Drug-producing implantable matrix devices and methods - Patents.com

Abstract

Described herein are variations of implantable matrices that produce pharmaceuticals for the treatment of chronic diseases. For example, the implantable matrix can include a vascular graft of human acellular vasculature coated with cells. The cells can be engineered to produce pharmaceuticals capable of treating chronic diseases. The cell-seeded matrix can be implanted adjacent to a patient's blood vessels so that pharmaceuticals produced by the cells are readily released into the patient's bloodstream. Additionally, or alternatively, the cell-seeded matrix can treat chronic diseases in an environment local to the implantation site. Also described herein are methods for making and using such implantable matrices.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 438,444, filed January 11, 2023, which is incorporated by reference in its entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates to implantable pharmaceutical-producing matrices and methods for making same. [Background technology]

[0003] Patients with chronic diseases such as cancer, multiple sclerosis, rheumatoid arthritis, hemophilia A (i.e., factor VIII (FVIII) deficiency or classical hemophilia), Crohn's disease, ulcerative colitis, and ankylosing spondylitis often rely on pharmaceuticals for treatment. Current pharmaceutical technology is primarily based on the infusion delivery of cytokines, hormones, antibodies, or recombinant proteins produced in cell, yeast, or bacterial cultures. Typically, these pharmaceuticals are administered via infusion therapy, delivering the treatment directly into the bloodstream; however, this procedure can lead to complications such as phlebitis and infection at the injection site. Furthermore, intravenous therapy sessions can be time-consuming, requiring up to two hours once or twice a week, potentially leading to lifelong dependency on these treatments. Furthermore, these repeated treatments can cause significant financial burdens for patients. Therefore, there is a need for new and improved delivery of pharmaceuticals to patients with chronic diseases. Accordingly, the present disclosure provides devices and methods for producing therapeutic molecules (drugs) via implantable matrices within a patient's body. Summary of the Invention

[0004] In some embodiments, a medicament-producing implantable matrix is ​​provided, comprising a matrix containing at least one cell population.

[0005] In some embodiments, an implantable pharmaceutical-producing matrix is ​​provided that includes a framework comprising collagen and a matrix comprising at least one cell population, the matrix being at least one of attached to and within the framework.

[0006] In some embodiments, a method of forming an implantable matrix that produces a pharmaceutical agent is provided, comprising combining at least one cell population with a dissolved matrix, casting the combination into a framework, and polymerizing the dissolved matrix.

[0007] In some embodiments, a device is provided that includes a tubular support graft comprising a matrix and at least one cell population, the tubular support graft configured to be placed concentrically around a vascular graft.

[0008] In some embodiments, a device is provided that includes a tubular support graft configured to be placed concentrically around a tubular vascular graft, and a matrix comprising at least one cell population and disposed on an outer surface of the tubular support graft.

[0009] In some embodiments, the present disclosure further relates to a method for forming a tubular support graft, the method including: placing a support mandrel within a tubular mold, wherein a first end of the support mandrel and the first end of the tubular mold are fluidly sealed by a first end cap; injecting a mixture of hydrogel and cell suspension into a volume formed between a surface of the support mandrel and the tubular mold; fluidly sealing a second end of the support mandrel and the second end of the tubular mold through a second end cap; and removing the second end cap and tubular sleeve after the mixture has hardened to form the tubular support graft.

[0010] In some embodiments, a method is provided for forming a cell-populated tubular support graft for a tubular vascular graft, comprising attaching the tubular support graft to a support mandrel, rotating the support mandrel at a predetermined speed, depositing droplets of a mixture of hydrogel and at least one cell population onto the outer surface of the support mandrel via a three-dimensional printer, and, after hardening, removing the cell-populated tubular support graft from the support mandrel.

[0011] Some variations of at least the above-described embodiments may further include one and / or another variation, and in some variations may further include more than one of the following elements, features, functionality, structures, materials, steps, and / or descriptions, and in some variations (when not mutually exclusive) substantially all of the following elements, features, functionality, structures, materials, steps, and / or descriptions, resulting in still further variations of embodiments of the present disclosure: - at least one cell population produces one or more molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a; - at least one cell population produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulins, testosterone, human Htt, or p42; - at least one cell population produces interleukin-10; - at least one cell population produces sirolimus or tacrolimus; - at least one cell population comprises mesenchymal stem cells; - at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells; the matrix comprises at least one selected from the group consisting of hydrogels and biodegradable polymers; the matrix comprises a hydrogel containing thrombin and fibrinogen, the thrombin being at a concentration of about 0.1 units / mL to about 1 mg / mL, or the matrix comprises a hydrogel comprising thrombin and fibrinogen, wherein the fibrinogen can be at a concentration of about 3 mg / mL to about 100 mg / mL; the matrix comprises a Fas receptor-activating molecule; - the Fas receptor-activating molecule comprises a Fas ligand; - the matrix is ​​implanted in at least one body region of the patient selected from the group consisting of the knee, arm, leg, hip, elbow, wrist, spine, stomach, blood vessels, and intestines; - that the matrix can be formed at the desired implantation site by injecting cells in a hydrogel adhesive solution; the matrix comprises at least one selected from the group consisting of hydrogels and biodegradable polymers, the biodegradable polymer is selected from the group consisting of polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, and poly(caprolactone); and / or o the hydrogel is at least one of a natural hydrogel and a synthetic hydrogel; - the vascular graft may comprise one or more selected from the group consisting of fibrin, collagen, alginate, gelatin, chitosan, dextran, hyaluronic acid, or PEG; - the vascular graft may comprise a combination of PEG and chitosan, PEG and gelatin, PEG and hyaluronic acid, PAM and gelatin, or PVA and gelatin; - the tubular support graft may comprise one or more selected from the group consisting of fibrin, collagen, alginate, gelatin, chitosan, dextran, hyaluronic acid, or PEG; - that the tubular support graft may comprise a combination of PEG and chitosan, PEG and gelatin, PEG and hyaluronic acid, PAM and gelatin, or PVA and gelatin; - the tubular support graft is formed by one of tubular molding, casting, flat tissue culture, electrospinning, and three-dimensional printing; - the length of the tubular support graft is less than the length of the tubular vascular graft; - the length of the tubular support graft is greater than the length of the tubular vascular graft; - The length of the tubular vascular graft is 1cm to 100cm. - forming the tubular support graft includes placing the tubular support graft on the exterior surface of the vascular graft; - forming the tubular support graft includes implanting a vascular graft carrying the tubular support graft into the patient's arm; - forming the tubular support graft includes implanting a vascular graft carrying the tubular support graft adjacent the patient's leg.

[0012] These and other objects, features, and advantages of at least some of the embodiments of the present disclosure will become even more apparent with reference to the following drawings, the brief description thereof immediately following, and the detailed description thereafter. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows an implantable matrix having a tissue substrate, a hydrogel substrate, cells, and additives, according to some variations. [Figure 2] FIG. 2 shows an implantable matrix with tissue substrate, cells, and additives, according to some variations. [Figure 3] FIG. 3 shows an implantable hydrogel matrix, cells, and additives, according to some variations. [Figure 4]FIG. 4 shows an implantable hydrogel matrix, support mesh, cells, and additives, according to some variations. [Figure 5] Figure 5A shows an implantable matrix with a tissue frame and an inner hydrogel, and Figure 5B shows an implantable matrix with a tissue frame and an inner hydrogel. [Figure 6] FIG. 6 shows an exemplary schematic diagram of a method for preparing a tissue substrate for use in an implantable matrix. [Figure 7] FIG. 7 shows an exemplary schematic diagram of a variation of a method for attaching a tissue substrate to a hydrogel substrate for use as an implantable matrix. [Figure 8] FIG. 8 shows an exemplary schematic of a variation of a method for three-dimensional printing of a tubular cell-populated hydrogel (i.e., a tubular support graft). [Figure 9] FIG. 9 shows an exemplary schematic diagram of a variation of a method for placing a tube of cell-populated hydrogel (i.e., a tubular support graft) onto a vascular graft, according to some variations, wherein the tubular support graft comprises hydrogel, cells, and additives. [Figure 10] 10 shows an exemplary schematic diagram of a tubular cell-populated hydrogel (tubular support graft), according to some variations. The tubular support graft can be a cell-hydrogel composite with cells within the hydrogel. [Figure 11] FIG. 11 shows a tubular vascular graft with a cell mass coating (tubular support graft) anastomosed to a patient's arteries and veins. [Figure 12] Figure 12A shows a patch of implantable matrix attached to an implantation site of interest, including vertebrae, intestine, and joint surfaces. Figure 12B shows a patch of implantable matrix attached to an implantation site of interest, including vertebrae, intestine, and joint surfaces. Figure 12C shows a patch of implantable matrix attached to an implantation site of interest, including vertebrae, intestine, and joint surfaces. [Figure 13]13 shows an exemplary schematic of a variation of a method for generating a tubular cell-assembled hydrogel (i.e., a tubular support graft) by pouring a hydrogel mixture into the space between a cylindrical mandrel and a tubular mold. Once the hydrogel has cured / polymerized, the tubular support graft and mandrel can be removed from the mold. DETAILED DESCRIPTION OF THE INVENTION

[0014] definition The term "a" or "an" refers to one or more of that entity, i.e., it can refer to multiple referents. As such, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. Furthermore, reference to "an element" by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements are present, unless the context clearly requires that one and only one element is present.

[0015] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device or method used and defines that value or the variation that exists between measured samples. Unless otherwise stated or apparent from the context, the term "about" means within a 10% range above or below the reported numerical value (except where such numerical value would exceed 100% or be less than 0% of the possible values). When used in conjunction with a range of values ​​or series of values, the term "about" applies to both endpoints of the range or each consecutively recited value unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used interchangeably.

[0016] Non-limiting examples of various aspects and variations of the present invention are described herein and illustrated in the accompanying drawings.

[0017] As generally described herein, a drug-producing implantable matrix can include a matrix populated with drug-secreting cells. The matrix can include, for example, a cell-loaded hydrogel attached to or coated on a patch of decellularized tissue. The cell-populated hydrogel matrix can facilitate localized drug delivery via seeded cells, which can avoid an immune response to the implantable matrix. Drug-secreting cells can be engineered, for example, to produce a drug (i.e., a drug or agent) to treat a specific chronic disease. A biovascular drug-producing implantable matrix can be anastomosed to a patient's vein or artery so that the secreted drug is released into the patient's bloodstream. The decellularized tissue substrate of the implantable matrix can facilitate attachment (e.g., suturing, gluing) and anastomosis of the matrix to the implantation site. In some variations, the matrix can be formed as a coating on the outer surface of a vascular graft and anastomosed to the blood circulation via vascular surgery. For example, as shown in FIG. 1, implantable matrix 100 can include cells 104 in hydrogel 108 coated on tissue patch 102. The cells can be mixed in a hydrogel precursor solution and can include two or more cell populations. For example, in some variations, cells 104 can include drug-producing cells and IL-10-expressing or immunosuppressant-producing cells, which mitigate an immune response to implantable matrix 100. This co-delivery of cell populations can locally protect the drug-producing cells from an immune response. As shown in FIG. 2, implantable matrix 200 can alternatively include tissue patch 202, which can include cells 204 seeded directly on its outer surface. This biovascular implantable matrix can facilitate systemic treatment of chronic diseases by allowing drug-secreting cells 104, when seeded on the surface, to reside in close proximity to the patient's circulatory system. Referring again to FIG. 1, in some variations, implantable matrix 100 may further include additives 106 that may be seeded into the hydrogel matrix.The additives may, for example, further reduce immune response to the cells, or may generally support the physical properties of the hydrogel 108 and / or tissue patch 102, or may generally support cell growth in vivo.

