 l]-[0047], In healthy tissues, MMPs participate in the degradation of damaged collagen and procollagen during the formation of stable collagen fibers. For example, fully formed collagen fibers are protected from MMP activity. Because of the potential delay and low efficiency of collagen network formation in EDS patients, EDS connective tissues are likely to be more susceptible to MMP activity. Reducing the activity of MMP-9 in EDS tissues may allow for additional time for the collagen network to be formed in these patients. Identifying MMP-9 inhibition as a potential therapeutic intervention, rather than other MMP inhibitors was an unexpected finding. There is no treatment for EDS. In certain embodiments disclosed herein, evidence that specific inhibition of MMP-9 can restore collagen defects observed in EDS connective tissues and therefore can provide a restorative treatment for this condition was surprising. Because many other drugs that are approved for the treatment of other conditions possess MMP-9 inhibitory activity, models disclosed herein provide an option to screen and identify the therapeutics suitable for treating EDS based on their MMP inhibitory activity.

[0052] Further to paragraphs [0048]-[0051 ] above, embodiments of the disclosure herein relate to methods of treating or reducing side effects of EDS and/or hEDS in a subject. In accordance with these embodiments, MMP-9 can be targeted using inhibitors to inhibitor or completely block MMP-9 activity or expression. In some embodiments, an MMP-9 inhibitor can include an agent such as a chemical or biological agent to inhibit MMP-9 and inhibit or eliminate EDS or EDS progression or ameliorate a side effect of EDS and/or hEDS. In some embodiments, the inhibitor can include a genetic inhibitor of MMP-9 expression or editing system such as CrispR/Cas system targeted to reduce or eliminate MMP-9 expression using this or other gene editing technology. In certain embodiments, some inhibitors contemplated of use to treat or ameliorate EDS and/or hEDS are disclosed herein in Tables 1, 2 and/or 3 below. In other embodiments, inhibitors of use to specifically inhibit or reduce MMP-9 activity, transcription or translation are contemplated. In other embodiments, an inhibitor of MMP-9 in combination with another treatment to treat or ameliorate EDS and/or a symptom or side effect of EDS and/or hEDS are contemplated. In some embodiments, the MMP-9 inhibitor combination treatments of a connective tissue disorder can include, but is not limited to, at least one of MMP-9 Inhibitor CAS ID 206549-55-5, Andecaliximab CAS ID 1518996-49-0, GI254023X CAS ID 260264-93- 5, Metformin CAS ID 657-24-9, Minocycline CAS ID13614-98-7, Disulfiram CAS ID 97-77-8, Lovastatin CAS ID 75330-75-5, Simvastatin, Avasimibe, Fluvastatin, Fenofibric acid, Batimastat, Marimastat, or a combination thereof; and at least one other agent able to treat a connective tissue disorder or side effect thereof. In certain embodiments, the MMP-9 inhibitor or agent includes, but is not limited to, at least one of MMP-9 Inhibitor CAS ID 206549-55-5, Andecaliximab CAS ID 1518996-49-0, GI254023X CAS ID 260264-93-5, Metformin CAS ID 657-24-9, Minocycline CAS ID13614-98-7, Disulfiram CAS ID 97-77-8, Lovastatin CAS ID 75330-75-5, or the like or a combination thereof. In some embodiments, other drugs with known MMP-9 inhibitory activity, of use for EDS treatment include, but are not limited to, at least one of metformin, lovastatin, simvastatin, avasimibe, fluvastatin, and fenofibric acid, or the like or a combination thereof. In other embodiments, other drugs with known MMP-9 inhibitory activity include but are not limited to batimastat and marimastat.

Table 1: Some Selective MMP Inhibitors

Name Cas ID MMP Selectivity ICso

Funalenone 259728-61 -5 MMP-1 170 pM ARP 100 704888-90-4 MMP-2 12 nM Halofunginone 17395-31-2 MMP-2 UK 356618 230961-08-7 MMP-3 5.9 nM MMP-8 Inhibitor I 236403-25-1 MMP-8 4 nM “MMP-9 Inhibitor” 206549-55-5 MMP-9 5 nM Andecaliximab 1518996-49-0 MMP-9 3 nM GI254023X 260264-93-5 MMP-9 2.5 nM MMP408 1258003-93-8 MMP-12 2 nM CL-82198 118890-36-9 MMP-13 10 pM WAY-170523 307002-73-9 MMP-13 17 nM Metformin 657-24-9 MMP -2/9 Minocycline 13614-98-7 MMP -2/9 Disulfiram 97-77-8 MMP -2/9 MMP-3 Inhibitor VIII 208663-26-7 MMP-3/12 23 nM/13 nM MMP-2/3 Inhibitor II CID 7328863 MMP-2/3 1.5 pM/520 nM Lovastatin 75330-75-5 MMP-1/2/3/9 PD166793 199850-67-4 MMP-2/3/13 7 nM/7 nM/8 nM

*(-) denotes no available information.

