Methods for cell delivery in decellularized heart valves using injectable hydrogels

Abstract

Provided herein are methods of preparing an engineered heart valve for implantation into a subject in need (e.g., a pediatric patient). For example, a porcine pulmonary valve can be decellularized and then recellularized with cells from the subject. Hydrogels can be used to facilitate cell delivery and growth within the sub-endothelial layer (e.g., spongiosa) of a heart valve, which is essential for long-term success of the implantation.

Description

[0001] METHODS FOR CELL DELIVERY IN DECELLULARIZED HEART VALVES USING INJECTABLE HYDROGELS

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 571,286, filed on March 28, 2024. The entire contents of the foregoing application are incorporated herein by reference.

[0004] TECHNICAL FIELD

[0005] This invention relates to the field of heart valve tissue engineering.

[0006] BACKGROUND

[0007] Diseases of the heart valves remain one of the main causes of death worldwide, due the limited treatment and replacement options. If repairing the diseased valve fails or is not possible, the only remaining option is to replace it, and the number of heart valve replacements is expected to double within the next 30 years. Replacement valves can be divided into two categories: mechanical valves and bioprosthetic valves. The mechanical valves have a high durability, however the risk of thrombosis is high, requiring a lifetime of anti-coagulation therapy. On the other hand, bioprosthetic valves are less susceptible to coagulation, but they suffer from calcification and degradation, which significantly limits their durability. Additionally, both options do not facilitate remodelling by the body or growth, a limitation of major significance in pediatric patients.

[0008] Currently, the most common valve replacements in children are cryopreserved allografts. These implants have mechanical characteristics that are close to native valves initially, but the cryopreserved allografts lose function over time. There is also a limited supply of cryopreserved allografts available at any given time, and they do not allow growth or remodelling. Pediatric patients require regular valve replacements to keep up with their body’s growth and development. It is of the utmost importance to develop a replacement valve which can grow and remodel, and thereby to eliminate the need for multiple replacement operations. SUMMARY

[0009] The present disclosure is related to methods to decellularize human heart valves (e.g., allograft heart valves) or non-human animal heart valves (e.g., porcine pulmonary valves), which can subsequently be recellularized, e.g., via injection of hydrogel (e.g., GelMA- HAMA,)-encapsulated stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells), with or without static surface seeding of endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells) from a subject (e.g., a human patient or a model animal). Using autograft or allograft cells, the reseeded valves may be implanted in patients in need thereof without incurring excessive immune response, thrombosis, or loss of function over time. The supply of animal heart valves is also not limited. For pediatric patients, such tissue engineered heart valves (TEHVs) that may grow with the body without repeated replacement, thereby significantly improving the quality of life.

[0010] As disclosed herein, a xenograft TEHV was developed, using decellularized porcine pulmonary leaflets that were recellularized with recipient cells. These leaflets were recellularized in vitro by injection of shMSC-encapsulated in GelMA-HAMA and with or without static seeding of shEPC on the leaflet surfaces. The decellularization protocol presented here is capable of decellularizing porcine pulmonary valves, whilst keeping the structural and biochemical integrity of the tissues intact. The recellularized valves were successfully implanted in an ovine model. After four-six weeks in vivo, the explants showed no functional abnormalities during echocardiography and the histological organization of the leaflets resembled the native leaflet closely. The results also showed that the shMSCs inside the decellularized valve were alive and well integrated. Ingrowth from pro and anti inflammatory macrophages from the environment was observed. These features are essential for the regeneration of the implant. Despite previous attempts with the former gel, achieving a homogeneous cell distribution was not yet accomplished. To address this, various injectable hydrogels were tested. Semilunar porcine heart valves were decellularized to evaluate the feasibility of these hydrogels. Primary isolated valvular interstitial cells (VICs) from Dorset sheep were combined with an in-house developed injectable hydrogel utilizing click-like polymerization. The encapsulated cells were injected into the interstitial layer of the decellularized valves, cultured for 0, 5, and 7 days in vitro, and subsequently processed for histological analysis. This study demonstrates that injectable hydrogels can successfully introduce cells into the spongiosa layer of the valve leaflet, achieving a good spatial and homogeneous cell distribution, thus overcoming previous challenges. Two main injection sites were identified: via the nodulus and in combination with the wall, both facilitating cell introduction into the scaffold. The optimal injection site for the leaflet is dependent on the specific gel used.

[0011] This method for TEHV remains a plausible candidate for future clinical use, and may require further laboratory investigation and refinement of recellularization techniques. The availability of porcine tissue together with the use of autologous or allograft cells make this approach promising for translation to the clinic. More work can be done to further optimize the recellularization method and to map out potentially harmful elements of the valves. These elements are currently the main reasons for the initial unfavourable clinical results in humans.

[0012] Thus, the present disclosure provides new techniques for creation of a tissue engineered heart valve leaflet based on a modified technique for decellularization of native heart valve leaflet; new recellularization methods using a variety of hydrogels, including photocrosslinkable, shear-thinning and self-assembling hydrogels; and new techniques for heart valve leaflet harvest and implantation.

[0013] As used herein, the term “decellularization” refers to the process of removing cells and / or cellular debris from a tissue. In some embodiments, the decellularization process prepares tissue, such that cells are removed from the valve, leaving behind a cell-free (acellular) scaffold which has excellent mechanical and biological properties. In some embodiments, the decellularization process prepares a tissue, such that it is available to accept new cells into its biological scaffold. In some embodiments, decellularized valves from a donor (e.g., pig) can activate immune responses in a recipient (e.g., human or sheep), however, this recellularization technique can be used independently from the decellularization protocol. In some embodiments, recellularization of the decellularized valves can be subsequently performed using injectable hydrogels . In some embodiments, a beneficial regenerative immune response was induced either by the gel, the cells or the gel - cells mixture, this is yet to be determined. It is clear that repopulation via either immune cells or recellularization within the scaffold matrix is essential for the decellularized matrix to become a living matrix and to gain growth potential. In some embodiments, decellularized valves have no remodeling capacity for the ECM unless or until native cells have infiltrated. In some embodiments, valves are decellularized by detergents. The detergent can be any type of detergent including, but not limited to, non-ionic, anionic, detergents for the use of cell lysis, and combinations thereof. As used herein, the term “recellularization” refers to the process of repopulating at least a portion of a tissue, scaffold, or other bioengineered construct with cells. In some embodiments, recellularization is seeding or injecting cells in vitro. In some embodiments, recellularization is seeding or injecting cells in vitro is to promote the capacity of the engineered tissue to remodeling ECM and host cellularization in vivo. In some embodiments, recellularization involves repopulating the tissue (e.g., a heart valve) in vivo with host cells.

[0014] As used herein, the term “implant” refers to placing a new tissue to replace a damaged or diseased valve. In some embodiments, implant involves placing a new heart valve into the heart to replace a damaged or diseased valve. The new valve can be either a mechanical valve made of metal or carbon, or a biological valve made from animal or human tissue.

[0015] As used herein, the term “explanf ’ refers to the removal of a previously implanted tissue (e.g., a heart valve). In some embodiments, explanted tissue is examined for the coverage with cells present on the surface and in the inner layers. In some embodiments, explanted tissue is examined for inflammation. In some embodiments, explanted tissue is examined for thrombosis formation.

[0016] The term “culturing” as used herein refers to the growth, maintenance, storage and passaging of cells. Cell culture techniques are well understood and often involve contacting cells with particular media to promote growth. In the present case, cells contacted with or exposed to a tissue (e.g., a decellularized heart valve) during culture may continue to grow and / or proliferate and / or differentiate.