[0018] Alternatively, the drug-producing cells described herein can be contained in an implantable pouch or similar device and delivered to the implantation site of interest.

[0019] The implantable matrices described herein have features that improve many aspects of chronic disease treatment. For example, treatment via an implantable matrix requires only a single procedure to implant the matrix, potentially sparing patients from the complications, time, and expense of ongoing pharmaceutical treatment. Furthermore, as described in detail herein, the implantable matrix can provide additional support at the implantation site and can provide local protection for drug-producing cells.

[0020] Although the pharmaceutical production systems described herein may be described with reference to the local or systemic treatment of chronic diseases (e.g., cancer, multiple sclerosis, rheumatoid arthritis, hemophilia, Crohn's disease, ulcerative colitis, ankylosing spondylitis, etc.), it should be understood that such systems may additionally or alternatively be configured to treat other diseases or disorders, such as genetic disorders.

[0021] Various aspects of the drug-producing implantable matrix variations and methods of making and using them are described in further detail below. Section 1 discusses implantable matrix devices. Section 2 discusses cellular secretion of drugs and (optional) additives for implantable matrices. Section 3 discusses manufacturing of implantable matrices. Section 4 discusses implantation of the matrices. Section 5 discusses examples of implantable matrix devices and methods described herein.

[0022] Section 1: Embeddable Matrices 1 , implantable matrix 100 may generally include a tissue substrate 102 and a hydrogel 108 for delivering drug-producing cells 104 to an implantation site and supporting their growth and proliferation in vivo. In some variations, the implantable matrix may further include a hydrogel adhesive substrate and / or additives 106. As discussed in detail herein, in some variations, the implantable matrix may include either the tissue substrate 102 or the hydrogel 108.

[0023] Generally, an implantable matrix can be populated with at least one cell. In some variations, the substrate can have a thickness of about 1 μm to about 5 cm, e.g., about 1 μm to about 5 mm, about 10 μm to about 1 mm, about 50 μm to about 500 μm, or about 100 μm to about 250 μm. In some variations, the surface area of ​​the matrix substrate can be, e.g., about 10 μm. 2 ~about 10cm 2 , about 100μm 2 ~about 1cm 2 , about 1mm 2 ~approx. 500mm 2 , or about 100 mm 2 ~250mm 2 is about 1 μm 2 ~Approx. 100cm 2 Although the figures referred to herein show the implantable matrix as a rectangular patch or a tubular graft, it should be understood that the matrix can be any suitable shape that aids in the systemic delivery of a drug, e.g., a circle, a triangle, an oval, an irregular shape, or a pouch configured to contain drug-producing cells.

[0024] tissue base material As shown in FIG. 2 , the implantable matrix 200 may include a tissue substrate 202. In some variations, the tissue substrate 202 may be used to deliver surface-seeded cells 204 proximal to a patient's circulatory system so that medicinal agents secreted by the cells 204 are readily delivered into the bloodstream. In some variations, the tissue substrate 202 may also include surface-seeded additives 206. The tissue substrate 202 may be a patch of decellularized tissue and may be biodegradable and biocompatible to temporarily support the growth of the seeded cells at the implantation site as it is enzymatically degraded by the patient's body. For example, a collagen-based matrix may be enzymatically degraded and mediated through natural means by proteins such as collagenase. The degradation process of the matrices described herein may be on the order of days, weeks, months, or years. In some variations, the tissue substrate 202 may be an artificial acellular substrate or a decellularized tissue substrate. In some variations, vascular tissue, skin, or tendon may be decellularized to generate the tissue substrate 202. For example, a regenerative vascular conduit can be decellularized to generate a tissue matrix 202 (e.g., a Human Acellular Vessel). In some variations, the tissue matrix 202 can be collagenous. Collagen and other mammalian-derived protein-based polymers can provide an effective matrix for cell growth because they contain numerous cell signaling domains present in the extracellular matrix in vivo. Furthermore, the mechanical properties of collagen (toughness, stiffness, inelasticity) can aid in the embedding of an implantable matrix. Collagen matrixes can be produced through natural means without chemical modification. Various methods have been developed to synthesize matrixes with enhanced mechanical properties, such as chemical crosslinking, UV or thermal crosslinking, and / or blending with other polymeric agents. In some variations, the artificial acellular tissue matrix may be made from, for example, fibrin, collagen, alginate, gelatin, chitosan, dextran, hyaluronic acid, PEG, PEG / chitosan, PEG / gelatin, PEG / hyaluronic acid, PAM / gelatin, or PVA / gelatin.Typically, the cells 204 and / or additives 206 can be seeded onto the outer surface of the tissue substrate 202. Typically, the thickness of the tissue substrate 202 can be about 1 μm to about 5 cm, for example, about 10 μm to about 1 cm, about 100 μm to about 5 mm, or about 250 μm to about 1 mm. In some variations, the thickness of the tissue substrate 202 is approximately equal to the thickness of a decellularized blood vessel, such as a HAV. For example, the thickness of the tissue substrate 202 can be about 500 μm.

[0025] In variations in which the tissue substrate is a rectangular patch, the tissue substrate may have a side length of about 1 mm to about 10 cm, such as about 1 cm to about 9 cm, about 2 cm to about 8 cm, or about 3 cm to about 7 cm. The side lengths of the tissue substrate may be the same or different. For example, the tissue substrate may have a width of about 2 cm and a length of about 2 cm, or a width of about 2 cm and a length of about 5 cm. In some variations, the width of the tissue substrate may be approximately equal to the circumference of a decellularized blood vessel, such as a HAV.

[0026] Hydrogel matrix Referring to FIG. 3 , an implantable matrix 300 can include a hydrogel matrix 302 populated with cells 404 and optionally seeded with additives 306. Hydrogels generally comprise three-dimensional (3D) processed (e.g., crosslinked or polymerized) networks of polymers. They are useful for tissue engineering due to their tunable biochemical and biophysical properties for controlling cell function (e.g., adhesion, proliferation, differentiation, etc.) and can be designed as artificial extracellular matrix (ECM) substrates to provide spatial orientation and promote cellular interactions with their surroundings. Hydrogels can be formed by crosslinking polymer chains through physical or chemical methods. For example, physical hydrogels are ionically induced, formed by molecular entanglement and secondary forces such as hydrogen bonding, crystallite formation, electrostatic interactions, and hydrophobicity, which are often reversible. An advantage of physical hydrogels is their biocompatibility due to the absence of chemical crosslinkers that can cause cytotoxicity.

[0027] The hydrogel matrix, e.g., hydrogel matrix 302, can include naturally occurring biomaterials, synthetic polymers, or a combination thereof. In some variations, the hydrogel can include two or more layers of hydrogel matrix. In some variations, hydrogel matrix 302 can be a microbead hydrogel, which increases the efficiency and reproducibility of large-scale manufacturing of the implantable matrices described herein. In some variations, hydrogel matrix 302 can be a cell-assembling hydrogel adhesive or cohesive that naturally adheres to the implantation site. Hydrogel adhesives can exhibit strong chemical interactions, such as strong van der Waals interactions, to achieve adhesion. A unique advantage of hydrogel adhesives is their adaptability to tissue migration. In some variations, the hydrogel matrix can be an injectable hydrogel.

[0028] Any polymer capable of forming a hydrogel upon processing (e.g., crosslinking or polymerization) can be used in the hydrogel matrix 302 according to the present invention. In some variations, the polymer can be a linear or branched polymer. In some variations, the polymer can be a dendrimer. In some variations, the polymer can be a homopolymer or a copolymer comprising two or more monomers. The copolymer can be a block copolymer, a graft copolymer, a random copolymer, a blend, a mixture, and / or an adduct of polymers. Typically, polymers according to the present invention are organic polymers. In some variations, the polymer can be modified with one or more moieties and / or functional groups. Any moiety or functional group can be used according to the present invention. In some variations, the hydrogel can comprise a synthetic polymer. Advantages of synthetic hydrogels for tissue engineering applications include tunability of mechanical properties and high reproducibility in both small- and large-scale manufacturing. The synthetic environment can also allow for viability of cells within the hydrogel as they remodel the surrounding microenvironment at the implantation site. Bioactive molecules such as proteins, enzymes, and growth factors can be incorporated into synthetic hydrogel matrices to mediate specific cellular functions. Synthetic hydrogel matrices can also be chemically modified to impart beneficial properties such as altered porosity and stiffness, improved stability, biocompatibility, and degradability, and tailored mechanical strength for various cellular applications.Non-limiting examples of synthetic polymers for use in the present invention include polyglycolic acid (PGA), polylactic acid (PLA), polylactic-co-glycolic acid (PLGA), poly(caprolactone) (PCL), polyethylene (PET), polycarbonate (PC), polyanhydrides, polyhydroxy acids, polypropylfumarate, polycaprolactone, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, and the like. Hydrogels include natural polymers such as vinyl alcohol, polyvinyl acetate (PVA), polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polyurea, polystyrene, polyamine, poly(ethylene glycol), polyacrylamide (PAM), poly(aspartic acid), poly(2-hydroxyethyl methacrylate), poly(1,3-dioxane-2-one), poly(sebacic anhydride), poly(β-hydroxyalkanoates), polycaprolactam, polylactide, polyglycolide, and derivatives and / or combinations of any of these. In some variations, the hydrogels contain natural polymers such as carbohydrates, proteins, nucleic acids, and lipids. Some advantages of natural hydrogels include low toxicity and high biocompatibility. Furthermore, the natural environment can influence cellular behavior through signaling cascades initiated by binding events with cell surface receptors. Further examples of natural polymers for use in the present invention include, but are not limited to, fibrin, collagen, alginate, agarose, gelatin, chitosan, dextran, hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and any derivatives and / or combinations thereof. In some variations, the hydrogel may include an antifibrinolytic agent (e.g., fibrinogen) and / or a procoagulant (e.g., thrombin). Such agents may be sourced from human plasma. In some variations, the natural polymer may be synthetically produced or partially synthetically produced.In some variations, the hydrogel may include a combination of synthetic and natural polymers, such as, for example, PEG and chitosan, PEG and gelatin, PEG and hyaluronic acid, PAM and gelatin, PVA and gelatin, or PAM and gelatin. In some variations, the hydrogel includes one or more polymers approved for human use by the U.S. Food and Drug Administration. In some variations, the hydrogel matrix 302 may include combinations of the foregoing polymers or other polymers, where at least one polymer is capable of forming a hydrogel. Those skilled in the art will recognize that the polymers listed herein represent a non-exhaustive, exemplary list of polymers that may be included in the implantable matrix.

[0029] Typically, the thickness of hydrogel matrix 302 can be from about 1 μm to about 5 cm, such as from about 10 μm to about 1 cm, from about 100 μm to about 5 mm, or from about 250 μm to about 1 mm. In some variations, a thinner matrix may be preferred to support oxygen-demanding seeded cells, such as beta cells. In some variations, a thicker matrix may be preferred to support a larger number of seeded cells.