Table 2. Broad Spectrum MMP Inhibitors

Name CAS ID MMP Selectivity

Batimastat 130370-60-4 MMP-1/2/3/8/9 Marini astat 154039-60-8 MMP- 1/2/7/9/14 Doxycycline (Hyclate) 24390-14-5 MMP-1/2/7/8/9/12/13* MMP Inhibitor V 223472-31-9 MMP-2/3/8/9/12/13 GM 6001 142880-36-2 MMP- 1 Z2/3/7/8/9/ 12/ 14/26 NNGH 161314-17-6 MMP- 1/2/311110/12/14/20 Actinonin 13434-13-4 MMP- 1 Z2/3/7/8/9/ 10/12/13

* Sources differ on the specificity for doxycycline Table 3. Exemplary Collagen production inhibitors

Name CAS ID Activity

SC 79 305834-79-1 Akt Activator

TGF-pi Growth Factor

CGS-21680 124431-80-7 Adenosine A2A receptor agonist

[0053] In certain embodiments, and further to paragraphs [0048]-[0052], above, it is contemplated that MMPs participate in the degradation of damaged collagen and procollagen during the formation of stable collagen fibers. For example, fully formed collagen fibers are protected from MMP activity. Because of the potential delay and low efficiency of collagen network formation in a subject having EDS, EDS connective tissues can be more susceptible to MMP activity and MMP adverse effects. By reducing the activity of MMP-9 in EDS tissues, this may allow for additional time for the collagen network to be formed in the EDS subject making them less susceptible to MMP activity.

[0054] In some embodiments, and further to paragraphs [0048]-[0053], pharmaceutical compositions are contemplated. In accordance with these embodiments, pharmaceutical compositions can include isolated exosomes (e.g., isolated neuronal and/or astrocytic exosomes), lipid-based nanoparticles (LNPs), viral particles, nucleotides (e.g., siRNA, RNAi, DNA encoding siRNA or RNAi) described herein. In some embodiments, pharmaceutical compositions herein can include isolated exosomes (e.g., isolated neuronal and/or astrocytic exosomes), LNPs, viral particles, and/or nucleotides described herein and at least one pharmaceutically acceptable excipient or carrier. As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of a subject without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. As used herein, the term “pharmaceutically acceptable carrier” can refer to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use herein, include, but are not limited to, buffers that are well known in the art, and can be phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; and/or non-ionic surfactants.

[0055] In some embodiments, and further to paragraphs [0048]-[0054] above, pharmaceutical compositions for use herein can be formulated for parenteral administration, such as intravenous, intracerebroventricular injection, intra-ci sterna magna injection, intra-parenchymal injection, intra-renal, intradermal, subcutaneous, direct introduction to a tumor or a combination thereof. In some embodiments, pharmaceutical compositions for use herein can be formulated for local delivery to one or more tumors. In some embodiments, pharmaceutical compositions for use herein be formulated for parenteral administration can include pharmaceutically acceptable carriers including sterile liquids, such as water and oil, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. Saline solutions and aqueous dextrose, polyethylene glycol (PEG) and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. In some embodiments, pharmaceutical compositions for use herein can further include additional agents, for example preservatives, buffers, tonicity agents, antioxidants and stabilizers, nonionic wetting or clarifying agents, viscosity -increasing agents, and the like. In some embodiments, pharmaceutical compositions described herein can be packaged in single unit dosages or in multidosage forms.

[0056] In some embodiments, and further to paragraphs [0048]-[0055], formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which can contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which can include suspending agents and thickening agents. In accordance with some embodiments herein, aqueous solutions can be suitably buffered (preferably to a pH of from 3 to 9). The preparation of suitable parenteral formulations for use herein under sterile conditions can be readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0057] In some embodiments, and further to paragraphs [0048]-[0056], pharmaceutical compositions herein can further include one or more pharmaceutically acceptable salts. Nonlimiting examples of pharmaceutically acceptable salts include acid addition salts (formed from a free amino group of a polypeptide with an inorganic acid, or an organic acid. In some embodiments, the salt formed with the free carboxyl groups is derived from an inorganic base, or an organic base. In some embodiments, any of the pharmaceutical compositions herein can be used in therapeutic applications, for example, treating or reducing progression of EDS and/or hEDS, which are also disclosed herein.

[0058] In some embodiments, and further to paragraphs [0048]-[0057], a subject to any of the methods disclosed herein can be any subject for whom treatment or therapy is desired. In some embodiments, a subject can have or can be suspected of having EDS, hEDS or a combination thereof. In other embodiments, a subject can be a mammal. In some embodiments, a subject can be a human. In yet other embodiments, a human subject such as an adult, child, adolescent, toddler, young adult or infant or fetus in need of treatment methods disclosed herein can be identified by routine medical examination, e.g., laboratory tests, biopsy, magnetic resonance imaging (MRI) scans, ultrasound exams, and the like.

[0059] Certain embodiments disclosed herein, and further to paragraphs [0048]-[0058], concern kits containing at least one composition including, but not limited to, an MMP-9 inhibitors of use to treat EDS and/or hEDS in a subject and a container for storage, transport and/or use thereof.