[0017] In one aspect, the disclosure is related to a method of delivering a plurality of cells or cell components into a sub-surface layer of a tissue, comprising: (a) encapsulating a plurality of cells into a hydrogel; and (b) delivering the hydrogel encapsulating the plurality of cells to the sub-surface layer of the tissue, thereby delivering the plurality of cells into the subsurface layer of the tissue. In some embodiments, the tissue is a heart valve tissue, and the sub-surface layer of the issue is a sub -endothelial layer (e.g., spongiosa) of the heart valve tissue. In some embodiments, the tissue is a decellularized tissue. In some embodiments, the plurality of cells comprise primary cells or cell lines, in some embodiments, the plurality of cell components comprise a protein (e.g., a growth factor or a cytokine), a nucleic acid, a carbohydrate, and / or a lipid, optionally the plurality of cell components comprise extracellular vesicles. In some embodiments, the plurality of cells comprise stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells). In one aspect, the disclosure is related to a method of delivering a therapeutic agent into a sub-endothelial layer (e.g., spongiosa) of the heart valve tissue, comprising: (a) encapsulating the therapeutic agent into a hydrogel; and (b) delivering the hydrogel encapsulating the therapeutic agent to the sub-endothelial layer (e.g., spongiosa) of the heart valve tissue, thereby delivering the therapeutic agent into the sub-endothelial layer (e.g., spongiosa) of the heart valve tissue. In some embodiments, the therapeutic agent comprises a peptide, a protein, a growth factor, a cytokine, and / or a chemokine. In some embodiments, the hydrogel is a photocrosslinkable hydrogel, a shear-thinning hydrogel, a click-based hydrogel, or a self-assembling hydrogel. In some embodiments, the hydrogel is a gelatin methacrylate-hyaluronic acid methacrylate (GelMA-HAMA) hydrogel, an PNP shearthinning hydrogel, or a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel.

[0018] In one aspect, the disclosure is related to a method of recellularizing a decellularized heart valve, comprising: (a) delivering a hydrogel encapsulating a plurality of stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells) to a subendothelial layer (e.g., spongiosa) of the decellularized heart valve; and / or (b) contacting a plurality of endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells) with a surface of the decellularized heart valve, thereby recellularizing the decellularized heart valve. In some embodiments, the hydrogel is a gelatin methacrylate-hyaluronic acid methacrylate (GelMA-HAMA) hydrogel. In some embodiments, the hydrogel comprises about l%-50% GelMA (gelatin methacrylate), about 0.01%-10% HAMA (hyaluronic acid methacrylate), and about 0.01%-10% PI (2-hydroxy-4’- (2-hydroxyethoxy)-2-methylpropiophenone). In some embodiments, the hydrogel is a P- aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel. In some embodiments, the hydrogel has a concentration of about 1% to about 20% P-aminoacrylate synthetic polyethylene glycol (PEG). In some embodiments, the plurality of stem cells or interstitial cells are encapsulated into the hydrogel at a concentration of about 0.1 million to about 100 million cells per milliliter (mL), e.g., by crosslinking under UV light; and in some embodiments, the plurality of endothelial cells are contacted with the surface of the decellularized heart valve at a density of about 0.01 million to about 10 million per cm2. In some embodiments, the hydrogel encapsulating the plurality of stem cells or interstitial cells is injected to the spongiosa of the decellularized heart valve via the arterial wall (e.g., the pulmonary artery wall at the leaflet insertion site) and / or nodulus of the decellularized heart valve. In some embodiments, the surface of the decellularized heart valve is incubated with fibronectin before contacting with the plurality of endothelial cells. In some embodiments, the method described herein further comprises culturing the plurality of stem cells or interstitial cells after step (a), and / or culturing the plurality of endothelial cells after step (b).

[0019] In one aspect, the disclosure is related to a method of decellularizing a tissue, comprising: (a) contacting the tissue with a first solution comprising about 0.001%-5% SDC (sodium deoxycholate) and about 0.001%-5% SDS (sodium dodecyl sulfate) for about 1-24 hours (e.g., 12 hours); (b) optionally repeating step (a) for 1-5 cycles with a fresh first solution for each cycle; (c) contacting the tissue with a second solution comprising about 0.001%-5% sodium azide for about 1-24 hours (e.g., 12 hours); and (d) optionally repeating step (c) for 1-5 cycles with a fresh second solution for each cycle, thereby decellularizing the tissue. In some embodiments, the second solution further comprises about 0. l%-10% PSG (penicillin / streptomycin / glutamine). In some embodiments, the method described herein further comprises washing the tissue to remove the first and / or second solutions. In some embodiments, the decellularizing substantially maintains the structural and / or biochemical integrity of the tissue. In some embodiments, no cells are present in the tissue after the decellularizing, e.g., as determined by haematoxylin and eosin (HE) staining. In some embodiments, the tissue is a heart valve tissue.

[0020] In one aspect, the disclosure is related to a decellularized tissue obtained using the method described herein.

[0021] In one aspect, the disclosure is related to a method of implanting an engineered heart valve in a subject, comprising: (a) obtaining a decellularized heart valve; (b) recellularizing the decellularized heart valve, thereby obtaining the engineered heart valve; and (c) implanting the engineered heart valve into the subject.

[0022] In one aspect, the disclosure is related to a method of implanting an engineered heart valve in a subject, comprising: (a) decellularizing a heart valve isolated from a mammal; (b) recellularizing the decellularized heart valve, thereby obtaining the engineered heart valve; and (c) implanting the engineered heart valve into the subject. In some embodiments, the mammal is a pig and the heart valve is a porcine pulmonary valve. In some embodiments, the subject is a human subject (e.g., a pediatric patient) or a model animal (e.g., sheep). In some embodiments, the decellularized heart valve is recellularized with cells isolated from the subject. In some embodiments, the implanted engineered heart valve exhibits a normal function, e.g., normal opening and closing with no observable regurgitation of the blood flow as determined by echocardiography, at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, or 50 years after implantation. In some embodiments, the implanted engineered heart valve does not induce inflammation or rejection.

[0023] In one aspect, the disclosure is related to an engineered heart valve comprising a first population of exogenous cells in a sub -endothelial layer (e.g., spongiosa) of a heart valve tissue. In some embodiments, the heart valve tissue is a pulmonary valve or an aortic valve. In some embodiments, the plurality of exogenous cells comprises stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells). In some embodiments, the engineered heart valve described herein further comprises a second population of exogenous cells on a surface of the heart valve tissue, in some embodiments, the second population of exogenous cells comprise endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells). In some embodiments, the first and / or second populations of exogenous cells are isolated from a human subject (e.g., a pediatric patient) or a model animal (e.g., sheep). In some embodiments, the heart valve tissue is a porcine pulmonary valve.

[0024] In one aspect, the disclosure is related to a composition comprising a gelatin methacrylate-hyaluronic acid methacrylate (GelMA-HAMA) hydrogel, an PNP shearthinning hydrogel, or a P-aminoacrylate polyethylene glycol (PEG)-based hydrogel encapsulating a plurality of cells.

[0025] In some embodiments, the disclosure is related to use of a gelatin methacrylatehyaluronic acid methacrylate (GelMA-HAMA) hydrogel, an PNP shear-thinning hydrogel, or a P-aminoacrylate polyethylene glycol (PEG)-based hydrogel for delivering a plurality of cells into a tissue.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0027] DESCRIPTION OF DRAWINGS

[0028] FIGS. 1 A-1C show overview of the decellularization process. FIG. 1 A shows fresh pulmonary valve on Day 0. FIG. IB shows decellularized pulmonary valve on Day 5. FIG. 1C shows trimmed and sterilized leaflets.

[0029] FIG. 2 shows concentrations of insoluble and soluble collagens per milligram wet weight.

[0030] FIG. 3 shows concentrations of GAG per milligram wet weight.

[0031] FIGS. 4A-4C show mechanical properties of fresh and decellularized valves, for the circumferential and radial fiber direction. FIG. 4A shows ultimate tensile stress (UTS) results. FIG. 4B shows stiffness results. FIG. 4C shows strain to failure to results.

[0032] FIG. 5 shows histological analysis of fresh and decellularized leaflets. Arrow 1 indicates ventricularis. Arrow 2 indicates spongiosa. Arrow 3 indicates fibrosa. Sections were sliced at 15 pm thickness and stained with HE and imaged using brightfield microscopy. Scalebar = 100 pm.

[0033] FIG. 6 shows pictures of explanted pulmonary valves, showing top and inside view of the implanted valve. Left: explanted pulmonary valve of sheep 475 at 6 weeks, leaflet was seeded with shMSC only. Middle: sheep 744 at 4 weeks, leaflet was seeded with shMSC and shEPC. Right: sheep 714 at 4 weeks, leaflet was seeded with shMSC and shEPC.

[0034] FIG. 7 shows histological analysis of the root and leaflet of explanted and native leaflets of sheep 475. Results for pre-implant was not available. Scalebar = 100 pm.

[0035] FIG. 8 shows histological analysis of the root and leaflet of pre-implanted, explanted and native leaflets of sheep 744. Scalebar = 100pm.

[0036] FIG. 9 shows histological analysis of the root and leaflet of pre-implanted leaflet of sheep 714. Results for explant and native were not available. Scalebar = 100 pm.