[0030] As shown in Figure 4, in some variations, implantable matrix 400 can be a cell-populated hydrogel matrix comprising hydrogel 402 containing cells 404 and optional additives 406. Additionally, implantable matrix 400 can be mechanically supported by mesh 408. Generally, hydrogel and mesh substrates combine the advantages of hydrogels (e.g., lubricity, biocompatibility, anti-biofouling properties) with the advantages of mesh substrates (e.g., relatively greater stiffness, toughness, strength). Mesh 408 can be fabricated from a biodegradable and biocompatible polymer. Some non-limiting examples of polymers suitable for fabricating a mesh supporting a hydrogel matrix include PLA (polylactic acid), PGA (polyglycolide), PLGA (polylactic-co-glycolic acid), polyurethane, polyesterurethaneurea (PEUU), poly(etheresterurethane)urea (PEEUU), silicone, polyaryletherketone, polyetherketoneketone, polyetherblockamide, polytetrafluoroethylene (PTFE), polyoxymethylene, polyethyleneterephthalate, polypropylene Examples of suitable materials include polycaprolactone (PCL), poly-4-hydroxybutyrate, polycarbonate, poly(ester carbonate urethane) urea (PECUU), copolymers thereof, derivatives thereof, and combinations thereof. In some variations, the mesh may be at least partially non-polymeric and may comprise compositions including, but not limited to, stainless steel, gold, silver, platinum, titanium and titanium alloys, tantalum, cobalt chromium alloys, carbon fiber (graphite or diamond), hydroxyapatite, and other calcium phosphate materials. In some variations, the mesh 408 may be fabricated from any combination of the foregoing materials.

[0031] Referring again to FIG. 3, in some variations, the hydrogel matrix 302 of the implantable matrix 300 may be molded into a rectangular shape. In these variations, the hydrogel matrix 302 may include a side length of about 1 mm to about 10 cm, such as about 50 mm to about 5 cm, about 100 mm to about 2.5 cm, about 500 mm to about 2.25 cm, or about 1 cm to about 2 cm. The side lengths of the rectangular hydrogel matrix may be the same or different. Alternatively, the hydrogel matrix 302 may be molded into a tubular shape (e.g., a tubular support graft), as shown in FIG. 9, which illustrates a method 900 of placing a tubular cell population hydrogel matrix (tubular support graft) concentrically around a vascular graft. In these variations, the tubular hydrogel matrix 302 may have a length of about 1 cm to about 100 cm, such as about 5 cm to about 60 cm, or about 10 cm to about 40 cm. The tubular hydrogel substrate 308 can have an inner diameter of about 1 mm to about 50 mm, e.g., about 2 mm to about 30 mm, or about 3 mm to about 20 mm. The tubular hydrogel substrate 308 can have an outer diameter of about 1 mm to about 50 mm, e.g., about 2 mm to about 30 mm, or about 3 mm to about 20 mm. Generally, the inner diameter of the tubular hydrogel 308 can be larger than the outer diameter of the decellularized blood vessel, e.g., the HAV. In some embodiments, the inner and / or outer diameter of the tubular hydrogel substrate can taper (e.g., increase or decrease) from the first end to the second end. For example, the tubular hydrogel matrix 302 can include an inner diameter of 8 mm and an outer diameter of 10 mm at the first end and an inner diameter of 7 mm and an outer diameter of 9 mm at the second end, with the inner and outer diameters decreasing at a constant rate from the first end to the second end.

[0032] In alternative variations, the hydrogel matrix 302 may include circular, triangular, and oval, irregular shapes, or may comprise a pouch configured to contain pharmaceutical-producing cells. Those skilled in the art will recognize that the hydrogel substrate shapes listed herein represent a non-exhaustive, exemplary list of suitable shapes for the hydrogel substrate.

[0033] As shown in FIG. 1 , the implantable matrix 100 can be a rectangular patch comprising a hydrogel matrix 108 attached to a tissue substrate 102. The hydrogel 108 can be coated or poured onto the tissue substrate 102. Typically, the hydrogel 108 can be attached to the outer surface of the tissue 102. In some variations, the hydrogel 108 can be a hydrogel adhesive, which can be disposed directly on the tissue substrate 102 or on a separate hydrogel. Methods for forming an implantable matrix comprising a tissue substrate, a hydrogel substrate, and a hydrogel adhesive are described in further detail herein. In some variations, the hydrogel 108 and the tissue 102 can be polymerized via methods described in further detail herein.

[0034] 5A-5B, implantable matrix 500 can include a tissue-based frame (i.e., frame 510) surrounding hydrogel 502. Frame 510 can include a rigid material, such as collagen, to facilitate implantation (e.g., suturing, gluing, etc.) of implantable matrix 500 at the implantation site. For example, frame 510 can be a rigid collagenous frame that can be sutured to the patient's tissue during implantation of implantable matrix 500. Frame 510 can be rectangular, circular, triangular, irregular, or any other suitable shape that surrounds hydrogel 502. Rectangular frame can include side lengths ranging from about 1 mm to 10 cm. For example, rectangular frame can be about 1 cm x 5 cm, about 2 cm x 2 cm, or about 2.5 cm x 2 cm.

[0035] 9, in some variations, the implantable matrix 914 may be a cell-populated hydrogel sleeve 907 (e.g., a tubular support graft) disposed around a tubular tissue substrate 902 (e.g., a tubular vascular graft). The length, width, and / or thickness of the tubular hydrogel 907 may be greater than, less than, or equal to the length, width, and / or thickness of the vascular graft 902. Methods for attaching the vascular graft 902 and the tubular support graft 907 are described in further detail herein.

[0036] For example, Figure 9 shows a method of concentrically arranging a cell population hydrogel tube around a vascular graft. The vascular graft 902 may have a diameter of about 1 to about 20 mm, such as about 2 mm to about 16 mm, about 4 mm to about 12 mm, or about 6 mm to about 8 mm. The vascular graft 902 may have a length of about 1 mm to about 10 mm, such as about 2 mm to about 8 mm, or about 3 mm to about 7 mm. The vascular graft 902 may have an inner diameter of about 1 mm to about 50 mm, such as about 2 mm to about 30 mm, or about 3 mm to about 20 mm. The vascular graft 902 may have an outer diameter of about 1 mm to about 50 mm, such as about 2 mm to about 30 mm, or about 3 mm to about 20 mm. In some variations, the inner and outer diameters of the vascular graft 902 may be approximately equal to the inner and outer diameters of a decellularized blood vessel, such as a HAV. In some embodiments, the inner and / or outer diameter of the tubular hydrogel substrate can be tapered (e.g., increase or decrease) from the first end to the second end. For example, the vascular graft 902 can include an inner diameter of 8 mm and an outer diameter of 10 mm at the first end and an inner diameter of 7 mm and an outer diameter of 9 mm at the second end, with the inner and outer diameters decreasing at a constant rate from the first end to the second end.

[0037] In alternative variations, the tissue substrate may include circular, triangular, and oval, irregular shapes, or may include pouches configured to contain pharmaceutical-producing cells. Those skilled in the art will recognize that the tissue substrate shapes listed herein represent a non-exhaustive, exemplary list of shapes suitable for tissue substrates.

[0038] Section 2: Cellular secretion of drugs and additives Generally, cells for use in the present invention can secrete medicinal agents to treat patients with chronic diseases. Live cells can be seeded onto and / or into any one of the matrix substrates described herein to form an implantable matrix capable of secreting medicinal agents. For example, as shown in FIG. 1, cells 104 and optional additives 106 can be mixed into a hydrogel precursor solution, and a hydrogel matrix 108 can be bonded to a tissue substrate 102 to form an implantable matrix 100. As another example, in FIG. 2, a tissue substrate 202 can be seeded with cells 204 on its surface.

[0039] Any type of cell can be used according to the present invention. Non-limiting examples of cells suitable for use in the present invention include mammalian cells (e.g., human cells, primate cells, mammalian cells, rodent cells, etc.), bird cells, fish cells, insect cells, plant cells, fungal cells, bacterial cells, and hybrid cells. In some variations, cell types can include endothelial cells, mesenchymal stem cells, and / or hematopoietic cells such as megakaryocytes or platelets. In some variations, endothelial cells can be human liver sinusoidal endothelial cells (LSECs). In some variations, cells can include monocytes. In some variations, cells can include organoids. In some variations, cells for use in the present invention can be generated from mesenchymal stem cells, endothelial progenitor cells, and / or hematopoietic stem cells. Additionally or alternatively, cells can be cultured from human endothelium, including but not limited to tissue from the surface layer of arteries and / or veins, endocardium, and / or glomerulus of kidney. In some variations, exemplary cell types suitable for use in the present invention include primary cells and / or cell lines derived from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g., monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, sperm cells, egg cells, hepatocytes, epithelial cells from the lung, intestine, intestinal tract, liver, skin, etc., and / or hybrids thereof may be seeded onto and / or within a matrix according to the present invention. In some variations, the implantable matrix may contain cells that produce anti-inflammatory substances to mitigate the effects of infiltration of host immune cells. Anti-inflammatory substance-producing cells may include, but are not limited to, neutrophils, macrophages, monocytes, hepatocytes, lymphocytes, mast cells, and any combination thereof. Anti-inflammatory substances may include, but are not limited to, interleukins and / or transforming growth factor-β (TGF-β). Any of the foregoing cell populations may contain an intracellular source of ionized calcium.Those skilled in the art will recognize that the cells listed herein represent an exemplary, but not comprehensive, list of cells that can be encapsulated within matrices according to the present invention.

[0040] In some variations, a single matrix may contain populations of the same or different cell types. In some variations, a single implantable matrix may contain two or more different types of cells. For example, an implantable matrix may contain one or more populations of drug-secreting cells and one or more populations of cells that secrete drugs that suppress an immune response to the implantable matrix. Some non-limiting examples of immunosuppressive cell types include interleukin-10-expressing cells, tacrolimus-producing cells, sirolimus-producing cells, and MHC class I and II knockout mesenchymal stem cells. Such cells may provide local protection for drug-producing cells within or on the implantable matrix. In some variations, a single implantable matrix may contain any number of cell types. In some variations, the cells may be evenly distributed throughout the matrix substrate. In some variations, the cells may be distributed on the surface of the substrate. For example, the cells may be distributed on the exposed surface of a tissue substrate containing HAV. In some variations, the cells may be encapsulated within a substrate, such as a hydrogel substrate. In some variations,

[0041] Typically, cell populations for use in the present invention can be genetically engineered cell populations. The cells can be engineered to produce one or more pharmaceutical agents, such as cytokines, hormones, antibodies, proteins, fusion proteins, or any other pharmaceutical agent used to treat chronic diseases. For example, cells for use in the present invention can be engineered to produce one or more cytokine, antibody, or inhibitor-type drug molecules, such as, but not limited to, ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a. Additionally or alternatively, the cells can produce, or can be engineered to produce, one or more therapeutic protein, peptide, or hormone molecules, such as, but not limited to, Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or the 23aa peptide (P42). In some variations, the secreted therapeutic protein or peptide may be anti-inflammatory. Furthermore, in some variations, one or more proteins secreted by the cell population may be a fusion protein. Cell engineering methods are described in further detail herein and generally include any viral, non-viral, CRISPR-Cas9, or TALEN-transfection / gene delivery (AAV and similar)-mediated gene delivery method. Furthermore, in some variations, cells for use in the present invention may produce, or be engineered to produce, growth factors, interferons, interleukins, chemokines, monokines, hormones, angiogenic factors, drugs, and / or antibiotics.