EXAMPLES

[0060] The following examples are included to illustrate certain embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered to function well in the practice of the claimed methods, compositions, and apparatus. However, those of skill in the art should, in light of the present disclosure, appreciate that changes can be made in some embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1

[0061] In one exemplary method, a mouse model was developed to study hEDS. The mouse model is a unique quantitative mouse xenograft model of hEDS that closely aligns with human disease including skin abnormalities of hEDS. These models rely on the use of hEDS patient’s fibroblasts to generate a patch of human hEDS skin on an immune deficient mouse. This model was generated based on a graft assay system previously described. Briefly, a silicone grafting chamber is surgically inserted underneath of the skin and onto the muscle fascia of an immunodeficient mouse (either NOD/SCID or nude). Chambers are made of Momentive LSR 2650, two-component liquid silicone rubber for injection molding processes. The chambers are reusable and autoclavable. The chamber contains three manually punched 2 mm holes in the dome to allow for gas exchange during the time the chamber is in place under the skin. A circular piece of skin that is approximately the same diameter as the inner diameter of the chamber is removed from the back of the mouse, exposing the muscle fascia. The skin surrounding the incision is separated using forceps, and the flange of the grafting chamber is inserted underneath. A cell slurry including about 5xl0⁶ healthy control keratinocytes and about 5xl0⁶ fibroblasts isolated from an hEDS patient is introduced into the grafting chamber (Fig. 1). The cell medium used to generate the cell slurry maintains hydration for the cells and exposed muscle fascia. After 7 days, the graft chamber is removed under the anesthetic conditions, and the grafts are allowed to heal for the next 5-8 weeks.

[0062] Fig. 1 represents a schematic of xenografting experimental layout. 1A) Xenograft schematic. Injury inflicted on the backs of immunocompromised mice may be with healed using human cells. (Fig. 1A) Healed wounds are then comprised of diseased (EDS) or healthy human skin. IB) Graft process illustration showing a grafting process including a grafting chamber used to position human cells within inflicted wound. After cells settle on top of muscle fascia, the grafting chamber is removed. The human skin graft may be harvested upon healing.

3D Skin Equivalent (in vitro) Model

[0063] To complement this in vivo model, an in vitro cell-based model of hEDS was developed by generating 3D skin equivalents using hEDS human fibroblasts embedded in thiolated- hyaluronan (HA-SH) fibrin gel scaffolds (Fig. 2). [0064] FIGS. 2A-2C represent exemplary schematics for 3D skin equivalent airlift cell culture system 200. 2A) Human fibroblasts 207 are embedded in a thiolated-hyaluronan (HA-SH) conjugated fibrin gel scaffold 206 crosslinked by polyethylene glycol diacrylate (PEGDA). WT human keratinocytes 208 are subsequently seeded on top of the HA-SH conjugated fibrin gel scaffold 206 and cultured submerged in transwell cell culture inserts. 201 2B) Fibrin gel constructs are then cultured at the liquid-air interface at the lower membrane 202. Loaded vs Relaxed Gels influence distinct fibroblast phenotypes and function at this stage. The addition of indicated adjustable spacer 205 between well and lip 209 of the insert 201 is necessary to physically raise the transwell insert and prevent the lower membrane 202 from resting on the bottom of the well 203, ultimately enabling culture media 204 to diffuse across the membrane 202 from below. In certain embodiments, an acellular layer of the gel scaffold (not shown) can be included between gel scaffolds and permeable lower membranes to facilitate cell attachment and anchor gel scaffold to lower membranes. Commercially available spacers were found to not properly hold the culture inserts in place, leading the inserts slipping and/or uneven positioning within the well. The disclosed, non-exhaustive embodiment includes fibroblasts embedded within the gel scaffold and keratinocytes layered on top of the gel scaffold. In certain embodiments (not shown), an acellular layer of the gel scaffold (e.g., a gel scaffold that does not include embedded cells) can be provided between the permeable membrane and gel scaffold with embedded fibroblasts. Thickness of the acellular layer can vary but a thickness less than half a thickness of a gel scaffold harboring cells or a quarter or a tenth of the thickness or less can be sufficient. 2C) highlights the limitations of standard approaches that lack a custom airlift spacer and fail to reproduce the EDS phenotype.

[0065] FIGS. 3A-3D depicts a technical schematic for the custom airlift spacer. Dimensions shown are in millimeters. Designed for use with Falcon transwell culture inserts (12-well platform, Corning 353180). This design features ledges on each side of holes through which each insert spans, to properly hold insert arms. This design includes round opening to facilitate easy media deposition and aspiration. Spacers can be 3D printed using polylactic acid (PLA) filament and disinfected with ethanol and UV light.