[0037] FIG. 10 shows a schematic workflow of decellularization, recellularization, and implantation of sheep MSCs and ECFCs in decellularized pig heart valves.

[0038] FIG. 11 shows an overview of decellularization process.

[0039] FIG. 12 shows cellular encapsulation and injection using the PNP hydrogel. A non- homogeneous cell distribution (left) and disruption of the ECM (right) were observed. FIG. 13 A shows cellular encapsulation and injection using the GelMA-HAMA hydrogel after 1 month in vitro culture. The mid area of the leaflets show cell throughout the scaffold.

[0040] FIGS. 13B-13E show immunofluorescence images of the tip region or belly region of the leaflets.

[0041] FIG. 14A shows HE staining of VIC-injected decellularized pulmonary valve leaflet using a f-aminoacrylate synthetic hydrogel after 7 days in vitro.

[0042] FIGS. 14B-14C show histological overview of native aortic and pulmonary valve leaflets recellularized with valvular interstitial cells using a click hydrogel and cultured for 0, 5, and 7 days (“TO,” “T5” and “T7,” respectively) in vitro. All leaflets come from the same batch. The native and decellularized aortic leaflets are embedded in OCT, and the other leaflets were embedded in paraffin.

[0043] FIGS. 14D-14E show DAPI staining results of the transverse cross sections of the leaflets.

[0044] FIGS. 14F-14G show HE staining results of the transverse cross sections of the leaflets. Black dots are staining debris.

[0045] FIG. 15 shows injection sites for a hydrogel (left) and colored hydrogel (right) to visualize the penetration of the gel.

[0046] FIGS. 16-17 show histological analysis of hydrogel-injected leaflets at different injection sites (via both the nodulus and the root).

[0047] FIG. 18 shows a combination of histological overviews of explanted GelMA-HAMA injected leaflets from sheep 475, sheep 744, sheep 714, and a native sheep leaflet. The results for sheep 475 are further depicted in FIG. 7. The results for sheep 744 are further depicted in FIG. 8. The results for sheep 714 are further depicted in FIG. 9. A pre-implant is shown in the bottom row and an explant is shown in the top row.

[0048] FIGS. 19A-19B show pictures of explanted leaflet (FIG. 19A) and cross section of the leaflet (FIG. 19B).

[0049] FIGS. 19C-19H show the belly section (FIGS. 19C, 19E and 19G) and the tip area (FIGS. 19D, 19F, and 19H).

[0050] DETAILED DESCRIPTION

[0051] Diseases affecting the heart valves remain one of the main causes of death worldwide. Patients will often need a valve replacement, for which two implant options are available: mechanical valves and bioartificial valves. The materials from which these implants are made often lead to blood clotting and patients have to take anti-clotting medication or immunosuppressive medication for the remainder of their lives. Most importantly, implants also do not grow with the body, and children need to have their valve replaced every few years. The heart valve tissue engineering field tries to grow living tissues in the lab, which resemble the normal human valve and can grow with the body. Normal semilunar valves contain three leaflets, and the place where they are attached to the arterial wall is called the root. Each leaflet has three distinct layers, which have a specific composition. A potential source of material in tissue engineering are decellularized valves. Cells are removed from the valve, leaving behind a cell-free (acellular) scaffold which has excellent mechanical and biological properties. Decellularized pig valves have even been implanted in children, however, the immune system was activated by the implant due to the persistence of some porcine proteins, Decellularized homograft valves have recently shown to be similar or outperform standard tissue conduit valves, however, they didn’t recellularize so they cannot accommodate the growth.

[0052] To enable a decellularized heart valve to grow, the present disclosure describes a new approach for recellularizing heart valves and tested the effectiveness in three sheep. Pig pulmonary valves, which regulate the blood flow from the heart to the lungs, were removed from the animals and decellularized using detergents. Detergents break down the cells residing in the valves so they can be washed away. This method did not affect the biological and mechanical properties of the pulmonary valves. These valves were recellularized with cells isolated from the sheep itself, which was to receive the implant. A normal leaflet surface is covered with cells, which are called valvular endothelial cells (VECs), and cells also reside on the inside of the leaflet, e.g., the valvular interstitial cells (VICs). Endothelial-like cells were isolated from the sheep’s blood, and interstitial-like cells were isolated from the sheep’s bone marrow. The interstitial-like cells were mixed with a hydrogel and injected at the base of the pulmonary leaflet, where the valve leaflet connects to the artery wall. One recellularized leaflet was then implanted into the sheep from which the cells had been retrieved. The sheep was sacrificed after six weeks, when the animal was anesthetized and the pulmonary valve was explanted. Two sheep received autologous interstitial-like cells mixed with a hydrogel and injected at the base of the pulmonary leaflet, where the valve leaflet connects to the artery wall with the addition of endothelial-like cells were seeded on the surfaces of the leaflet. One recellularized leaflet was then implanted into the sheep from which the cells had been retrieved. These sheep were sacrificed after four weeks, when the animal was anesthetized and the pulmonary valve was explanted. For all sheep, the opening and closing of the leaflet was observed with echocardiography at implant, after 10 days and before explant. These echocardiographs showed the pulmonary valve opened and closed normally, with the implanted leaflet having no adverse effect on valve function. A close inspection of the explanted valve confirmed there was no scar tissue, although a fresh bloodclot had formed during the explant procedure. The micro-architecture of the pre-implant, explant and normal sheep leaflets was observed by slicing section of several micrometres thick and staining the tissue matrix and cells. The stained sections were inspected using a microscope. The pre-implant leaflet from one sheep seemed to be absent of cells but this issue is explained below. The explanted leaflets were completely covered with cells present on the surface and in the inner layers. Furthermore, the reseeded leaflet looked similar to a normal sheep pulmonary leaflet. Altogether, the decellularization method did not substantially affect the integrity of the valve, and the short-term results in the sheep model indicate the leaflets are incorporated by the body and function normally. These results confirms that recellularization of decellularized heart valves is a promising approach for heart valve tissue engineering, especially when the recellularization method is developed further.

[0053] Using an injectable hydrogel, the present disclosure demonstrates the ability to introduce cells throughout all layers of decellularized valve scaffold with great spatial control. This technique was validated for aortic and pulmonary valve leaflets. By in vitro assays, valvular interstitial cells showed widespread cell distribution inside decellularized valvular leaflets after 0, 3,5 and 7 days of culture without leakage of cells or gel. By in vivo assays, single leaflet implants showed good leaflet function up to six weeks without cell losses. In addition, mononuclear cells were attracted to the interior of the scaffold from the blood stream environment. This seeding method could contribute to the ultimate goal of a living heart valve, with the capability of growth and remodelling.

[0054] The novelty of the present disclosure is summarized as follows. Decellularized homo- or xenograft leaflets hold great promise as base for TEHV. Clinical studies using decellularized homografts have demonstrated a similar or even superior performance in short to mid-term (similar) to conventional cryopreserved homografts as well as some cellular ingrowth. Current decellularized xenograft heart valves only show surface cellularization but no cells reach the spongiosa middle layer. Decellularized homograft heart valves have shown some cellular ingrowth inside the tissue but only in specific regions and this is insufficient to remodel the valve. For the long-term success of a tissue engineered heart valve, homogeneous cellular ingrowth into the sub-endothelial (middle) layer of the leaflet is essential. Hydrogels have been developed and used for many years. In combination with TEHVs, they have been used on the surfaces of a decellularized leaflet. However, hydrogels have not been used on the sub-surface (inside) layers of decellularized heart valves. Moreover, hydrogels have not been used inside any decellularized matrix. After many trials with different types of hydrogels, the present disclosure provides methods to inject cells into the sub-endothelial (middle) layer of the leaflet in a homogeneous matter, throughout the full extent of the leaflet (i.e., all the way from the insertion point of the leaflet on the arterial wall to the tip region). This technology can be used independently of the decellularization method, and most likely in combination with different types of hydrogels, cells and other bioactive substances, including growth factors, drugs, etc.

[0055] Tissue engineered heart valves

[0056] A tissue engineered heart valve (TEHV) is an appealing valve replacement option, especially for pediatric patients. In the tissue engineering approach, scaffolds are seeded with cells to create living tissue with the ability to grow and remodel. Prior researchers have tried to mimic the anatomical structure and function of a heart valve. A semilunar valve consists of three leaflets, which are each attached to a sinus who together form the valvular root. A normal semilunar leaflet is made up of three distinct layers: the fibrosa, composed of dense collagen type-1 fiber sheets with an anisotropic orientation; the spongiosa core, composed of glycosaminoglycans (GAGs) and proteoglycans (PGs); and finally the ventricularis, a combination of collagen and elastin fibers arranged in a fibrous mesh. Normally, a leaflet is populated by valvular endothelial cells (VECs), which populate the surfaces of the valve leaflet and valvular interstitial cells (VICs), which synthesize and degrade ECM.