[0042] Any of a variety of cell culture media capable of supporting the growth of one or more cell types or cell lines can be used to grow and / or maintain the cells of the present invention, including complex media and / or serum-free culture media. Typically, cell culture media contain buffers, salts, energy sources, amino acids (e.g., natural amino acids, unnatural amino acids, etc.), vitamins, and / or trace elements. Cell culture media may optionally contain a variety of other components, including, but not limited to, carbon sources (e.g., natural sugars, unnatural sugars, etc.), cofactors, lipids, sugars, nucleosides, animal-derived components, hydrolysates, hormones, growth factors, surfactants, indicators, minerals, activators of specific enzymes, inhibitors of activators of specific enzymes, enzymes, organic substances, and / or small molecule metabolites.

[0043] The conditions under which cells are encapsulated within the matrix can be optimized to maximize cell viability. For example, cell viability can be increased if the matrix contains a low polymer concentration. Cell culture medium conditions (e.g., pH, ionic strength, nutrient availability, temperature, oxygen availability, osmolality, etc.) can also be adjusted and / or modified to maximize cell viability within the matrix. Cell viability can be measured by monitoring one or more indicators, including, but not limited to, intracellular esterase activity, cell membrane integrity, metabolic activity, gene expression, and protein expression. For example, live cells exposed to a fluorescent esterase substrate (e.g., calcein AM) fluoresce green as a result of intracellular esterase activity that hydrolyzes the esterase substrate to a green-fluorescent product. As another example, dead cells exposed to a fluorescent nucleic acid stain (e.g., ethidium homodimer-1) fluoresce red because their cell membranes are compromised, allowing penetration of the high-affinity nucleic acid stain.

[0044] As shown in FIG. 1 , implantable matrix 100 can optionally include one or more additives 106 that aid in integration of the implantable matrix at the implantation site, growth of cells within the implantable matrix, and / or local therapy for treating hemophilia A or similar disorders. Additives 106 can be dispersed on or within matrix 100 during the manufacturing process. Additionally, cells 104 can secrete or be engineered to secrete additives 106. Some non-limiting examples of additives 106 include hemostatic agents, growth factors, interferons, interleukins, chemokines, monokines, organoids, hormones, angiogenic factors, drugs, crosslinking agents, enzymes, antibiotics, bioelectronics, organic filler particles, and / or inorganic filler particles. For example, matrix 100 can be supplemented with angiogenic growth factors, such as VEGF, PDGF, HGF, or FGF, to promote angiogenesis in implantable matrix 100. In yet another example, matrix 100 may be loaded with inorganic and / or organic fillers to improve its mechanical properties. In some variations, matrix 100 may be supplemented with any suitable Fas receptor-activating molecule, such as Fas ligand (e.g., CD178), to induce immune privilege for cells within the matrix. In some variations, additive 106 may be encapsulated in a biomolecule that distributes throughout matrix 100 to modulate its mechanical properties and / or the release of additive 106 in vivo. For example, the release rate of a biomolecule-encapsulated additive from the matrix substrate may be predicted and / or controlled via enzymatic degradation of the capsule. As another example, additive 106 may include a cleavable crosslinker or enzyme to aid in the degradation of matrix 100 at the implantation site. As yet another example, additive 106 may be a bioelectronic additive that can be used to stimulate cellular secretion or distribution of other additives from matrix 100.

[0045] Section 3: Fabrication of implantable matrices Generally, the process for creating an implantable matrix can include engineering cells to produce one or more pharmaceutical agents of interest and creating a three-dimensional matrix in which the engineered cells populate. In some variations, one or more tissue substrates can also be seeded with drug-producing cells to protect the pharmaceutical-producing cells from an immune response. In some variations, one or more matrix substrates can also be supplemented with one or more additives described herein.

[0046] Activation of cell secretion The cells can be genetically engineered to produce one or more drugs for treating chronic diseases. Cellular drug secretion can treat chronic disease patients systemically and / or locally at the implantation site. In some variations, the cells can be engineered to additionally produce one or more of the additives described herein. In some variations, multiple cell populations within the implantable matrix can each produce one or more drugs and / or one or more additives. The engineered cells can produce drugs continuously or via gene promoter or light-induced initiation, such as demeclocycline and minocycline, cumate, tamoxifen (or other estrogen receptor drugs), tetracycline or its derivatives (e.g., doxycycline). Any viral, non-viral, CRISPR-Cas9, or other suitable gene editing technology can be used to engineer cells for drug secretion. For example, gene targeting reagents derived from the CRISPR-Cas9 system can be used to target one or more genes that control the production of the drug of interest. Gene targeting reagents derived from the CRISPR-Cas9 system can include Cas9 or dCas9 fused to a viral transcription activation domain, and the transcriptional efficacy of gene targeting reagents derived from the CRISPR-Cas9 system can be enhanced with a synergistic activation module, including, for example, HSF1 and / or p65. In some variations, gene editing technology can upregulate the transcription of genes involved in drug production, thereby overexpressing the drug produced by cells. Such technology can involve, for example, a doxycycline-inducible overexpression system. In some variations, the upregulation of target genes can be single or multiple. In some variations, the activation of a cell population involves the stimulation of des-amino-D-arginine vasopressin (DDAVP), which can lead to cellular secretion of FVIII. Furthermore, in some variations, bioelectronics can be used to stimulate the diffusion of drugs from an implantable matrix.

[0047] cultured cells An effective amount of high-quality cells is important for use in therapeutic applications such as local factor replacement therapy. Cells for use in the present invention can generally be cultured in a preferred artificial environment to produce larger cell populations. Cells according to the present invention can be grown and / or maintained using any of a variety of cell culture media capable of supporting the growth of one or more cell types or cell lines, including complex media and / or serum-free culture media. Cells can be cultured until confluence is reached, which may take from about 24 hours to about one month. For example, cells can be cultured for about two days, about one week, about two weeks, or about three weeks. In some variations, the cell culture process can be repeated multiple times. The cell culture can be a two-dimensional or three-dimensional culture. In some variations, the cell culture can be a cell suspension. Agitation of the cell suspension can involve the use of a magnetic stirrer in a culture flask or a rotary spinner flask. Cell culture media may contain buffers (e.g., boric acid, ammonium carbonate, calcium carbonate, sodium carbonate, citric acid, glycine, Tris / glycine, ammonium phosphate, potassium phosphate, sodium phosphate, etc.), salts, energy sources, amino acids (e.g., natural amino acids, unnatural amino acids, etc.), vitamins, and / or trace elements. Cell culture media may optionally contain various other components, including, but not limited to, carbon sources (e.g., natural sugars, unnatural sugars, etc.), cofactors, lipids, sugars, nucleosides, animal-derived components, hydrolysates, hormones, growth factors, surfactants, indicators, minerals, activators of specific enzymes, inhibitors of activators of specific enzymes, enzymes, organic matter, and / or small molecule metabolites. Typically, cell culture and environmental factors, such as pH, temperature, and carbon dioxide concentration, may be optimized in consideration of the cell line being cultured. In some variations, the cell suspension may be diluted to obtain the desired cell seeding density. In some variations, the cultured cells may be immortalized to prevent senescence using techniques such as vinyl chloride immortalization with genes such as SV40 T antigen or human telomerase. In some variations, the cells may be cryopreserved after culture and regenerated (e.g., thawed) before combining with the matrix.Generally, the cell culture can be sampled and centrifuged to obtain a cell pellet that can be mixed with the precursor solution before processing. Typically, the cell culture can be centrifuged for about 1 minute to about 20 minutes, such as about 5 minutes to about 10 minutes. In some variations, the cell culture can be centrifuged and resuspended in fresh growth medium as needed throughout the cell culture process.

[0048] Matrix fabrication In variations where the implantable matrix comprises a three-dimensional tissue in which cells are populated, methods for producing a cell-populated matrix may generally involve mixing hydrogel monomers with a cell suspension before polymerizing the hydrogel, or pipetting cells from culture medium onto the surface of a three-dimensional polymer network, and transferring the cell-loaded matrix to a growth medium.

[0049] Typically, chemical and / or mechanical tissue decellularization can be used to create tissue matrices. In some variations, blood vessels, skin, or tendons can be decellularized to create tissue matrices. For example, regenerative vascular conduits can be decellularized to create decellularized tissue matrices (e.g., human acellular blood vessels (HAVs)). The decellularization process generally includes freezing and thawing the tissue, exposing the tissue to chemical agents, cleaning the tissue, and sterilizing the tissue. In some variations, the decellularization and sterilization steps can be simultaneous. The complexity and length of the decellularization protocol can be proportional to the degree of geometric and biological preservation desired for the post-processed tissue (e.g., macrostructure, ultrastructure, matrix and basement membrane proteins, growth factors, etc.). Typically, tissues are decellularized to a degree that avoids eliciting a pro-inflammatory response (e.g., M1 macrophage phenotype), meaning that DNA, phospholipids, and / or mitochondrial material are present in sufficiently low concentrations or amounts in the resulting tissue. In some variations, the chemical agent to which the tissue is exposed may include one or more of an acid, a base, a solvent (e.g., acetone, alcohol, etc.), a detergent, a hypotonic solution, or a hypertonic solution. In some variations, the tissue may be further exposed to a biological agent such as an enzyme (e.g., nuclease, trypsin, dispase, chelating agent, etc.). In some variations, the decellularization process further includes mechanically removing unwanted tissue layers, applying pressure to the tissue, and / or electroporating the tissue.

[0050] In some variations, shaping the decellularized tissue substrate for use in the present invention may involve cutting a tissue sheet. Particularly useful tissue substrate shapes may include rectangular patches and tubular sleeves. More non-limiting examples of shapes suitable for tissue substrates may include circles, triangles, ellipses, or irregular shapes.

[0051] A method 600 for preparing HAVs for use in an implantable matrix is ​​shown in FIG. 6. In step 601, HAVs 602 are cut along their longitudinal axis to create flat patches 604. The HAVs 602 can have lengths of about 1 mm to about 10 cm, e.g., about 2 cm to about 8 cm, or about 3 cm to about 7 cm. Cutting the HAVs 602 can involve mechanical cutting, thermal ablation, radiofrequency ablation, cryoablation, another suitable cutting means, or a combination thereof. The patches 604 can have widths 608 approximately equal to the circumference of the HAVs 602, e.g., about 1 cm to about 3 cm, or about 1.5 cm to about 2.5 cm. In step 603, rectangular windows are cut in the tissue patch 604 to create tissue frameworks 606 containing an empty area in their centers. In variations in which the tissue matrix comprises an artificial acellular matrix, the precursor solution can be poured into a frame-shaped mold to create the tissue matrix frame.