[0066] In certain exemplary methods, 3D skin equivalents are cultured submerged for 7 days, before being raised to the air-liquid interface using the custom spacer (Fig. 4). The airlift culture system allows the keratinocytes seeded on top to terminally differentiate upward, mimicking the behavior of native skin. Upon maturation, the gels can be released from the sides of the walls of the cell culture insert, resulting in fibroblast-mediated constriction of the constructs. This constriction has been found to induce increases in ECM remodeling through a mechanism known as macromolecular crowding (MMC). This timeframe of increased matrix remodeling is ideal for potential drug treatments.

[0067] Human dermal fibroblasts were isolated from hEDS patient punch biopsies and expanded in human dermal fibroblast medium (HDF-M) including minimal essential media (MEM), fetal bovine serum (FBS), non-essential amino acids, L-glutamine, antibiotic agent, anti -mitotic agent, and 2-mercaptoethanol. Fibroblasts were passaged at 80% confluency to maintain proliferative capacity. Fibroblasts up to passage 6 were harvested for 3D skin equivalents using 0.25% Trypsin-EDTA. Human epithelial keratinocytes (HEKn, ATCC PCS- 200-010) obtained from neonatal foreskin were expanded in EpiLife® media (Gibco MEPI500CA), supplemented with EpiLife® Defined Grown Supplement (EDGS; Gibco S0125). HEKn keratinocytes were cultured on collagen-coated polystyrene dishes (PureCol, Advanced Biomatrix). Keratinocytes were cultured to no more than 60-80% confluency to prevent proximity -induced differentiation. Keratinocytes up to passage 5 are harvested for 3D skin equivalents using Accutase™ (STEMCELL Technologies). Human dermal fibroblasts were then encapsulated in an interpenetrating network (IPN) containing a fibrin-based gel with a covalently crosslinked thiolated-hyaluronan (HA-SH). The scaffold was generated by combining 40 mg/mL fibrinogen (Sigma) with 25 U/mL thrombin in the presence of 200 mM calcium chloride (CaCL). The scaffold also contained 0.05% w/v HA-SH (Advanced Biomatrix) with a 0.5: 1-2: 1 crosslink molar ratio of polyethylene glycol diacrylate (PEGDA, Advanced Biomatrix). Hydrogels were prepared by combining pre-mixed aliquots of fibrinogen, aprotinin and HA-SH with pre-mixed aliquots of thrombin, CaCh and PEGDA. Optionally, 350-500 pL of this mixture can be directly added into 0.4-8 pm pore-sized Falcon transwell culture inserts (Coming 353180), using a 12-well culture to provide an acellular scaffold layer, acting to increase construct adhesion to the lower membrane and facilitate uniform construction upon detachment. The acellular layer was incubated at 37° C for 1 hour to polymerize. A separate scaffold mixture was prepared and added to cell suspensions in DMEM (Gibco) and fetal bovine serum (FBS, Gibco) containing fibroblasts with a final concentration of 100,000 fibroblasts/mL. 0.5 mL of the resultant mixture was deposited into the transwell inserts, either directly or atop the previously added acellular layer. The inserts rested on top of the custom-designed 3D- printed airlift spacer. The technical drawings of an embodiment of disclosed custom spacers with the top view, short side view, long side view, and isometric are shown in FIGS. 3A-3D, respectively. Plates were incubated in at 37° C for 1 hour to polymerize.

[0068] Reduced serum media (HDF-M) was added to each well and cultured for 5 days alone before adding keratinocytes. During this 5-day period, the media was transitioned from HDF-M to EpiLife® in intervals of 25% per day. Optionally, the transition can be done over about a day to about 2 days to hasten culture timeline. 500,000 HEKn keratinocytes were seeded on top of each gel. EpiLife* (e.g., a first media) media was placed above and below the transwell inserts and the culture system was left undisturbed for 48 hours. 48 hours after seeding keratinocytes on the gels, media transition from EpiLife® to full-thickness CnT-Prime media (CellnTec) (e g., a second media) began. Media was transitioned in 25% intervals every day until the media was 100% airlift media.

[0069] In another exemplary method, this system was then cultured having a liquid-air interface, with media below the transwell inserts (1.6-1.8 mL, depending on construct thickness) and exposing the gel scaffold surface to air. At the time of airlift, the hydrogel scaffold was mechanically detached from the walls of the transwell insert to allow for fibroblast-mediated contraction within the gel scaffold. Detachment in this example, involved probing the sides of the hydrogel (where the hydrogel interfaces with the inner walls of the insert) with for example, a pipette tip or by any tool such as a spatula, a knife, or other tool capable of separating gel scaffold from insert side walls or by agitation. Following airlift and detachment, the 3D skin equivalents were cultured at a liquid-air interface for about 10 to about 20 days, in this example, 14 days incubation for full maturity of the 3D skin equivalents. Following creation of the 3D skin equivalents, therapeutic agents can be tested for effect on the cells harbored in the scaffold. In one example, for 7 days, therapeutic agents were supplemented into the media followed by the analysis of collagen network changes using electron microscopy and imaging, or other advanced light microscopy techniques. A representative creation and culture timeline schematic is illustrated in FIG. 4A.