[0057] A potential natural source of TEHV scaffolds could be decellularized native valves, and decellularized porcine valves have even been used clinically. These valves have similar biological and mechanical characteristics to a human valve. Decellularization to remove xenogeneic cells decreases the chances of inducing an immune response. However, the results have not always been encouraging. A decellularized porcine pulmonary valve (Matrix P plus valve), when implanted in pediatric patients, caused a severe fibrogenic and inflammatory response, which was likely due to insufficient decellularization and exposure of xenogenic ECM. The absence of cells at implantation also means there is no endothelial layer present, and at least one study has indicated the decellularized leaflet surface is susceptible to thrombosis. Additionally, an acellular graft has no remodelling capacity for the ECM unless or until native cells have infiltrated. To address these issues, researches have focused on recellularizing implants in vitro. One potential cell source for reseeding valves are mesenchymal stem cells (MSC). These can be isolated from bone marrow and prior research has indicated they react similarly to VICs when exposed to mechanical stress. A cell source for valve endothelization are the epithelial progenitor cells (EPC) also called endothelial colony forming cells (ECFCs), which originate from peripheral blood, and have shown an ability to endothelize valve constructs.

[0058] Methods to improve recellularization that have extensively been investigated and reviewed include surface coatings with cell attractants such as chemokines, polymers, biomolecules and gels and pre-seeding strategies on the outside of the valve leaflet. These efforts have improved cellularization on the surface of the leaflet but not the cell migration inside the scaffold. Dynamic cell seeding of the leaflets in bioreactors under hemodynamic conditions further improved the cell seeding efficiencies by the activation of repair and tissue growth mechanisms. Yet, cellular infiltration of the interstitial layer was by all efforts parsimonious.

[0059] An alternative proposed approach to increase host recellularization of the leaflets involves homologous fibroblast population of fibrin constructs in vitro to produce collagen matrices, which subsequently are decellularized. In vivo studies in a sheep model have shown improved cellular ingrowth after 6 months.

[0060] In addition, it is known that animals endothelialize easier and that this re- endothelialization does not occur in human. For this reason, the methods described herein may have an even greater value when animal models are translated to human trials.

[0061] Tissue engineered heart valves (TEHVs) offer a promising alternative strategy for heart valve disease, combining engineered materials as scaffolds and stem cells to create living valve replacements. One strategy involves the use of decellularized donor heart valves as scaffolds, removing native cells and other bioactive components while preserving the structural proteins, known as the extracellular matrix (ECM). In recent years, progress has been made in decellularization strategies and decellularized xenograft or homograft valve tissues are attractive scaffold materials for TEHVs. However, the subsequent repopulation of the tissues by host cells in vivo has challenges including limited cell ingrowth and non- homogeneous cell distribution. The long-term success of TEHVs depends on the scaffold transformation after implantation into living tissue through the interactions between scaffold properties and the regenerative capacity of the host. The present disclosure developed a novel method to introduce cells inside the spongiosa (middle) layer of decellularized heterograft valve scaffolds prior to implantation.

[0062] Preparation of engineered heart valves

[0063] There are two semilunar valves the aortic valve and the pulmonary valve. These valves are named for their half-moon shape and are located between the ventricles and the major arteries leaving the heart. Aortic valve is positioned between the left ventricle and the aorta, it allows blood to flow from the heart into the aorta and prevents backflow into the left ventricle. Pulmonary valve is located between the right ventricle and the pulmonary artery, it permits blood to flow from the heart into the pulmonary artery and prevents backflow into the right ventricle. A semilunar valve (e.g., aortic valve or pulmonary valve) consists of three leaflets, which are each attached to a sinus who together form the valvular root. A normal semilunar leaflet is made up of three distinct layers: the fibrosa, composed of dense collagen type-1 fiber sheets with an anisotropic orientation, the spongiosa core, composed of glycosaminoglycans (GAGs) and proteoglycans (PGs), and finally the ventricularis, a combination of collagen and elastin polymers fibers arranged in a fibrous mesh. Normally, a leaflet is populated by valvular endothelial cells (VECs), which populate the endothelial surfaces parts of the valve leaflet and valvular interstitial cells (VICs), which synthesize and degrade ECM.

[0064] Tissue engineered heart valves (TEHVs) are an appealing option for valve replacement, as current replacement options do not facilitate growth or remodelling. A potential source of TEHV scaffolds are porcine decellularized valves, which have similar biological and mechanical characteristics to native valves. A lack of cells makes the leaflet surface susceptible to thrombosis, and when implanted in pediatric patients, the exposed xenogenic extracellular matrix may cause a severe immune response.

[0065] The present disclosure is related to a novel recellularization method to resolve these issues and the effectiveness of this approach was tested in an ovine model (n=3). Porcine pulmonary valves were decellularized during 3 cycles of 12 hours in demineralized water (demi-PEO) with 0.5% sodium deoxycholate and 0.5% sodium dodecyl sulphate (SDS), followed by 3 cycles of 24 hours in demi-PEO supplemented with 1% Penicillin / Streptomycin / Glutamine and 0.5% sodium azide. Overall, the tissues maintained their mechanical and biological properties, although a significant decrease in the amount of glycosaminoglycans was observed. Additionally, the histology of decellularized leaflets indicated the collagen arrangement was slightly disturbed when compared to fresh tissue. The cells for recellularization were obtained from the recipient sheep. Using a density gradientbased centrifugation protocol, endothelial progenitor cells (shEPC) and mesenchymal stem cells (shMSC) were successfully isolated from blood and bone marrow, respectively. shMCS were encapsulated in gelatin-methacrylate hyaluronic acid methacrylate (GelMA-HAMA) and injected into the spongiosa via the pulmonary artery wall at the leaflet insertion line, and shEPC were seeded on the valve surfaces. A single reseeded leaflet was implanted into the sheep via left-thoracotomy using cardiopulmonary bypass, and the animals were sacrificed after 4-6 weeks. Echocardiography was performed at implant, 10 days after surgery and at explant before heparinization and anesthetizing. The echocardiograms showed no abnormalities and gross inspection of the explanted valves showed fresh thrombi on the leaflets, which had likely occurred during the explant procedure. Histological investigation of pre-implant specimens showed little to no cells were present on the leaflet surface and in the spongiosa. However, the explanted leaflets were fully endothelialized and cells populated the spongiosa. The overall structure of the xenogenic leaflets closely resembled that of the native sheep leaflets.

[0066] As a first step, the decellularized valves were analyzed to assess whether they maintained their structural and biological integrity during the decellularization procedure. A detergent-based protocol was used, using several cycles combining 0.5% SDS and 0.5% SDC, followed by 0.5% sodium azide. The success of the decellularization protocol was based on the absence of cells in the decellularized leaflets. Previous researches had confirmed that only a neglectable amount of DNA was present after decellularization of aortic valves with the protocol described herein. A slight decrease was observed in the UTS and strain-to- failure after decellularization, in both the circumferential and radial fiber directions. Also, the decellularization protocol minimally increased the stiffness in the circumferential direction, whereas in the radial direction the stiffness decreased. Although none of these differences were determined to be significant (p > 0.05), the decellularization method did show some effect on the structural integrity and biomechanical properties of the leaflets. Additionally, the biochemical composition of the leaflets was altered. Both insoluble and soluble collagen was reduced during the process, but differences were not statistically significant (p > 0.05). The GAG content was significantly affected (p < 0.05), e.g., about 40% of GAG was lost. Additionally, histological analysis indicated that the decellularization protocol was successful in removing cells. The changes in the mechanical characteristics were reflected in the histological organization of decellularized leaflets, which showed slight distortions in the collagen arrangement compared to fresh tissues. Overall, these results are in agreement with previously published work on the effects of detergent-based decellularization protocols.