[0052] In some variations, the implantable matrix can include a matrix attached to a tissue substrate. For example, the implantable matrix can include a hydrogel substrate and a tissue substrate. FIG. 7 generally illustrates a method 700 for attaching a hydrogel to a tissue frame. In step 701, a frame 705 and a hydrogel precursor solution 711 are poured into a mold 710. In step 703, the contents of the mold are subjected to treatment with a heat source 714 so that the precursor solution forms a hydrogel matrix substrate. The resulting hydrogel 702 can be bonded to the tissue frame 705 by any treatment method suitable for crosslinking / polymerizing the precursor solution 711. For example, the frame 705 can include porous collagen, and the precursor solution can include fibrinogen and thrombin. After incubation, the precursor solution 711 can form a fibrin hydrogel that adheres to the porous collagen tissue frame 705 via polymerization.

[0053] Generally, a hydrogel substrate can be formed through the preparation of a precursor solution (eg, a dissolved matrix) and subsequent processing of the precursor solution.

[0054] The percentage of polymer precursor and / or polymer in the precursor solution can be a percentage that allows for the formation of a matrix via the processes described herein. In some variations, the concentration of the polymer and / or polymer precursor in the precursor solution can be from about 0.01 units / mL to about 1000 units / mL, such as from about 0.1 units / mL to about 500 units / mL, from about 1 unit / mL to about 100 units / mL, from about 10 units / mL to about 80 units / mL, or from about 20 units / mL to about 60 units / mL. For example, the concentration of the polymer and / or polymer precursor in the precursor solution can be from about 0.01 mg / mL to about 1000 mg / mL, such as from about 0.1 mg / mL to about 500 mg / mL, from about 1 mg / mL to about 100 mg / mL, from about 10 mg / mL to about 80 mg / mL, or from about 20 mg / mL to about 60 mg / mL. As another example, the concentration of thrombin in the precursor solution can be from about 0.1 mg / mL to about 100 mg / mL, and the concentration of fibrinogen in the precursor solution can be from about 3 mg / mL to about 100 mg / mL. Similarly, the percentage of polymer precursor and / or polymer in the precursor solution can range from about 1 wt.% to about 60 wt.%, about 1 wt.% to about 50 wt.%, about 1 wt.% to about 40 wt.%, about 5 wt.% to about 30 wt.%, about 5 wt.% to about 20 wt.%, or about 5 wt.% to about 10 wt.%. In some variations, the percentage of polymer precursors and / or polymers in precursor solutions suitable for forming hydrogels according to the present invention can be about 1 wt / w%, about 2 wt / w%, about 3 wt / w%, about 4 wt / w%, about 5 wt / w%, about 6 wt / w%, about 7 wt / w%, about 8 wt / w%, about 9 wt / w%, about 10 wt / w%, about 20 wt / w%, about 30 wt / w%, about 40 wt / w%, about 50 wt / w%, about 60 wt / w%, or more. In some variations, the percentage of polymer precursors and / or polymers in precursor solutions suitable for forming hydrogels according to the present invention can be approximately 5 wt / w%. Generally, the absorption capacity of the hydrogel matrix increases with increasing polymer concentration in the precursor solution.

[0055] In some variations, the precursor solution may include a buffer such as boric acid, ammonium carbonate, calcium carbonate, sodium carbonate, citric acid, glycine, Tris / glycine, ammonium phosphate, potassium phosphate, sodium phosphate, or any other suitable buffer. In some variations, the precursor solution may include a dispersant. In some variations, the precursor solution may include one or more stabilizers such as calcium carbonate, calcium bisulfite, calcium citrate, calcium phosphate, calcium chloride, magnesium chloride, magnesium sulfate, calcium gluconate, or magnesium gluconate. In some variations, the prepared precursor solution may include a first polymer and / or polymer precursor and a second polymer and / or polymer precursor, and the concentrations of the first and second polymers and / or polymer precursors may be different. In some variations, the precursor solution may include multiple polymers and / or polymer precursors, each having a different concentration. In some variations, the precursor solution may include two or more precursor solutions. In some variations, each of the prepared multiple precursor solutions may include a different polymer precursor and / or polymer, or different concentrations of polymer precursor and / or polymer.

[0056] The precursor solution can be mixed with cells before processing. Generally, the prepared precursor solution can be kept on ice or in a temperature-controlled environment before and / or during mixing with the cells. Cells can be seeded into the precursor solution using any suitable method. For example, a pipette can be used to transfer the cells into a flask containing the precursor solution. The contents of the flask can then be pipetted up and down to mix the solution to facilitate seeding. In some variations, the precursor solution can be sterilized before mixing with the cells. The sterilization process can include filtration techniques such as membrane filtration, Seitz filtration, sintered glass filtration, or candle filtration. In some variations, two or more separate precursor solutions can be independently filtered and then mixed. In an alternative variation, the hydrogel substrate can be seeded with cells after processing, or before or after processing.

[0057] Generally, the percentage of cells in the precursor solution can be a percentage that allows for the formation of a matrix according to the present invention. In some variations, the percentage of cells in the precursor solution can be about 0.1 w / w% to about 80 w / w%, about 1.0 w / w% to about 50 w / w%, about 1.0 w / w% to about 40 w / w%, about 1.0 w / w% to about 30 w / w%, about 1.0 w / w% to about 20 w / w%, about 1.0 w / w% to about 10 w / w%, about 5.0 w / w% to about 20 w / w%, or about 5.0 w / w% to about 10 w / w%. In some variations, the percentage of cells in a precursor solution suitable for forming a matrix according to the present invention can be approximately 5 w / w%. In some variations, the concentration of cells in a precursor solution suitable for forming a matrix according to the present invention can be about 1 x 10 5 cells / mL~1×10 8 cells / mL, or approximately 1 x 10 6 cells / mL~1×10 7 The cell density may be in the range of cells / mL.

[0058] The conditions under which cells are encapsulated within the hydrogel can be optimized to maximize cell viability. For example, cell viability can be increased if the matrix contains a low polymer concentration. Cell culture medium conditions (e.g., pH, ionic strength, nutrient availability, temperature, oxygen availability, osmolality, etc.) can also be adjusted and / or modified to maximize cell viability within the matrix. Cell viability can be measured by monitoring one or more indicators, including, but not limited to, intracellular esterase activity, cell membrane integrity, metabolic activity, gene expression, and protein expression. For example, live cells exposed to a fluorescent esterase substrate (e.g., calcein AM) fluoresce green as a result of intracellular esterase activity that hydrolyzes the esterase substrate to a green-fluorescent product. As another example, dead cells exposed to a fluorescent nucleic acid stain (e.g., ethidium homodimer-1) fluoresce red because their cell membranes are compromised, allowing penetration of the high-affinity nucleic acid stain.

[0059] The precursor solution or cell-loaded precursor solution can be poured into a mold so that upon processing, an implantable matrix is ​​formed in the shape of the mold. The mold can be any suitable three-dimensional shape, such as a rectangular prism, a hexagonal prism, a pentagonal prism, a cylinder, etc. The mold can be comprised of any material. In some variations, the maximum dimension of the mold can be about 1 μm to about 10 m. For example, the maximum dimension of the mold can be about 100 μm to about 1 m, about 1 mm to about 10 cm, or about 100 mm to about 1 cm. In some variations, the mold can be treated before use to modify its surface properties. For example, the mold can be made hydrophilic via plasma cleaning, chemical derivatization of the surface, or any other suitable method. In some variations, the mold can be treated with a bioreagent, such as a surfactant.

[0060] In some variations, the implantable matrix can be formed via three-dimensional printing or electrospinning. For example, the cell-loaded hydrogel can be used as ink in a three-dimensional printer to print the implantable matrix. As another example, a three-dimensional printer can be used to deposit a cell-seeded hydrogel adhesive onto a substrate.

[0061] The precursor solution or cell-loaded precursor solution can be processed to form an implantable matrix. Processing of the precursor solution can be achieved by physical or chemical methods and can be controlled by various environmental factors, such as temperature, pH, and / or the addition of chelating ions. In some variations, the precursor solution can include one or more enzymes to aid processing. For example, the precursor solution can contain fibrinogen and thrombin, which are important for the enzymatic polymerization of fibrinogen to form a fibrin hydrogel. In some variations, processing of the precursor solution can include thermal curing. For example, the precursor solution can be processed by incubation in a temperature-controlled environment for an appropriate period of time. The temperature-controlled environment can have a temperature of about -15°C to about 100°C, such as about 0°C to about 60°C, about 15°C to about 50°C, or about 30°C to about 40°C. For example, a precursor solution containing thrombin and fibrinogen can be incubated at a temperature of about 37°C. The predetermined time period can be from about 1 second to 10 days, e.g., from about 30 seconds to about 10 hours, from about 10 minutes to about 12 hours, or from about 15 minutes to about 1 hour. For example, a precursor solution containing thrombin and fibrinogen can be incubated for about 20 minutes. As another example, a hydrogel adhesive precursor solution can be allowed to cure for several hours to several days to establish strong cohesion and interfacial adhesion. In some variations, treating the precursor solution can include photopolymerization in the presence of a photoinitiator via exposure to ultraviolet (UV) light. In some variations, the treatment process can include irradiation with beta, gamma, or X-ray radiation. In some variations, treating can include subjecting the precursor solution to repeated freeze-thaw cycles. In some variations, treating can include using chemicals such as hydrocarbon dialdehyde or dihalide derivatives.

[0062] In some variations, fabricating a hydrogel matrix can include depositing layers of hydrogel throughout a processing process. Deposition can be accomplished by any suitable method and can include any number of hydrogel layers. For example, depositing a hydrogel layer can involve adding a first precursor solution to a mold, at least partially crosslinking the first precursor solution, adding a second precursor solution to the mold, and at least partially crosslinking the deposited solution in the mold.

[0063] In some variations, fabricating the implantable matrix can include depositing a hydrogel matrix onto a mesh matrix. A general method for depositing a hydrogel onto a mesh substrate can include pouring a hydrogel precursor solution onto the mesh substrate in a mold and then treating the contents of the mold to adhere the two substrates. Typically, the hydrogel can gel around the mesh. In some variations, prior to step 1, the mesh can be treated with molecules that covalently bond to both the mesh substrate and the hydrogel, resulting in a strong bond between the two. Molecules used to treat the mesh can include, for example, crosslinking molecules (e.g., silanes or silane coupling agents, such as (3-aminopropyl)triethoxysilane (APTES) and 3-(trimethoxysilyl)propyl methacrylate (TMSPMA)). Alternatively, the mesh substrate can be primed with a mixture including a photoinitiator (e.g., benzophenone), and treatment can involve curing the precursor solution with UV light, resulting in grafting of hydrogel polymer chains onto the mesh network. In some variations, a precursor solution may be painted onto the mesh. Painting can be accomplished using any suitable painting technique. The hydrogel solution may include a copolymer of hydrogel monomers and a coupling agent and may behave like a viscous liquid or a regular paint. The functional groups on the coupling agent may have tunable kinetics of interacting with each other and with complementary functional groups on the mesh substrate to harden the hydrogel paint and form strong bonds after application of the hydrogel paint to the mesh.