[0070] FIG. 4B represents exemplary images of the in vitro system 200 described herein. An insert 201 can include a lower membrane 202 and can be configured to fit within the well 203 such that the walls of the insert 201 are sufficiently distanced from the walls of the well 203 that culture media 204 does not extend up the space between the insert 201 and the well 203. An adjustable spacer 205 can be provided between the well 203 and a lip or protrusion 209 of the insert 201 to raise and/or lower the insert 201 within the well 203. The insert 201 can be loaded with a gel scaffold 206 with fibroblasts 207 and keratinocytes 208 embedded within the gel scaffold 206. When the insert 201 is lowered within the well 203, the gel scaffold 206, fibroblasts 207, and keratinocytes 208 are completely submerged by the culture media 204. When the insert 201 is fully raised the gel scaffold 206, fibroblasts 207, and keratinocytes 208 can be exposed the culture media 204 only via the liquid-air interface at the lower membrane 202.

[0071] FIG. 5 represents exemplary images of morphological analyses of generated in vivo (xenograft) model. Human specific anti-vimentin antibody staining confirms the presence of human tissue in the engrafted area (left panel). H&E staining of engrafted tissue (center panel). Electron microscopy of collagen fibers in engrafted dermis (right panel).

[0072] FIG. 6 represents a recapitulation of molecular characteristics of EDS, or hEDS in bioengineered 3D skin equivalents generated from in this example, primary fibroblasts. hEDS and healthy fibroblasts were cultured in an HA-fibrin-based in vitro system described herein followed by introducing human healthy keratinocytes on top of gel scaffold as described in FIG. 4B. A representative 3D skin equivalent is illustrated in the left panel. Transmission electron microscopy (TEM) illustrated structure of collagen networks found in the healthy skin equivalent (middle panel) compared to EDS skin equivalent (right panel). A disorganized and loose collagen network is prominent and common in the EDS tissue image.

[0073] FIGS. 7A-7C represent in 7A, a schematic representation of an application of fibroblasts derived from induced pluripotent stem cells (iPSCs) through an organoid-based methodology (organoid-derived fibroblasts) in in vitro 3D models of hEDS. This approach led to enhanced collagen production within 3D models disclosed herein, indicative of iPSC-induced cellular rejuvenation. 7A) EDS patient-derived fibroblasts can be reprogrammed into induced pluripotent stem cells (iPSCs), where the reprogramming process facilitates cellular rejuvenation and provides a renewable source of patient-specific cells or other cells from a donor subject, as applicable. Generated EDS iPSCs can subsequently be differentiated into fibroblasts through a skin organoid-based differentiation approach. Here, organoid-derived EDS fibroblasts are suitable for in vitro and in vivo modeling of EDS, enabling applications in diagnosis, drug discovery and therapeutic testing. 7B) Healthy donor-derived fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) utilizing, for example, an RNA-based reprogramming method known in the art, followed by differentiation into iPSC-derived fibroblasts through an organoid-based process. Three-dimensional skin equivalents, generated from original primary fibroblasts (top) and corresponding iPSC-derived fibroblasts (bottom), were subjected to hematoxylin and eosin (H&E) staining to confirm the formation of correct skin layers. 7C) Transmission electron microscopy (TEM) analysis was performed to examine the collagen network structure in skin equivalents derived from primary fibroblasts (top) and iPSC-derived fibroblasts (bottom). The iPSC-derived fibroblast-based equivalents exhibited enhanced collagen production, highlighting functional utility of organoid-derived fibroblasts in modeling EDS (e.g. hEDS) or other connective tissue disorders and potentially surpassing performance of primary fibroblasts. Alternative cell rejuvenation methods would lead to similar enhanced collagen production in this model, demonstrating that this model is advantageous for 3D modeling of collagen-related diseases.

[0074] FIG. 8 illustrates images of morphological analyses of an in vitro (3D skin equivalent) model disclosed herein using organoid-derived fibroblasts. Human specific anti-loricrin and anti-vimentin antibody staining(s) verify the formation of human epidermis and dermis, respectively (left panel). H&E staining of 3D skin equivalents (center panel). Electron microscopy of collagen fibers in 3D skin dermis (right panel).

Example 2

Electron Microscopy

[0075] In another exemplary method, in vitro 3D skin equivalents were obtained and underwent drug screening to assess effects of various drugs on collagen network organization in EDS tissues. This model represents one screening method for assessing drug efficacy on collagen networks affected by hEDS. Mature 3D Skin Equivalents were cultured in media supplemented with Andecaliximab for one week.