[0067] In order to fully establish the present decellularization protocol for use in a clinical setting, the decellularized tissues must be free from residual antigens and detergents. A prior study on the release profile of SDC and SDS during 24-hour washing cycles of decellularized heart valves revealed that critical amounts of detergent remained present after the first washing cycle. Furthermore, a report of the clinical performance of the Matrix-P and Matrix- P-Plus valves in humans found limited host cell -grafting as one of the limiting factors. These authors argued that the incomplete removal of the decellularization agents might be the cause of the limited cell engraftment. Moreover, the presence of the alpha-Gal epitope is considered as one of the main causes of clinical failure of porcine tissues implanted into humans, as presence of this epitope induces a severe immune response. Overall, decellularization of tissues greatly decreases the presence of alpha-Gal, but the reduction is not guaranteed to prevent out an immune response.

[0068] The in vivo performance of the decellularized valves was investigated in an ovine model (n=3). A total of three leaflets were successfully implanted, of which two had been explanted at the time of writing. From each animal, blood and bone marrow were harvested to isolate shEPC and shMSC, respectively. Consequently, each animal received an implant containing autologous cells, which were maintained in vivo for 4-6 weeks. Echocardiograms obtained at implant and explant all showed that the replacement leaflet functioned well in vivo, and no abnormalities were observed. The explanted valves did show signs of thrombus formation, although it could not be determined where these thrombi formed, e.g., during the explant procedure or even post-mortem. Of the two pre-implant samples available, only one contained cells in the spongiosa and neither showed the presence of cells on the leaflet surface. At explant, both leaflets had been re-endothelialized and cells were present in the spongiosa. Additionally, signs of ECM remodelling were visible as increased organization of collagen fibers and the characteristic tri-layer structure remained comparable to the native histology. Initially experiments showed that the explants were not as evenly populated by cells compared to native tissue. Improvements were achieved by further optimizing the composition of the hydrogel and improved homogeneous distribution were obtained. In addition, it is noteworthy that the leaflet, which did show thrombus at explant and reduced endothelial coverage, did not receive pre-implant endothelial progenitor cells.

[0069] 1. Decellularization

[0070] Natural biostructures, e.g. a tissue or an organ, can be obtained from a donor of the same species as the subject, e.g., a human tissue or organ for a human recipient. The natural biostructure can also be obtained from a different species which includes, but is not limited to, monkeys, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep. As described herein, the decellularized porcine pulmonary valve can be used as the three-dimensional scaffold to reconstruct an engineered heart valve using stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells) in a sub-endothelial layer (e.g., spongiosa), and / or endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells) on one or both surfaces. In one embodiment, the stem cells, interstitial cells, and / or endothelial cells described herein are isolated from the peripheral blood or bone marrow of a subject. The engineered heart valve can be implanted back into the subject for further development. Biostructures, e.g., an engineered heart valve, can bedecellularized by removing the entire cellular and tissue content from a native heart valve (e.g., a porcine heart valve isolated from a pig). The decellularization process comprises a series of sequential extractions. One key feature of this extraction process is that harsh extraction that may disturb or destroy the complex infra-structure of the biostructure, be avoided. In some embodiments, the process may involve removal of cellular debris and solubilization of the cell membrane. This can be followed by solubilization of the cytoplasmic components and the nuclear components.

[0071] Preferably, the biostructure, e.g., a heart valve, is decellularized by removing the cell membrane and cellular debris on the surfaces and the sub-surface layer of the heart valve, using gentle mechanical disruption methods. The gentle mechanical disruption methods must be sufficient to disrupt the cellular membrane. However, the process of decellularization should avoid damage or disturbance of the biostructure's complex infra-structure, e.g., ventricularis, spongiosa, and fibrosa of the heart valve. Gentle mechanical disruption methods include scraping the surface of the heart valve, freezing cycles, agitating the heart valve, or stirring the heart valve in a suitable volume of fluid, e.g., distilled or demineralized water. In one embodiment, the gentle mechanical disruption method includes magnetically stirring (e.g., using a magnetic stir bar and a magnetic plate) the heart valve in a suitable volume of distilled or demineralized water until the cell membrane is disrupted and the cellular debris has been removed from the heart valve.

[0072] After the cell membrane has been removed, the nuclear and cytoplasmic components of the biostructure are removed. This can be performed by solubilizing the cellular and nuclear components without disrupting the infra-structure. To solubilize the nuclear components, ionic or non-ionic detergents or surfactants may be used. Examples of ionic detergents or surfactants include, but not limited to, sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), sodium cholate, and sarkosyl. In general, the head group of ionic detergents either have a positive or negative charge as well as a hydrophobic tail, and are chemically stronger than non-charged detergents. They are a great choice if needed to bind to proteins and alter their structure. However, it should be noted that due to their strength, ionic detergents more often than not involve some level of protein denaturation.

[0073] In contrast to ionic, non-ionic detergents have no charge in their head group, and can be further separated into two groups: polyoxyethylene and glycosidic compounds. There are differences among the varieties of detergents in either group. However, the main difference is polyoxyethylene compounds contain neutral head groups and a tail of hydrophobic chains, whereas glycosidic detergents usually use a sugar as a head base, such as glucose, and have an alkyl polymer tail. Examples of non- ionic detergents or surfactants include, but are not limited to, the Triton ™ series, available from Rohm and Haas of Philadelphia, Pa., which includes Triton™ X-100, Triton™ N- 101, Triton™ X-l 14, Triton™ X-405, Triton™ X-705, and Triton™ DF-16, available commercially from many vendors; the Tween® series, such as monolaurate (Tween® 20), monopalmitate (Tween® 40), monooleate (Tween® 80), and poly oxethylene-23 -lauryl ether (Brij™ 35), polyoxyethylene ether W-I (Poly ox™), and the like, sodium cholate, deoxycholates, CHAPS, saponin, n-Decyl P-D-glucopuranoside, n- heptyl P-D glucopyranoside, n-Octyl- a-D-glucopyranoside and Nonidet P-40. To ensure the proper removal of all cellular remnants, an additional treatment with DNAse or RNAse might be added.

[0074] One skilled in the art will appreciate that a description of compounds belonging to the foregoing classifications, and vendors may be commercially obtained and may be found in "Chemical Classification, Emulsifiers and Detergents", McCutcheon's, Emulsifiers and Detergents, 1986, North American and International Editions, McCutcheon Division, MC Publishing Co., Glen Rock, N.J., U.S.A, and Judith Neugebauer, A Guide to the Properties and Uses of Detergents in Biology and Biochemistry, Calbiochem, Hoechst Celanese Corp., 1987. In one preferred embodiment, the ionic surfactant used for decellularization is SDS, SDC, or both.

[0075] The concentration of the detergent may be altered depending on the type of biostructure being decellularized. For example, for delicate tissues, e.g., heart valve, the concentration of the detergent should be decreased. Preferred concentrations ranges of the detergent can be from about 0.001% to about 5% (w / v). More preferably, about 0.05% to about 1.0% (w / v). Even more preferably, about, 0.1% (w / v) to about 0.8% (w / v). In some embodiments, the detergent used herein has a concentration of about 0.5% (w / v).

[0076] Preferred concentrations of these range from about 0.001 to about 5% (w / v), with about 0.05 to about 0.1% (w / v) particular preferred. The cytoskeletal component, comprising consisting of the dense cytoplasmic filament networks, intercellular complexes and apical micro cellular structures, may be solubilized using alkaline solution, such as, ammonium hydroxide. Other alkaline solution consisting of ammonium salts or their derivatives may also be used to solubilize the cytoskeletal components. Examples of other suitable ammonium solutions include ammonium sulphate, ammonium acetate and ammonium hydroxide. In a preferred embodiment, ammonium hydroxide is used.

[0077] The concentration of the alkaline solutions, e.g., ammonium hydroxide, may be altered depending on the type of biostructure being decellularized. For example, for delicate tissues, the concentration of the detergent should be decreased. Preferred concentrations ranges can be from about 0.001 to about 5% (w / v). More preferably, about 0.005 to about 0.1% (w / v). Even more preferably, about, 0.01% (w / v) to about 0.08% (w / v). The decellularized structure may be stored at a suitable temperature until required for use. Prior to use, the decellularized structure can be equilibrated in suitable isotonic buffer or cell culture medium. Suitable buffers include, but are not limited to, phosphate buffered saline (PBS), saline, MOPS, HEPES, Hank's Balanced Salt Solution, and the like. Suitable cell culture medium includes, but is not limited to, RPMI 1640, Fisher's, Iscove's, McCoy's, Dulbecco's medium, and the like.