[0064] In some variations, injectable hydrogel matrices can be stabilized by physical crosslinks, including hydrogen bonding, hydrophobic interactions, ionic interactions, ligand-ion coordination, dipole-dipole interactions, and host-guest complexes. Physically crosslinked hydrogels typically provide a benign environment for cells and bioactive molecules. They generally exhibit relatively low mechanical strength, and their morphology and properties are easily altered by external stimuli. On the other hand, chemically crosslinked hydrogels formed by irreversible covalent bonds have demonstrated relatively high mechanical strength and stability. In some variations, injectable hydrogel matrices are stabilized by physical crosslinks and can be further strengthened by limited chemical crosslinking (e.g., Diels-Alder reaction, Michael addition, Schiff base reaction, enzyme-mediated, photopolymerization, etc.). For example, complexing agents such as cyclodextrins can be added to the hydrogel precursor solution to bind to the polymer precursor / polymer via host-guest interactions, followed by UV-initiated polymerization of the complexing agent to strengthen the hydrogel network. In some variations, the injectable hydrogel substrate may be further treated by an external stimulus after the implantable matrix is ​​injected into a user. The external stimulus may include temperature, pH, light, electric / magnetic fields, ultrasound, biomolecular species such as enzymes, etc. For example, the user's body temperature may induce further crosslinking of the implantable matrix within the user's body.

[0065] In some variations, the cell-populated hydrogel matrix can be a tubular support graft (i.e., a tubular hydrogel matrix) that can be concentrically positioned over a tubular vascular graft (i.e., a tubular tissue substrate). FIG. 10 shows a tubular support graft 1000 including a cell-seeded hydrogel matrix 1002. The hydrogel matrix 1002 is tubular and formed between a mold mandrel rod 1004 and a mold 1006. FIG. 13 shows a series of cross-sectional views of the tubular support graft during the manufacturing process. In step 1301, the mandrel rod 1304 is inserted into the mold 1306, and then the mixture of hydrogel precursor solution and cells 1302 is poured into the mold 1306. Non-limiting examples of materials for the mold can include glass, silicone, plastic, and metal. Typically, the mold 1306 can be treated prior to use, for example, with a Pluronic® treatment. The mandrel rod 1304 may have an outer diameter of about 1 mm to about 10 cm, such as about 2 mm to about 1 cm, about 3 mm to about 50 mm, about 4 mm to about 10 mm, or about 5 mm to about 8 mm. In some variations, a plug may be used to seal a first end of the mold 1306 to the central mandrel rod 1304 inside the mold 1306 before the precursor solution 908 is poured into the mold 1306. The precursor solution and cell mixture 1302 may include, for example, fibrinogen, thrombin, calcium, and cells. In some variations, a plug may be used to seal a second end of the mold 1306 to the central mandrel 1304 completely inside the mold 1306. In step 1303, mold 1306 and its contents are subjected to heat (e.g., incubated) from heat source 1310 to polymerize precursor solution 1302 and produce a tubular cell mass hydrogel (tubular support graft) 1302. The incubation process can take from about 1 minute to about 1 hour, such as from about 5 minutes to about 1 hour, or from about 15 minutes to about 20 minutes. Incubation can be performed in a temperature-controlled environment having a temperature of 20°C to about 40°C, e.g., from about 25°C to about 38°C, from about 22°C to about 40°C, or from about 30°C to about 37°C.In step 1305, the mandrel rod 1304 is removed from the mold 1306, releasing the polymerized tubular support graft 1302 from the mold. In some variations, the tubular support graft 1302 may be transferred to a final mandrel, which may be larger than the mandrel rod 1304 used during the initial manufacture of the tubular support graft 902. For example, the initial support mandrel 1304 may have an outer diameter of about 7 mm, and the final support mandrel may have an outer diameter of about 8 mm.

[0066] In alternative embodiments, tubular support grafts and / or tubular vascular grafts may be fabricated by electrospinning a tubular matrix followed by cell seeding and / or via 3D printing or bioprinting, pouring, molding, flat cell culture, folding / suturing, etc.

[0067] For example, a tubular support graft can be fabricated by three-dimensionally printing a bioink 808 containing cells 810 (and optionally additives 806) onto a support mandrel 804. FIG. 8 shows a series of cross-sectional views of a method 800 for manufacturing a tubular support graft. In step 801, a tubular support graft 802 (i.e., a tubular cell-populated hydrogel) is deposited onto a support mandrel rod 804, the mandrel 804 is rotated at a predetermined speed, and a hydrogel adhesive (bioink) 808 containing cells 810 (and optionally additives 806) is deposited onto the rotating mandrel 804. The support mandrel 804 can have an outer diameter of about 1 mm to about 10 cm, e.g., about 2 mm to about 1 cm, about 3 mm to about 50 mm, about 4 mm to about 10 mm, or about 5 mm to about 8 mm. The predetermined speed can be, for example, about 1 rpm to about 1,000 rpm, such as about 10 rpm to about 200 rpm, about 30 rpm to about 150 rpm, or about 50 rpm to about 100 rpm. Non-limiting examples for depositing the hydrogel adhesive 808 can include pipetting, pouring, or three-dimensional printing the cell-loaded hydrogel adhesive onto the surface of the mandrel. In some variations, three-dimensional printing the cell-loaded hydrogel adhesive can include using a three-dimensional bioprinter loaded with the cell-loaded hydrogel adhesive. In step 803, the tubular support graft 814 is cured by a heat source 812. In step 805, the tubular support graft 814 is removed from the support mandrel 804 and stored until use. In some variations, the tubular support graft 814 may be stored on a final support mandrel that may be larger than the support mandrel 804 used during the initial manufacture of the tubular support graft, which may facilitate concentric placement of the tubular support graft around the tubular vascular graft (i.e., tubular tissue substrate). Generally, prior to implantation of the tubular support graft and the implantable matrix comprising the tubular vascular graft, the tubular support graft may be placed concentrically around the tubular vascular graft prior to implantation of the construct.

[0068] 9 illustrates a method of disposing a tubular support graft around a tubular vascular graft. In step 901, a final tubular support mandrel 910, over which a tubular support graft 907 is placed, contacts a vascular mandrel 911, over which a tubular vascular graft 902 is placed. The tubular support graft 907 may include drug-secreting cells 904 and, optionally, an additive 906. In some variations, the vascular mandrel 911 may be inserted at least partially within the lumen of the final tubular support mandrel 910, such that the tubular support graft 907 is placed over the tubular vascular graft 902. In step 903, the tubular support graft 907 is disengaged from the final support mandrel and slid over and across the outer surface of the tubular vascular graft 902, resulting in an implantable matrix of drug-producing biological vessels.

[0069] Any one of the aforementioned matrix substrates may be loaded with additives as described herein. Generally, loading or loading a matrix may involve immersing the matrix in an aqueous solution containing the additive or dispersing the additive in a precursor solution. For example, an implantable matrix may be loaded with tissue thromboplastin to promote clotting at the implantation site. As another example, an implantable matrix may be loaded with angiogenic growth factors, such as VEGF, PDGF, HGF, or FGF, to promote vascularization within the matrix. In some variations, the release rate of the loaded additive may be determined by the diffusion of the additive into the tissue surrounding the implantation site and may be accelerated using a biodegradable hydrogel matrix. For example, the drug release rate may be tailored by tailoring molecular interactions (e.g., covalent bonds, electrostatic interactions, hydrophobic interactions) between the hydrogel and one or more loaded additives. In some variations, the mechanical properties of the matrix may be adjusted by loading the matrix with inorganic and / or organic fillers. Filler particles may be retained within the implantable matrix via physical and / or chemical bonds or by mechanical immobilization. The physical or chemical interaction between the polymer matrix and the filler particles can result in a more stable composite system.

[0070] Implantable matrix culture The implantable matrix may be stored in growth medium to maintain and allow cell proliferation until implantation. In some variations, cell culture medium may be placed on the implantable matrix within the mold. In some variations, an implantable matrix fabricated using a mold may be removed from the mold and transferred into a separate container containing cell culture medium. In some variations, only a portion of the implantable matrix may be cultured after fabrication. For example, an implantable matrix may include a tubular cell population support graft and a tubular vascular graft, but only the tubular support graft may require culture until implantation. In this example, the cultured tubular support graft may be attached to or slid onto the tubular vascular graft immediately prior to implantation.

[0071] Section 4: Embedding Embeddable Matrices The following provides an overview of various aspects of the use of implantable matrices.

[0072] As described above, the implantable matrix can be attached to a user's implantation site of interest so that the seeded cells can secrete a medicinal agent to treat a chronic condition systemically within the patient's body and / or locally at the implantation site. A general method of matrix implantation can include surgically accessing the implantation site and attaching the matrix to the implantation site. Any suitable surgical procedure, including invasive and minimally invasive surgical techniques, can be used to access the implantation site. In some variations, more than one implantation site can be accessed within a patient. The implantation site can include at least a portion of a tendon, organ (e.g., stomach, intestine), muscle, joint (e.g., knee, hip, elbow, wrist), blood vessel, limb (e.g., arm, leg), or any internal site in the patient requiring treatment. Generally, the implantable matrices described herein can be sutured, glued, injected, or attached to the implantation site by any other suitable means.

[0073] In some variations, the adhesive used to attach the implantable matrix to the implantation site may be a biodegradable and absorbable agent such as fibrin glue or a cyanoacrylate (eg, cyanoacrylic acid).

[0074] In variations in which the implantable matrix includes a tissue matrix, the matrix can be sutured, glued, or otherwise attached to the implantation site via the tissue matrix. For example, a HAV frame surrounding the cell-loaded hydrogel matrix can be used to suture the implantable matrix to the implantation site.

[0075] 11, implantable matrix 1100 including vascular support graft 1102 can be anastomosed to one or more blood vessels (e.g., artery 1104, vein 1106) of a patient so that medicinal agents secreted by the cells are in proximity to the patient's bloodstream and can easily circulate throughout the patient's body for systemic therapy. For example, cells can be seeded in or on coating 1108 and engineered to secrete IFN-beta-1a and / or ocrelizumab to systemically treat multiple sclerosis in the patient.

[0076] In variations in which the implantable matrix includes an injectable hydrogel matrix, the implantable matrix can be injected into the implantation site. In some variations, the injectable implantable matrix can be delivered to an irregularly shaped site. In some variations, the injectable implantable matrix can conform to the shape of the user's implantation site during and / or after implantation. In some variations, the implantation site can include a cartilaginous joint or a synovial joint. In some variations, injecting the implantable matrix with the injectable hydrogel matrix can be a minimally invasive procedure. In some variations, injecting the implantable matrix with the injectable hydrogel matrix can involve using a syringe.

[0077] In variations in which the implantable matrix includes a functionalized tubular support graft and a tubular vascular graft, the functionalized tubular support graft may be attached to or slid onto the tubular vascular graft immediately prior to implantation. In some variations, the tubular support graft may surround the tubular vascular graft. In some variations, the combination of the tubular support graft and the tubular vascular graft may be polymerized via any suitable polymerization mechanism prior to implantation.

[0078] Any number of implantable matrices containing drug-secreting cells treating one or more chronic diseases can be implanted at any number of implantation sites within a user's body. More than one type of attachment mechanism can be used to attach the implantable matrix to the implantation site. In some variations, more than one implantable matrix can be implanted per site. For example, as shown in FIGS. 12A-12C , multiple implantable matrices 1200 can be attached to implantation sites of interest, including vertebrae 1202, intestine 1204, and joint lining 1206. A therapeutically implantable matrix for implantation into vertebrae 1202 and / or joint lining 1206 can treat chronic inflammation with seeded anti-inflammatory drug-producing cells. A therapeutically implantable matrix for implantation in and / or on the intestine can treat Crohn's disease with seeded infliximab- and / or vedolizumab-producing cells.