[0076] FIGS. 9A-9F illustrates effects of Andecaliximab (MMP-9 Inhibitor) on patient-specific in vitro 3D skin models under electron microscopy analysis. Transmission Electron Microscopy (TEM) demonstrated the restoration of highly-organized collagen networks (arrows) in both primary and organoid-derived hEDS models (9C-9F), comparable to that of healthy controls (9 A, 9B). Scale bars: A-E) 600 nm, F) 1 pm. [0077] FIGS. 10A-10D represent exemplary images of effects on collagen production in healthy controls and organoid-derived EDS cells of an exemplary MMP-2 inhibitor (ARP100) assessed using electron microscopy. Transmission Electron Microscopy (TEM) demonstrated a less pronounced (if any) restoration of collagen network organization in an /// vitro organoid- derived EDS model (10C, 10D), compared to that of healthy controls (10A, 10B). Scale bars are as follows: A) 1 pm, B) 2 pm, C) 1 pm, D) 1 pm.

[0078] FIGS. 11A-11B represent alternative images of effects on collagen production in healthy controls and organoid-derived EDS cells of an MMP-2 inhibitor (ARP 100) on experimental samples (1 IB) and control samples (11A). Arrows indicate restoration of highly- organized collagen networks Scale bars are as follows: A) 1 pm, B) 1 pm.

[0079] FIG 12 illustrates effects on collagen production in healthy controls and organoid- derived EDS cells of an MMP-1 inhibitor (Funalenone) and an MMP-9 inhibitor (Andecaliximab) on in vitro skin models derived from a single specimen that responded to both MMP-1 and MMP-9 inhibition, as analyzed using TEM. Skin equivalents were generated from primary hEDS fibroblasts and treated with the inhibitors over a 7-day period. TEM analysis revealed a highly organized collagen network restoration in response to MMP-1 (middle panel) and MMP-9 (right panel) inhibition in this specimen. In contrast, skin models from other hEDS patients demonstrated no response to MMP-1 inhibition (FIG. 13) but consistently demonstrated collagen network restoration following MMP-9 inhibition (FIG. 9), in comparison with the MMP-1 inhibition. Scale bars: A) 1 pm, B) 1 pm, C) 4 pm.

[0080] FIG. 13 represents exemplary images of effects of an MMP-1 inhibitor (Funalenone) on the collagen network in majority of hEDS skin models as analyzed using transmission electron microscopy (TEM). This data demonstrates a lack of response to MMP-1 inhibition. The in vitro 3D skin models were generated from primary fibroblasts isolated from an hEDS patient whose cells were also tested with an MMP-9 inhibitor in FIG. 9, where collagen network restoration was observed in response to MMP-9 inhibition (FIG. 9D). Scale bars: A) 1 pm, B) 1 pm.

[0081] As observed above, these findings unexpectedly identify MMP-9 inhibition as critical for collagen network improvement in hEDS as represented using an in vitro 3D model disclosed herein, with no comparable effects observed from other MMP inhibitors. Moreover, these results underscore the utility and translational value of 3D skin model disclosed herein as a robust platform for example, for evaluating therapeutic agents targeting extracellular matrix restoration in EDS and in hEDS and related collagen disorders and offering unprecedented insights into patient-specific responses to potential therapies.

All the compositions and methods disclosed and claimed herein may be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variation may be provided to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

What is Claimed:

1. A system comprising: a plurality of wells; a plurality of inserts having a protruding lip or edge wherein each insert is configured to fit within one well of the plurality of wells and remain suspended from a base of the plurality of wells by the protruding lip or edge; a plurality of spacers configured to fit between each of the plurality of wells and plurality of insert protruding lips or edge and configured to adjust a level of the insert from a base of the well; and a plurality of biodegradable gel scaffolds placed within the plurality of insert, wherein the plurality of gel scaffolds comprises a crosslinked matrix configured to allow at least one of cell adhesion, cell expansion, cell differentiation, and cell migration and at least one cell.

2. The system according to claim 1, further comprising one or more cells embedded within the gel scaffold.

3. The system according to claim 2, wherein the cells comprise primary cells, cultured cells from a subject or donor, or from a cell line.

4. The system according to claim 3, wherein the at least one cell comprises at least one of fibroblast, chondrocyte, keratinocyte, melanocyte, Langerhans cell, Merkel cell, induced pluripotent stem cell (iPSC), or combination thereof.

5. The system according to any one of claims 1-4, wherein the at least one cell is from a skin biopsy.

6. The system according to claim 5, wherein the skin biopsy is from a subject having Ehlers-Danlos Syndrome (EDS) or other connective tissue disorder.

7. The system according to claim 6, wherein the subject is a mammalian subject; optionally, a human subject.

8. The system according any one of claims 1-7, wherein the system is configured such that the plurality of inserts can be raised or lowered within the plurality of wells.

9. The system according to any one of claims 1-8, wherein each insert in the plurality of inserts comprises a permeable bottom to permit contact of the gel scaffolds contained therein with an aqueous solution.

10. The system according to any one of claim 1-9, configured such that exterior sidewalls of the plurality of inserts do not abut or are not in direct contact with interior sidewalls of the plurality of wells.

11. The system according to any one of claims 1-10, wherein the crosslinked hyaluronan is crosslinked with acrylate, diacrylate, methacrylate, maleimide, thiol, hexadecylamide, carbodiimide, succinimide, triazole, diimidazole, tyramine, formaldehyde, divinyl sulfone, or 1,4-butanediol diglycidyl ether (BDDE).