[0078] In one aspect, the tissue described herein can be decellularized by contacting the tissue with one or more ionic detergent, e.g., SDC (sodium deoxycholate), SDS (sodium dodecyl sulfate), or a combination thereof. In some embodiments, after treatment by these detergents, the tissue is treated with an antibacterial agent (e.g., sodium azide). In some embodiments, the tissue is treated with an antibacterial agent (e.g., sodium azide) together with one or more antibiotics (e.g., penicillin and / or streptomycin) and one or more essential amino acids (e.g., glutamine). In one specific embodiment, the tissue is first treated with SDC and SDS, followed by sodium azide and PSG.

[0079] In one aspect, the tissue described herein can be decellularized by (a) contacting the tissue with a first solution comprising about 0.001%-5% SDC (sodium deoxycholate) and about 0.001%-5% SDS (sodium dodecyl sulfate) for about 1-24 hours (e.g., 12 hours); (b) optionally repeating step (a) for 1-5 cycles with a fresh first solution for each cycle; (c) contacting the tissue with a second solution comprising about 0.001%-5% sodium azide for about 1-24 hours (e.g., 12 hours); and (d) optionally repeating step (c) for 1-5 cycles with a fresh second solution for each cycle. The concentration of each of SDS and SDC in the first solution can be about 0.001%-5% (w / v), e.g., about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, or about 1% to about 5%. More specifically, the concentration of each of SDS and SDC in the first solution can be about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1%. In some embodiments, step (b) involves repeating step (a) for at least 1, 2, 3, 4, or 5 cycles with a fresh first solution for each cycle. The concentration of sodium azide in the second solution can be about 0.001%-5% (w / v), e.g., about 0.001% to about 0.01%, about 0.01% to about 0.1%, about 0.1% to about 1%, or about 1% to about 5%. More specifically, the concentration of sodium azide in the second solution can be about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.1%. In some embodiments, step (d) involves repeating step (c) for at least 1, 2, 3, 4, or 5 cycles with a fresh second solution for each cycle. In some embodiments, the second solution further comprises about 0. l%-10% PSG (penicillin / streptomycin / glutamine). For instance, the concentration of PSG in the second solution can be about 0.1% to about 5%, about 0.1% to about 2%, or about 0.8% to about 1.5%. More specifically, the concentration of PSG in the second solution can be about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, or about 1.5%. More specifically, the stock concentration of penicillin in PSG can be about 1000-100000 units / ml, 3000-50000 units / ml, 5000-25000 units / ml, or 8000-15000 units / ml; the stock concentration of streptomycin in PSG can be about 1-100 mg / ml, about 1-50 mg / ml, about 1-25 mg / ml, about 1-15 mg / ml, about 5-15 mg / ml, or about 8-12 mg / ml; and the stock concentration of L- glutamine in PSG can be about 50-1000 mM, about 50-500 mM, about 100-300 mM, about 150-200 mM, or about 180-220 mM. In a particular embodiment, the concentrations of both SDS and SDC in the first solution are about 0.5%; and the concentrations of sodium azide and PSG in the second solution are about 0.05% and 1%, respectively. In some embodiments, each cycle in step (a) or step (c) is about 1-24 hours, e.g., about 1-18 hours, about 6-18 hours, or about 10-14 hours. In some embodiments, each cycle is about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours, or about 24 hours. In some embodiments, the tissue is washed by water by thoroughly shaking the tubes (where the tissue is put in) when the first or second solution is replaced. In some embodiments, the decellularization process is conducted at about 4°C on a rocking plate.

[0080] In some embodiments, after the tissue is decellularized using the methods described herein, the tissue is washed in PBS, dlfcO, or miliQ H2O for 1-48 hours (e.g., about 24 hours) at about 4°C. In some embodiments, the decellularized tissue is further sterilized. For example, the decellularized tissue can be sterilized in 70% ethanol for about 1-10 minutes (e.g., about 5 minutes), under about UV light for about 10-120 minutes (e.g., about 1 hour), and / or in about 10-50% PSG at about 4°C for about 6-24 hours (e.g., about 12 hours).

[0081] After sterilization, the decellularized tissue can be washed thoroughly by water to remove traces of detergents, ethanol, and / or antibiotics. In some embodiments, the remaining amount of detergents, ethanol, and / or antibiotics is less than 0.001%, less than 0.0001%, or less than 0.00001%. After washing, the decellularized tissue can be kept in PBS at about 4°C for less than about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days before recellularization.

[0082] In some embodiments, the decellularizing process substantially maintains the structural and / or biochemical integrity of the tissue, e.g., the collagen level, the GAG level, and / or mechanical properties of a heart valve. For example, the decellularizing process described herein can maintain the structural and / or biochemical integrity of the heart valve such that it is suitable for recellularization and implantation, as discussed below.

[0083] In some embodiments, the decellularizing process can remove at least 95%, at least 96%, at least 96%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.999%, at least 99.9999%, or at least 99.99999% cells from the native tissue. The remaining cells can be evaluated, e.g., by haematoxylin and eosin (HE) staining. The tissue used for decellularization may be a native tissue, a modified tissue, or a genetically engineered tissue.

[0084] Since the recellularization process can be used independently of the decellularization protocol, other (e.g., commercially available) decellularized heart valve matrices from human, sheep, porcine or bovine might be used as a starting matrix for the injection of the hydrogel (e.g., any of the injectable hydrogels described herein) with biomolecules (e.g. cells, cell derivatives, growth factors).

[0085] 2. Recellularization

[0086] Recellularization is heavily reliant on culture conditions to support cell survival and growth within scaffolds with considerations required for different cell types, cell medium composition, respective growth factors and supplements, and cell seeding densities. Tailoring recellularization strategies to control the stages of proliferation, differentiation, and subsequently, maturation of cells is also advantageous in regenerating tissues. One report identified growth factors that can help promote myogenic proliferation but inhibit differentiation including basic fibroblast growth factor and epidermal growth factor whereas for myotube formation, insulin-like growth factor- 1 and transforming growth factor-pi were suggested. Many of these angiogenic factors are required for other cell types, introducing the need for tailoring cell medium composition to support co-cultures. One report’s recellularization attempts with a canine larynx involved significant characterization of cells in different medium conditions and supplements to determine a universal cell culture medium conducive to the growth and differentiation capabilities of primary human myoblasts, human airway basal cells, and human umbilical vein endothelial cells (HUVECs). Hence, regeneration of composite tissues has an interdependency on culturing and maturation strategies to help accelerate tissue maturation and eventually tissue functionalization.

[0087] Biophysical stimuli are also a significant design consideration for bioreactor environments, requiring the inclusion of chemical, electrical and / or physical stimulation to support a growing tissue. For example, a decellularized muscle will require progenitor muscle cells to help form muscle however complete skeletal muscle development is reliant on functional innervation to mimic neuronal activity typically required in native muscle. One report employed electrical stimulation by immersing carbon rods in the bioreactor environment for the rat forelimb and established an electrical field stimulation to promote muscle cell alignment and differentiation. Previous work on engineered skeletal muscle and application of electrical stimulation has substantial evidence to suggest the positive influence of electrical stimulation on muscle cell differentiation, alignment, and ability to promote myofiber arrangements. This is significant for functional tissue development, as maturation of cell types to the matured phenotype is required for proper tissue development.

[0088] In one aspect, a decellularized heart valve can be recellularized by: (a) delivering a hydrogel encapsulating a plurality of stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells) to a sub-endothelial layer (e.g., spongiosa) of the decellularized heart valve; and (b) contacting a plurality of endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells) with a surface of the decellularized heart valve. In some embodiments, step (b) involves contacting a plurality of endothelial cells with one or both surfaces of the decellularized heart valve.

[0089] In some embodiments, recellularization of the heart valve requires delivery of a plurality of stem cells or interstitial cells to a sub-endothelial layer (e.g., spongiosa) of the decellularized heart valve, e.g., using any decellularizing methods described herein. In some embodiments, the stem cells are mesenchymal stem cells (MSCs) that are isolated from bone marrow of a subject (e.g., a pediatric patient). MSCs are a popular stem cell type commonly used in regenerative medicine and tissue engineering. Biologically, MSCs are stromal cells that exhibit a diversity of properties such as self-renewal, multi-lineage differentiation, immunomodulation and mediating cell-matrix interactions. The use of MSCs is particularly relevant to vascular regeneration through their capacity to enhance angiogenesis and neovascularization by mechanisms that include direct trans-differentiation into vascular cell types and by the secretion of paracrine factors such as vascular endothelial growth factor and basic fibroblast growth factor. The immunomodulatory function of MSCs is also well known and can help promote constructive tissue remodelling and tissue survival following in vivo transplantation by suppression of host immune responses. Altogether, these properties, combined with their ease of acquisition in clinical settings make MSCs an attractive cell source for tissue regeneration and recellularization. Details of MSCs can be found, e.g., in Karantalis, V. et al. "Use of mesenchymal stem cells for therapy of cardiac disease." Circulation Research 116.8 (2015): 1413-1430, which is incorporated herein by reference in its entirety.