[0079] In variations in which the implantable matrix comprises a hydrogel adhesive, the implantable matrix may be adhered to the implantation site without the use of an additional attachment mechanism.

[0080] One or more surgical tools may be used during implantation. Exemplary surgical tools include, but are not limited to, scissors, blades, forceps, clamps, needles, suction tubes, clips, cameras, cauteries, wrenches, depth probes, retractors, gauge indicators, and chisels. Additionally, one or more delivery devices may be used to implant the implantable matrix. The delivery device may generally contain or hold the implantable matrix during implantation. Some non-limiting examples of delivery devices include syringes, tubes, and clips. In some variations, the delivery device may be externally operable. For example, a clip may hold the implantable matrix as it is delivered to the implantation site and may be activated by a handle that remains outside the patient's body during implantation. [Example]

[0081] Section 5: Examples Example 1: Direct coating of hydrogels seeded with drug-producing cells onto vascular grafts Human fibrinogen powder was dissolved in PBS or culture medium at 37°C for 1-2 hours until the solution was clear and at the desired fibrinogen concentration of approximately 40.9 mg / mL. The fibrinogen solution was then sterile filtered in a biosafety cabinet. Sterile calcium chloride solution was added to the fibrinogen solution, and the mixture was placed on ice. Human plasma-derived thrombin solution was also placed on ice in a separate conical tube. Drug-producing cells were harvested from the culture flask and centrifuged at 200 rcf for 3 minutes. The supernatant was discarded, and the island-like pellet was gently resuspended in the fibrinogen solution on ice. While on ice, the thrombin solution was added to the fibrinogen solution, and the fibrinogen solution was mixed well by pipetting up and down. Next, a glass tubular mold with an inner diameter of 8 mm was capped at one end with a silicone plug and filled with sterile distilled water containing Pluronic® F127. A Pluronic® treatment was applied for 30 minutes, and the mold was briefly washed with distilled water. A cylindrical mandrel with an outer diameter of 5 mm was inserted into the lumen of the HAV vascular graft. The vascular graft with the mandrel in its lumen was inserted into a glass tubular mold. A mixture of cells, fibrinogen, CaCl2, and thrombin was poured into the space between the outer surface of the vascular graft and the glass mold. The entire system was placed in a sterile, closed container and incubated at 37°C for 15-20 minutes to allow the mixture to polymerize. After polymerization, the vascular graft with drug-producing cells bearing the hydrogel coating was removed from the mold and transferred to a bottle containing the desired culture medium.

[0082] Example 2: Vascular support drafts directly seeded with drug-producing cells Human fibrinogen powder was dissolved in PBS or culture medium at 37°C for 1-2 hours until the solution was clear and at the desired fibrinogen concentration of approximately 40.9 mg / mL. The fibrinogen solution was then sterile filtered in a biosafety cabinet. Sterile calcium chloride solution was added to the fibrinogen solution, and the mixture was placed on ice. Human plasma-derived thrombin solution was also placed on ice in a separate conical tube. Drug-producing cells were harvested from the culture flask and centrifuged at 200 rcf for 3 minutes. The supernatant was discarded, and the island-like pellet was gently resuspended in the fibrinogen solution on ice. While on ice, the thrombin solution was added to the fibrinogen solution, and the fibrinogen solution was mixed well by pipetting up and down. Next, a glass tubular mold with an inner diameter of 8 mm was capped at one end with a silicone plug and filled with sterile distilled water containing Pluronic® F127. The Pluronic® treatment was applied for 30 minutes, and the mold was briefly washed with distilled water. A mandrel rod with an outer diameter of 7 mm was inserted into the glass mold. One end of the mold was sealed using a piece of silicone tubing between the glass mold and the mandrel rod, and the mandrel mold was centered inside the mold. The drug-producing cell / fibrinogen / CaCl2 / thrombin mixture was quickly poured into the space between the glass mold and the mandrel rod. The second end of the glass mold was sealed, and the mandrel rod was perfectly centered with the second segment of silicone tubing. The mold and its contents were transferred to a sterile closure tray and incubated at 37°C for 15-20 minutes to polymerize the fibrin gel. After polymerization, the fibrin sleeve containing the drug-producing cells was removed from the mold by removing the mandrel rod. The drug-producing cell-loaded fibrin sleeve was then transferred to a new mandrel rod with an outer diameter of 8 mm by first gently sliding it from the original 7 mm mandrel to the 8 mm mandrel. Transferring the sleeve to a larger mandrel facilitates sliding the sleeve onto the HAV, which has an outer diameter of approximately 7 mm. The 8 mm mandrel carrying the sleeve was then transferred to a flask of culture medium and stored at 4°C until implantation of the sleeve.

[0083] Example 3: Attachment of hydrogels seeded with drug-producing cells to vascular support graft frameworks Drug-producing patches can be fabricated from hydrogels populated with VWF-secreting cells. Rectangular prism-shaped molds were treated with Pluronic® F-127 for 30 minutes and then briefly washed with distilled water.

[0084] Human plasma-derived fibrinogen powder was dissolved in PBS or culture medium at 37°C for 1-2 hours until the solution was clear and had a fibrinogen concentration of approximately 40.9 mg / mL. The fibrinogen solution was then sterile filtered in a biosafety cabinet. Sterile calcium chloride solution was added to the fibrinogen solution, and the mixture was placed on ice. Human plasma-derived thrombin solution was also placed on ice in a separate conical tube.

[0085] Drug-secreting cells were harvested from the culture flask and centrifuged at 500 rcf for 5 minutes. The supernatant was discarded, and the cell pellet was gently resuspended in fibrinogen solution on ice. While on ice, thrombin solution was added to the fibrinogen solution, and the fibrinogen solution was mixed well by pipetting up and down. The cell / fibrinogen / CaCl / thrombin mixture was quickly poured into a mold. The mold and its contents were transferred to a sterile, closed tray and incubated at 37°C for 15–20 minutes to allow the fibrin gel to polymerize. After the treatment period, the fibrin gel sheet containing drug-secreting cells was removed from the mold and transferred to a cell culture flask containing culture medium.

[0086] While various inventive embodiments have been described and illustrated herein, those skilled in the art will readily conceive of numerous other means and / or structures for performing the functions and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is deemed to be within the scope of the inventive embodiments described herein. Moreover, those skilled in the art will generally recognize that any and all parameters, dimensions, materials, and configurations described herein are meant to be examples, and that the actual parameters, dimensions, materials, and / or configurations will depend on the particular application in which the inventive teachings disclosed herein are used. Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific inventive embodiments described herein. It will therefore be understood that the foregoing embodiments are presented by way of example only, and that, within the scope of the claims based on this disclosure and their equivalents, inventive embodiments may be practiced otherwise than as specifically described and claimed. The inventive embodiments of the present disclosure also cover each individual feature, system, article, material, kit, and / or method described herein. Additionally, any combination of two or more such features, systems, articles, materials, kits, and / or methods is within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0087] The embodiments disclosed herein may also be combined with one or more features, as well as complete systems, devices, and / or methods, to yield still other embodiments and inventions. Furthermore, some embodiments may be distinguishable from the prior art by specifically lacking one and / or another feature disclosed in a particular prior art reference, i.e., the claims to such embodiments are distinguishable from the prior art by including one or more negative limitations.

[0088] Also, various inventive concepts may be embodied as one or more methods. The actions performed as part of a method may be ordered in any suitable way. Thus, embodiments may be constructed to perform actions in an order other than the order described, which may include performing some actions concurrently.

[0089] As used herein, the indefinite articles "a" and "an" in the specification and claims should be understood to mean "at least one" unless clearly indicated to the contrary. The terms "can" and "may" are used interchangeably in this disclosure and indicate that a reference to an element, component, structure, feature, functionality, object, advantage, operation, step, process, apparatus, system, device, result, or description has the ability to use, include, or produce, or otherwise represent, the features set forth in the description used (or referred to) for a particular embodiment.

[0090] The phrase "and / or," as used herein and in the claims, should be understood to mean "either or both" of the elements to which it is connected, i.e., elements that are sometimes conjunctive and other times disjunctive. Multiple elements listed with "and / or" should be construed in the same manner, i.e., "one or more" of the elements connected thereto. Other elements, whether related or unrelated to those explicitly identified elements, may optionally be present other than the elements expressly identified by the "and / or" clause. Thus, as a non-limiting example, a reference to "A and / or B," when used with open-ended language such as "comprising," may refer in one embodiment to A only (which may include elements other than B), in another embodiment to B only (which may include elements other than A), and in yet another embodiment to both A and B (which may include other elements), and so forth.

[0091] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, the disjunctive items "or" or "and / or" in a list shall be interpreted as inclusive, i.e., including at least one, but also more than one, of an element or list of elements, and may also include additional unlisted items. Terms clearly indicating the contrary, such as "only one of" or "exactly one of," or, as used in the claims, "consisting of," shall refer to the inclusion of exactly one element of an element or list of elements. Generally, as used herein, the term "or" shall only be construed as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusion, such as "either," "one of," "only on of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used in the field of patent law.

[0092] As used herein in the specification and claims, the phrase "at least one" in reference to a list of one or more elements shall be understood to mean at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. Also, this definition allows for elements other than those explicitly identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to the elements explicitly identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer in one embodiment to at least one, and optionally more than one, A, with the proviso that B is absent (and may include elements other than B); in another embodiment to at least one, and optionally more than one, B, with the proviso that A is absent (and may include elements other than A); in yet another embodiment to at least one, and optionally more than one, A, and at least one, and optionally more than one, B (and may include other elements); and so forth.

[0093] In the claims and the foregoing specification, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like, are to be understood to be open-ended, i.e., to mean inclusive without limitation. Only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively, as set forth in Section 2111.03 of the U.S. Patent Office Manual of Patent Examining Procedures.

Claims

1. 1. An implantable matrix producing a pharmaceutical agent, comprising: A pharmaceutical-producing implantable matrix comprising a matrix containing at least one cell population.

2. The at least one cell population 10. The implantable matrix of claim 1, producing one or more drug molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a.

3. The at least one cell population 10. The implantable matrix of claim 1, which produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or p42.

4. 10. The implantable matrix of claim 1, wherein said at least one cell population produces interleukin-10.

5. 10. The implantable matrix of claim 1, wherein the at least one cell population produces one or more of sirolimus or tacrolimus.

6. The implantable matrix of claim 1 , wherein said at least one cell population comprises mesenchymal stem cells.

7. 10. The implantable matrix of claim 1, wherein said at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells.

8. The implantable matrix of claim 1 , wherein the matrix comprises at least one selected from the group consisting of a hydrogel and a biodegradable polymer.

9. The implantable matrix of claim 1 , wherein the matrix comprises a Fas receptor-activating molecule.

10. 10. The implantable matrix of claim 9, wherein the Fas receptor activating molecule comprises a Fas ligand.

11. The implantable matrix of claim 1 , wherein the matrix comprises a hydrogel comprising thrombin and fibrinogen.

12. 12. The implantable matrix of claim 11, wherein the thrombin is at a concentration of about 0.1 units / mL to about 100 units / mL.

13. 12. The implantable matrix of claim 11, wherein the fibrinogen is at a concentration of about 3 units / mL to about 100 units / mL.