12. The system according to claim 11, wherein the crosslinked matrix comprises a crosslinked hyaluronan, alginate, collagen, dextran, chitosan, silk fibroin, sericin, or synthetic polyethylene glycol (PEG) polymer, or combinations thereof.

13. The system according to any one of claims 1-4, wherein the crosslinked hyaluronan is thiolated hyaluronan (HA-SH).

14. The system according to claim 12 or 13, wherein the hyaluronan is crosslinked with one or more of acrylate, diacrylate, thiol, hexadecylamide, carbodiimide, succinimide, triazole, diimidazole, tyramine, formaldehyde, divinyl sulfone, or 1,4-butanediol diglycidyl ether.

15. The system according to any one of claims 1-14, wherein the gel scaffold further comprises at least one of fibrinogen, aprotinin, thrombin, CaCh, culture media, serum, or combination thereof.

16. A method of culturing cells using the system according to claim 1 or 2, comprising adding a first media to one or more of the plurality of wells; providing the gel scaffold to one or more inserts of the plurality of inserts; embedding first set of one or more cells within the gel scaffold; and culturing the first set of cells, wherein the one or more plurality of inserts comprising the gel scaffold and cells are oriented such that the gel scaffold and cells are in contact with the media; optionally fully submerged in the media, and progressively transitioning the first media to a second media over a period.

17. The method according to claim 16, further comprising layering a second set of one or more cells on the gel scaffold.

18. The method according to claim 17, wherein the first set of one or more cells comprise at least one of fibroblast, chondrocyte, keratinocyte, melanocyte, Langerhans cell, Merkel cell, induced pluripotent stem cell (iPSC), or combination thereof; optionally obtained from a skin biopsy.

19. The method according to claim 17 or 18, wherein the skin biopsy is from a subject having Ehlers-Danlos Syndrome (EDS) or other connective tissue disorder.

20. The method according to any one of claims 16-19, wherein the subject is a mammal; optionally, a human.

21. The method according to any one of claims 16-20, wherein the second set of one or more cells is layered on the gel scaffold or several days after to embedding the first set of one or more cells within the gel scaffold.

22. The method according to claim 21, wherein the period is about one week.

23. The method according to claim 22, wherein the insert comprising the gel scaffold and embedded cells are raised in preparation for generating a 3D skin equivalent.

24. The method according to any one of claims 16-23, wherein the gel scaffold is detached from inner walls of the insert to generate a 3D skin equivalent.

25. The method according to claim 24, wherein the 3D skin equivalent is cultured at the liquid-air interface for about one day to about 28 days; optionally about one day to about 14 days; optionally for about 1 day to about 7 days.

26. The method according to any one of claims 24-25, further comprising introducing at least one therapeutic agent to the 3D skin equivalent.

27. The method according to claim 26, further comprising measuring effect of the at least one therapeutic agent on extracellular matrix represented by the 3D skin equivalent.

28. The method according to claim 26 or 27, wherein the at least one therapeutic agent increases proliferation or expansion of fibroblast cells, increases matrix deposition; induces fibroblast quiescence, enhances matrix remodeling, induces cell differentiation, or a combination thereof.

29. The method according to any one of claims 26-28, further comprising measuring effect of the one or more therapeutic agents on the 3D skin equivalent.

30. The method according to claim 29, wherein the subject is subsequently treated with one or more therapeutic agents that demonstrate an advantageous effect.

31. The method according to claim 30, wherein the advantageous effect comprises an increase proliferation or expansion of fibroblast cells, an increase matrix deposition, an induction in fibroblast quiescence, an enhancement of matrix remodeling, an induction of cell differentiation, or a combination thereof.

32. Use of the system according to claim 1 or 2 to prepare 3D skin equivalents comprising, adding a first media to one or more of the plurality of wells; providing the gel scaffold to one or more inserts of the plurality of inserts embedding first set of one or more cells within the gel scaffold; and culturing the first set of one or more cells, wherein the one or more plurality of inserts comprising the gel scaffold and cells are oriented such that the gel scaffold and cells are in contact with an aqueous solution; optionally, fully submerged in the aqueous solution; progressively transitioning the first media to a second media over a period; and detaching the gel scaffold from walls of the insert to generate a 3D skin equivalent.

33. The use according to claim 32, further comprising exposing the 3D skin equivalent to one or more therapeutic agents; and measuring effect of the one or more therapeutic agents on the 3D skin equivalent.

34. The use according to claim 33, wherein the subject is subsequently treated with at least one of the one or more therapeutic agents that demonstrates an advantageous effect on the 3D skin equivalent.

35. The use according to claim 33 or 34, wherein the one or more therapeutic agents comprise one or more MMP inhibitors.

36. A gel scaffold comprising hyaluronan crosslinked with polyethylene glycol (PEG) and further comprising at least one of fibrinogen, aprotinin, thrombin, or a combination thereof.