[0090] In some embodiments, recellularization of the heart valve requires contacting of a plurality of endothelial cells with a surface of the decellularized heart valve, e.g., using any decellularizing methods described herein. In some embodiments, the endothelial cells are endothelial progenitor cells (EPCs) that are isolated from peripheral blood of a subject (e.g., a pediatric patient). EPCs are also named endothelial colony forming cells. It was long believed that angiogenesis, the formation of new blood vessels in humans, could only occur by the new blood vessels sprouting out of pre-existing vessels. This paradigm changed after researchers detected endothelial cells of hematopoietic origin on the surface of left ventricular assist devices. Earlier studies had also described endothelial-like cells within the peripheral blood stream. Endothelial progenitor cells (EPCs) were then isolated from the circulation. EPCs are also capable of forming new blood vessels, mainly after their recruitment and migration into the ischemic tissue, a process later termed “vasculogenesis.” In contrast to angiogenesis, during vasculogenesis the formation of new blood vessels can occur in the absence of pre-existing blood vessels. Since EPCs do not need pre-existing blood vessels to work, interest in EPC biology has been growing continuously since their discovery. EPCs are now regarded as biomarkers in cardiovascular disease and also as a potential therapeutic tool or target. Details of EPCs can be found, e.g., in Grisar, J.C.., et al. "Endothelial progenitor cells in cardiovascular disease and chronic inflammation: from biomarker to therapeutic agent." Biomarkers in Medicine 5.6 (2011): 731-744, which is incorporated herein by reference in its entirety.

[0091] Additional cells discussed in the disclosure include the followings. Endothelial colony forming cells (ECFCs) are a subtype of endothelial progenitor cells with significant proliferative and angiogenic abilities. They are capable of forming colonies and regenerating endothelial cell populations, making them important for vascular repair and development, valvular interstitial cells (VICs) are the most prevalent cells in heart valve leaflets and are responsible for maintaining the extracellular matrix, which provides the mechanical properties of the heart valve. They originate from endothelial cells and are found in all three layers of the heart valve. Valvular endothelial cells (VECs) cover the surface of heart valve leaflets and play a key role in sensing mechanical stimuli and maintaining tissue homeostasis. They communicate with valvular interstitial cells to ensure the proper function and integrity of the heart valves.

[0092] Before recellularization, the heart valve can be incubated with a suitable buffer (e.g., PBS) supplemented with fibronectin at about 4°C to promote attachment of endothelial cells on the surface. In some embodiments, the fibronectin is diluted by about 1 : 10000 to about 1 : 100, e.g., 1 : 1000. Recellularization by stem cells or interstitial cells in the sub -endothelial layer (e.g., spongiosa) of the decellularized heart valve can be performed prior to, at substantially the same time, or after recellularization by endothelial cells on the surface of the decellularized heart valve. In some embodiments, the stem cells or interstitial cells are delivered to the subendothelial layer (e.g., spongiosa) via hydrogels (e.g., any of the hydrogels described herein). In some embodiments, the hydrogel is a GelMA-HAMA hydrogel and cells are encapsulated by crosslinking with UV light. In some embodiments, the hydrogel is a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel, and the hydrogel has a concentration of about 1% to about 20% P-aminoacrylate synthetic polyethylene glycol (PEG). For example, the concentration is about 1% to about 5%, about 5% to about 10%, or about 10% to about 20%. The cell density within the cell-gel mixture can be about about 0.1 million to about 100 million cells per ml, e.g., e.g., about 0.1 million to about 1 million per ml, about 1 million to about 10 million per ml, or about 10 million to about 100 million per ml. Specifically, the cell-gel mixture can be injected to the sub-endothelial layer (e.g., spongiosa) of the decellularized heart valve via the arterial wall (e.g., the pulmonary artery wall at the leaflet insertion site) or nodulus of the decellularized heart valve. In some embodiments, the valve is incubated for 1 hour to about 12 hours (e.g., about 2 hours) before the valve is recellularized by endothelial cells on the surface.

[0093] In some embodiments, all surfaces (e.g., both sides of the leaflet) of the heart valve are recellularized by contacting with endothelial cells. For example, the plurality of endothelial cells can be contacted with one surface of the decellularized heart valve at a time, at a density of about 0.01 million to about 10 million per cm2, such as about 0.01 million to about 0.1 million per cm2, 0.1 million to about 1 million per cm2, or 1 million to about 10 million per cm2. Due to the relatively low density of the cells, the recellularizing process is also called “seeding” or “reseeding.” The seeding can be performed by placing small droplets of cell suspension onto the surface of the heart valve (e.g., leaflet). The concentration of cells in the suspension can be range from 0.1 million to about 100 million per ml, e.g., about 0.1 million to about 1 million per ml, about 1 million to about 10 million per ml, or about 10 million to about 100 million per ml. In some embodiments, during seeding, the cells are suspended in a suitable buffer or cell culture medium (e.g., any of the suitable buffers or cell culture media described herein). In some embodiments, the suitable buffer or cell culture medium does not include any antibiotics, e.g., PSG. Each surface of the heart valve can be recellularized for about 1 hour to about 12 hours (e.g., about 2 hours) before the valve is flipped over to seed the other surface.

[0094] After recellularization, the reseeded valve can be incubated for an additional time before implantation. For example, the incubation time can be about 1 day to about 10 days, about 2 days to about 8 days, or about 4 days to about 7 days.

[0095] 3. Implantation

[0096] The engineered heart valve (e.g., any of the decellularized or recellularized heart valves described herein) may be incubated with suitable cells, in vitro, prior to use, to provide a ready -to-implant heart valve. In some embodiments, the engineered heart valve may be colonized by a subject's own cells, providing a matrix or scaffold for growth of the cells. Tissues that may be repaired or replaced by the engineered heart valve may be a heart valve or a portion thereof of the subject (e.g., a leaflet). Other components may be included in the engineered heart valve. For example, the engineered heart valve may include DNA, RNA, proteins, peptides or therapeutic agents for the treatment of disease conditions. The engineered heart valve may also include biodegradable and non-biodegradable components.

[0097] In some embodiments, the engineered heart valve described herein includes donor MSCs. Especially, MSCs can be used from another human subject due to their immunosuppressive capabilities. For example, the IMPACT study (form University Medical Center Utrecht) showed that the use of donor MSCs provided an environment to attract and stimulate autologous cells to regenerate in clinical trials. More details can be found, e.g., in Korpershoek, J. V., et al. "Efficacy of one-stage cartilage repair using allogeneic mesenchymal stromal cells and autologous chondron transplantation (IMPACT) compared to nonsurgical treatment for focal articular cartilage lesions of the knee: study protocol for a crossover randomized controlled trial." Trials 21 (2020): 1-11, which is incorporated herein by reference in its entirety.

[0098] In some embodiments, the engineered heart valve described herein includes cells (e.g., any of the stem cells, interstitial cells, and / or endothelial cells described herein) isolated from a subject (e.g., a human subject) who is the recipient of the engineered heart valve. The use of autologous cells from the subject can reduce excessive immune response and / or rejection of the engineered heart valve. For example, once implanted, the engineered heart valve may exhibit a normal function, e.g., normal opening and closing with no observable regurgitation of the blood flow as determined by echocardiography, at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, or 50 years after implantation. The engineered heart valve may also exhibit a normal structure as compared to a native heart valve, at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, or 50 years after implantation.

[0099] In some embodiments, the recellularized cells (e.g., any of the stem cells, interstitial cells, and / or endothelial cells described herein) at implant grow normally in the subendothelial layer (e.g., spongiosa) and / or surfaces of the engineered heart valve, at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, or 10 weeks post implantation.