14. 10. The implantable matrix of claim 1, wherein the matrix is ​​implanted in at least one body region of a patient selected from the group consisting of an arm, a leg, a knee, a hip, an elbow, a wrist, a spine, a stomach, a blood vessel, and an intestine.

15. The matrix of claim 1 , wherein the matrix can be formed at a desired implantation site by injecting the cells in a hydrogel adhesive solution.

16. 9. The implantable matrix of claim 8, wherein the biodegradable polymer is a polymer selected from the group consisting of polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, and poly(caprolactone).

17. 9. The implantable matrix of claim 8, wherein the hydrogel is at least one of a natural hydrogel and a synthetic hydrogel.

18. 1. An implantable matrix producing a pharmaceutical agent, comprising: a framework containing collagen; A matrix comprising at least one cell population, a matrix attached to and / or within said frame body.

19. The at least one cell population 19. The implantable matrix of claim 18, producing one or more molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a.

20. The at least one cell population 20. The implantable matrix of claim 18, which produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or p42.

21. 19. The implantable matrix of claim 18, wherein said at least one cell population produces interleukin-10.

22. 20. The implantable matrix of claim 18, wherein the at least one cell population produces one or more of sirolimus or tacrolimus.

23. 20. The implantable matrix of claim 18, wherein said at least one cell population comprises mesenchymal stem cells.

24. 20. The implantable matrix of claim 18, wherein said at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells.

25. 20. The implantable matrix of claim 18, wherein the matrix comprises at least one selected from the group consisting of a hydrogel and a biodegradable polymer.

26. 26. The implantable matrix of claim 25, wherein the biodegradable polymer is a polymer selected from the group consisting of polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, and poly(caprolactone).

27. 26. The implantable matrix of claim 25, wherein the hydrogel is at least one of a natural hydrogel and a synthetic hydrogel.

28. 20. The implantable matrix of claim 18, wherein the matrix comprises a hydrogel comprising thrombin and fibrinogen.

29. 29. The implantable matrix of claim 28, wherein the thrombin is at a concentration of about 0.5 units / mL to about 100 units / mL.

30. 29. The implantable matrix of claim 28, wherein the fibrinogen is at a concentration of about 3 units / mL to about 100 units / mL.

31. 20. The implantable matrix of claim 18, wherein the matrix is ​​implanted in at least one body region of a patient selected from the group consisting of an arm, a leg, a knee, a hip, an elbow, a wrist, a spine, a stomach, a blood vessel, and an intestine.

32. 1. A method of forming an implantable matrix that produces a pharmaceutical agent, comprising: combining at least one cell population with the dissolved matrix; pouring the combination into a frame; and polymerizing the dissolved matrix.

33. The at least one cell population 33. The implantable matrix of claim 32, producing one or more molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a.

34. The at least one cell population 33. The implantable matrix of claim 32, which produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or p42.

35. 33. The method of claim 32, wherein at least one population of said cells produces interleukin-10.

36. 33. The method of claim 32, wherein the at least one cell population produces one or more of sirolimus or tacrolimus.

37. 33. The method of claim 32, wherein the at least one cell population comprises mesenchymal stem cells.

38. 33. The method of claim 32, wherein the at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells.

39. 33. The method of claim 32, wherein the matrix comprises at least one selected from the group consisting of a hydrogel and a biodegradable polymer.

40. 40. The method of claim 39, wherein the biodegradable polymer is a polymer selected from the group consisting of polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, and poly(caprolactone).

41. 40. The method of claim 39, wherein the hydrogel is at least one of a natural hydrogel and a synthetic hydrogel.

42. 33. The implantable matrix of claim 32, wherein the matrix comprises a Fas receptor-activating molecule.

43. 43. The implantable matrix of claim 42, wherein the Fas receptor activating molecule comprises a Fas ligand.

44. 33. The method of claim 32, wherein the matrix comprises a hydrogel comprising thrombin and fibrinogen.

45. 45. The method of claim 44, wherein the thrombin is at a concentration of about 0.5 units / mL to about 100 units / mL.

46. 45. The method of claim 44, wherein the fibrinogen is at a concentration of about 3 units / mL to about 100 units / mL.

47. 33. The method of claim 32, wherein the matrix is ​​implanted in at least one body region of the patient selected from the group consisting of the knee, hip, elbow, wrist, spine, stomach, and intestine.

48. An apparatus, A tubular support graft comprising a matrix and at least one cell population, A device comprising a tubular support graft, said tubular support graft configured to be placed concentrically around a tubular vascular graft.

49. The at least one cell population 49. The device of claim 48, which produces one or more molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a.

50. The at least one cell population 49. The device of claim 48, which produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or p42.

51. 49. The device of claim 48, wherein at least one population of said cells produces interleukin-10.

52. 49. The device of claim 48, wherein the at least one cell population produces one or more of sirolimus or tacrolimus.

53. 49. The device of claim 48, wherein the at least one cell population comprises monocytes.

54. 49. The device of claim 48, wherein the at least one cell population comprises mesenchymal stem cells.

55. 49. The device of claim 48, wherein the at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells.

56. 49. The device of claim 48, wherein the matrix comprises at least one selected from the group consisting of a hydrogel and a biodegradable polymer.

57. 57. The device of claim 56, wherein the biodegradable polymer is a polymer selected from the group consisting of polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, and poly(caprolactone).

58. 57. The device of claim 56, wherein the hydrogel is at least one of a natural hydrogel and a synthetic hydrogel.

59. 49. The implantable matrix of claim 48, wherein the matrix comprises a Fas receptor-activating molecule.

60. 49. The implantable matrix of claim 48, wherein the Fas receptor activating molecule comprises a Fas ligand.

61. 49. The device of claim 48, wherein the matrix comprises a hydrogel comprising thrombin and fibrinogen.

62. 62. The device of claim 61, wherein the thrombin is at a concentration of about 0.5 units / mL to about 100 units / mL.

63. 62. The device of claim 61, wherein the fibrinogen is at a concentration of about 3 units / mL to about 100 units / mL.

64. 49. The device of claim 48, wherein the tubular support graft is formed by one of molding, casting, flat tissue culture, electrospinning, and three-dimensional printing.

65. 49. The device of claim 48, wherein the length of the tubular support graft is less than the length of the tubular vascular graft.

66. 49. The device of claim 48, wherein the length of the tubular support graft is greater than the length of the tubular vascular graft.

67. 49. The device of claim 48, wherein the length of the tubular vascular graft is between 1 cm and 100 cm.

68. An apparatus, a tubular support graft configured to be placed concentrically around the vascular graft; and A device comprising a matrix comprising at least one cell population disposed on the exterior surface of said tubular support graft.

69. 69. The device of claim 68, wherein the at least one cell population produces one or more molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a.

70. 69. The device of claim 68, wherein the at least one cell population produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or p42.

71. 69. The device of claim 68, wherein said at least one cell population produces interleukin-10.

72. 69. The device of claim 68, wherein the at least one cell population produces one or more of sirolimus or tacrolimus.

73. 69. The device of claim 68, wherein the at least one cell population comprises mesenchymal stem cells.

74. 69. The device of claim 68, wherein said at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells.

75. 69. The device of claim 68, wherein the tubular support graft comprises one or more selected from the group consisting of fibrin, collagen, alginate, gelatin, chitosan, dextran, hyaluronic acid, or PEG.

76. 69. The device of claim 68, wherein the tubular support graft comprises a combination of PEG and chitosan, PEG and gelatin, PEG and hyaluronic acid, PAM and gelatin, or PVA and gelatin.

77. 69. The device of claim 68, wherein the matrix comprises at least one selected from the group consisting of a hydrogel and a biodegradable polymer.

78. 78. The device of claim 77, wherein the biodegradable polymer is a polymer selected from the group consisting of polyglycolic acid, polylactic acid, polylactic-co-glycolic acid, and poly(caprolactone).

79. 78. The device of claim 77, wherein the hydrogel is at least one of a natural hydrogel and a synthetic hydrogel.

80. 69. The device of claim 68, wherein the matrix comprises a hydrogel comprising thrombin and fibrinogen.

81. 81. The device of claim 80, wherein the thrombin is at a concentration of about 0.5 units / mL to about 100 units / mL.

82. 81. The device of claim 80, wherein the fibrinogen is at a concentration of about 3 units / mL to about 100 units / mL.

83. 69. The device of claim 68, wherein the tubular support graft is formed by one of molding, casting, flat tissue culture, electrospinning, and three-dimensional printing.

84. 69. The device of claim 68, wherein the length of the tubular support graft is less than the length of the tubular vascular graft.

85. 69. The device of claim 68, wherein the length of the tubular support graft is greater than the length of the tubular vascular graft.

86. 69. The device of claim 68, wherein the length of the vascular graft is between 1 cm and 100 cm.

87. 1. A method of forming a functionalized tubular support graft for a vascular graft, comprising: attaching a tubular support graft to a support mandrel; rotating the support mandrel at a predetermined speed; depositing droplets of a mixture of hydrogel and at least one cell population onto the exterior surface of the tubular support graft using a three-dimensional printer; and removing said functionalized tubular support graft from said support mandrel after curing.

88. 88. The method of claim 87, wherein the at least one cell population produces one or more molecules selected from the group consisting of ocrelizumab, natalizumab, pembrolizumab, infliximab, vedolizumab, dabrafenib, lecanemab, or interferon beta-1a.

89. 88. The method of claim 87, wherein the at least one cell population produces one or more molecules selected from the group consisting of Factor VII, Factor VIII, Factor IX, Factor X, Von Willebrand factor, protein C, human albumin, human immunoglobulin, testosterone, human Htt, or p42.

90. 88. The method of claim 87, wherein said at least one cell population produces interleukin-10.

91. 88. The method of claim 87, wherein the at least one cell population produces one or more of sirolimus or tacrolimus.

92. 88. The method of claim 87, wherein the at least one cell population comprises monocytes.

93. 88. The method of claim 87, wherein the at least one cell population comprises mesenchymal stem cells.

94. 88. The method of claim 87, wherein said at least one cell population comprises major histocompatibility complex class I and II knockout mesenchymal stem cells.

95. 88. The device of claim 87, wherein the tubular support graft comprises one or more selected from the group consisting of fibrin, collagen, alginate, gelatin, chitosan, dextran, hyaluronic acid, or PEG.

96. 88. The device of claim 87, wherein the tubular support graft comprises a combination of PEG and chitosan, PEG and gelatin, PEG and hyaluronic acid, PAM and gelatin, or PVA and gelatin.

97. 88. The method of claim 87, further comprising implanting the vascular graft of the functionalized tubular support graft in the arm of a patient.

98. 88. The method of claim 87, further comprising implanting the vascular graft of the functionalized tubular support graft adjacent to an artery of the patient.

99. 88. The method of claim 87, wherein the vascular graft can comprise one or more selected from the group consisting of fibrin, collagen, alginate, gelatin, chitosan, dextran, hyaluronic acid, or PEG.

100. 88. The method of claim 87, wherein the vascular graft can comprise a combination of PEG and chitosan, PEG and gelatin, PEG and hyaluronic acid, PAM and gelatin, or PVA and gelatin.

101. 88. The method of claim 87, further comprising positioning the tubular support graft on the outer surface of the vascular graft.

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