37. The gel scaffold according to claim 36, wherein the hyaluronan is thiolated hyaluronan and the PEG is PEG diacrylate (PEGDA).

38. The gel scaffold according to claim 36 or 37, further comprising first set of one or more cells embedded within the gel scaffold and, optionally, second set of one or more cells layered on a surface of the gel scaffold.

39. The gel scaffold according to claim 38, wherein the first set of one or more cells comprise one or more of fibroblast, chondrocyte, keratinocyte, melanocyte, Langerhans cell, Merkel cell, induced pluripotent stem cell (iPSC), or combination thereof.

40. A method for treating Ehlers-Danlos Syndrome (EDS) in a subject comprising administering to the subject a composition comprising an agent for inhibiting or eliminating matrix metalloproteinase 9 (MMP-9) activity or expression in the subject and treating EDS in the subject.

41. The method according to claim 39, wherein the EDS comprises hypermobility EDS (hEDS).

42. The method according to claim 40 or 41, wherein the agent for inhibiting or eliminating MMP-9 activity or expression comprises a biologic agent, a chemical agent, and or gene editing.

43. The method according to any one of claims 40-42, wherein the MMP-9 inhibitor comprises at least one of MMP-9 Inhibitor CAS ID 206549-55-5, Andecaliximab CAS ID 1518996-49-0, GI254023X CAS ID 260264-93-5, Metformin CAS ID 657-24-9, Minocycline CAS ID13614-98-7, Disulfiram CAS ID 97-77-8, Lovastatin CAS ID 75330-75-5, Simvastatin, Avasimibe, Fluvastatin, Fenofibric acid, Batimastat and Marimastat or a combination thereof.

44. The method according to any one of claims 40-43, wherein the MMP-9 inhibitor comprises at least one of MMP-9 Inhibitor CAS ID 206549-55-5, Andecaliximab CAS ID 1518996-49-0, and GI254023X CAS ID 260264-93-5.

45. A composition comprising at least one MMP-9 inhibitor and at least a second agent that treats a connective tissue disorder or side effect of a connective tissue disorder; optionally, EDS or hEDS.

46. The composition according to claim 45, further comprising at least one pharmaceutically acceptable excipient.

47. The composition according to claim 45 or 46, wherein the MMP-9 inhibitor comprises at least one of MMP-9 Inhibitor CAS ID 206549-55-5, Andecaliximab CAS ID 1518996-49-0, GI254023X CAS ID 260264-93-5, Metformin CAS ID 657-24-9, Minocycline CAS ID13614- 98-7, Disulfiram CAS ID 97-77-8, Lovastatin CAS ID 75330-75-5, Simvastatin, Avasimibe, Fluvastatin, Fenofibric acid, Batimastat, Marimastat, or a combination thereof.

48. A method for treating a subject having EDS comprising: administering a composition comprising at least one MMP-9 inhibitor to the subject; and treating EDS in the subject.

49. The method according to claim 48, wherein the MMP-9 inhibitor comprises at least one of MMP-9 Inhibitor CAS ID 206549-55-5, Andecaliximab CAS ID 1518996-49-0, GI254023X CAS ID 260264-93-5, Metformin CAS ID 657-24-9, Minocycline CAS ID13614-98-7, Disulfiram CAS ID 97-77-8, Lovastatin CAS ID 75330-75-5, Simvastatin, Avasimibe, Fluvastatin, Fenofibric acid, Batimastat, Marimastat, or a combination thereof.

50. An animal model for examining or diagnosing EDS in a patient or testing novel therapeutic modalities for EDS treatment comprising creating a skin xenograft model of the EDS comprising obtaining a healing wound associated on a back of an immunodeficient animal wherein the healing wound comprises a combination of human keratinocytes and EDS-subject obtained cells; optionally, fibroblasts.

51. The animal model according to claim 50, further comprising, comparing collagen fibrils in an extracellular matrix derived from the patient with collagen fibrils in an extracellular matrix derived from healthy controls.

52. A method for diagnosing a connective tissue disorder in a subject comprising: obtaining skin cells from a subject suspected of having the connective tissue disorder; using the system according to claim 1 or 2 to generate a subject-specific 3D skin equivalent; analyzing the subject-specific 3D skin equivalent for at least one of proliferation, or expansion of fibroblast cells, matrix deposition; fibroblast quiescence, matrix remodeling, cell differentiation, or a combination thereof; and diagnosing a connective tissue disorder in the subject.

53. The method according to claim 52, wherein the subject is suspected of having EDS and diagnosing EDS in the subject.

54. The method according to claim 52 or 53, further comprising testing one or more therapeutic agents for enhancing at least one of proliferation or expansion of fibroblast cells, matrix deposition; fibroblast quiescence, matrix remodeling, cell differentiation, or a combination thereof using the subject-specific 3D skin equivalent; and treating the subject with the one or more therapeutic agents enhancing the at least one of proliferation or expansion of fibroblast cells, matrix deposition; fibroblast quiescence, matrix remodeling, cell differentiation, or a combination thereof.