[0100] Structural and biochemical properties of decellularized tissues

[0101] The extracellular matrix (ECM) is a dynamic molecular network arranged in a tissuespecific ultrastructure that not only acts to structurally support the respective tissue but also regulate various cell functions such as cell migration, proliferation, differentiation, survival, and function to maintain cellular homeostasis. These activities are consequently variable across the unique composition and functional requirements of respective tissues. Retaining such ECM properties provides tissue stability and a framework for recellularization to guide cell activity. Given the inherently complex structure and composition of the ECM, one that is not completely understood, it is difficult to synthesize and bioengineer such a construct. However, through decellularization, a native biologic ECM scaffold with retained ultrastructure and key proteins can be obtained. The significance of using an ECM scaffold is that it is a naturally occurring, highly conserved milieu of structural and molecular components important for cell growth and proliferation. Preserving the ECM can aid recellularization by providing biochemical cues to facilitate cellular attachment, viability, and tissue function.

[0102] The ECM comprises of structural glycoproteins such as fibronectin, glycosaminoglycans (GAGs), and laminin along with fibrous proteins such as collagen and elastin. These key constituents have been of focus in previous recellularization studies. Further, the distribution and relative composition of these key constituents is tissue specific. This factor can explain the variability in the functional, structural, and mechanical properties observed across tissues. This is significant because it introduces an added complexity to the decellularization and recellularization of composite tissues in particular. Methods of evaluating ECM constituents is also inconsistent and not defined by any criteria. Qualitative analyses include histological and immunohistochemistry (IHC)-based evaluations for most ECM components such as collagen, elastin, fibronectin, GAGs, and laminin whereas quantitative assays have been employed for collagen, elastin and GAG quantification. In evaluating recellularization through the lens of the ECM properties, one must consider the interdependency of the types of cells chosen for recellularization and their interactions with the ECM. The ECM is synthesized by the resident cell populations of respective tissues, which becomes more complex in composite tissue models. While extensive reviews and studies have been conducted on the effects of varying decellularization agents and their impacts on ECM proteins, the functions and roles of the listed ECM constituents in how they influence tissue recellularization has yet to be appreciated. Details can be found, e.g., in Adil, A. et al. "Recellularization of bioengineered scaffolds for vascular composite allotransplantation." Frontiers In Surgery 9 (2022): 843677, which is incorporated herein by reference in its entirety.

[0103] 1. Collagen

[0104] As the most abundant component of the ECM, collagen provides structural support in various isoforms with collagen type I primarily the most prominent isoform. Tissues containing basement membranes show collagen IV located in basement membranes of vascular structures due to its ligand affinity with ECs whereas collagen VI serves as a connector between GAGs and functional proteins to collagen I. Other isoforms such as collagen III can be found in submucosal ECM. Given the variations of collagen types and their respective positions across differing parts of the ECM, the complexity required to recreate an ECM can be seen. While ECM scaffolds obtained through decellularization may retain collagen to varying extents, understanding the types of collagens and their respective locations in the ECM is critical to guide recellularization efforts. The relative amounts and locations of collagen types is crucial for cell growth to occur. When recellularizing either isolated or composite tissues, each tissue compartment could be analyzed for its respective collagen types to elucidate maintenance of collagen and how recellularization may have been supported by it. Collagen content has been consistently measured across recellularization studies using either histological or quantitative assay kits, with observably maintained or increased collagen content in most studies. As shown in FIG. 2, after decellularization, the soluble and insoluble collagen content can be measured. In some embodiments, the amount of collagen content (soluble or insoluble) in a decellularized tissue (e.g., the decellularized heart valve described herein) using any of the decellularizing methods described herein is at least 50%, at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% as compared to that of a fresh tissue. In some embodiments, the amount of insoluble collagen content in a decellularized heart valve is at least 50 pg / mg, at least 60 pg / mg, at least 70 pg / mg, at least 80 pg / mg, at least 90 pg / mg, or at least 100 pg / mg. In some embodiments, the amount of soluble collagen content in a decellularized heart valve is at least 0.1 pg / mg, at least 0.2 pg / mg, at least 0.3 pg / mg, at least 0.4 pg / mg, at least 0.5 pg / mg, or at least 0.6 pg / mg. In some embodiments, decellularization using any of the methods described herein does not result in significant decrease of collagen content.

[0105] 2. GAG

[0106] GAGs are negatively charged, linear polysaccharide compounds often found covalently linked to core proteins of proteoglycans. These unbranched polysaccharides, GAGs, can be subdivided into two classes: sulfated GAGs (sGAGs) and non-sulfated GAGs. Sulfated GAGs include heparin sulfate, chondroitin sulfate, and keratin sulfate whereas nonsulfated GAGs include hyaluronic acid. GAGs are involved in cell proliferation, differentiation, growth, and adhesion along with cytokine and growth factor binding. For recellularization, retention of GAGs could be advantageous for retaining the tissue architecture as well as mechanical properties of a decellularized graft. GAGs have been detected in various studies using Alcian blue colorimetric assay kits (sGAG Dye Binding Assay, ALPCO, Salem, Northern Hampshire) or Blyscan™ Sulphated-GAG assay kit (Biocolor LTD, Carrickfergus, Northern Ireland), with the latter being the most common method.

[0107] Although their identified need for providing biochemical cues and regulating cell function, GAGs are sensitive to preserve due to their position in the cell membrane. Reduction of GAGs can impact viscoelastic properties of grafts and the ability to retain water in the ECM. Many GAGs are located in cellular membranes that are often solubilized by decellularization agents. In an investigation of the human ear scaffold, GAGs were significantly reduced in cartilage and skin tissues whereas in a different study on the rat forelimb, 40% of GAGs were retained. Similar decreases in GAG content were reported for decellularized skin / adipose tissue flaps, human and rat face grafts, and penile scaffolds. One report suggested that this GAG reduction does not impact GAG-related functions in such decellularized ECM scaffolds due to the evident ability of recellularization, where cell attachment and proliferation can be observed. During recellularization, selected cell types may secrete ECM factors that influence ECM composition and development.

[0108] As shown in FIG. 3, after decellularization, the GAG content can be measured. In some embodiments, the amount of GAG content in a decellularized tissue (e.g., the decellularized heart valve described herein) using any of the decellularizing methods described herein is at least 30%, at least 35% at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% as compared to that of a fresh tissue. In some embodiments, the amount of soluble collagen content in a decellularized heart valve is at least 0.2 pg / mg, at least 0.25 pg / mg, at least 0.3 pg / mg, at least 0.35 pg / mg, at least 0.4 pg / mg, at least 0.45 pg / mg, at least 0.5 pg / mg, at least 0.55 pg / mg, or at least 0.6 pg / mg. In some embodiments, the amount of soluble collagen content in a decellularized heart valve is less than 0.2 pg / mg, less than 0.25 pg / mg, less than 0.3 pg / mg, less than 0.35 pg / mg, less than 0.4 pg / mg, less than 0.45 pg / mg, less than 0.5 pg / mg, less than 0.55 pg / mg, or less than 0.6 pg / mg. In some embodiments, decellularization using any of the methods described herein may result in significant decrease of GAG content.

[0109] 3. Mechanical properties

[0110] Contingent upon the ECM and tissue architecture are the mechanical properties of scaffolds. Mechanical testing is critical in considering the close relationship of mechanotransduction in influencing cell fate and differentiation, particularly for stem cells that differentiate based on matrix stiffness and elasticity. Further, tissues’ distinct anatomic locations and function as part of a composite tissue dictate the biomechanical forces required in the design of bioreactor systems to mimic physiological conditions of respective tissues, and the types of mechanical tests used for evaluation of the scaffolds. Thus far, mechanical testing has been conducted in decellularized human and porcine ear, rat forelimb, and porcine fasciocutaneous flap scaffolds. Some mechanical properties of these tissues, such as ultimate tensile stress (UST), stiffness, and strain to failure can also be determined. One report performed the most extensive functional evaluations in the rat forelimb by assessing bone mineral density and content using peripheral dual-energy X-ray absorptiometry. Young’s modulus, bending stiffness and strength, and fracture energy were also evaluated. The range of motion in wrist and digit joints was well maintained post-decellularization. In porcine skin flap tissues, biaxial mechanical testing was also used to determine tissue stiffness changes. For muscle regeneration and functionality, myofiber morphometric measurement and isometric contractile force measurement was used. The latter technique is commonly employed for muscle function and muscle testing studies, a translatable component for recellularization studies focusing on muscle regeneration. Other examples include ball burst tests and tensile strength tests used in decellularized human ear scaffolds, and Young’s modulus measurements used in decellularized porcine ear scaffolds.

[0111] No mechanical analyses post-recellularization were conducted in any of these studies. This may be attributable to the challenges in regenerating tissues to their functional states in composite tissues after recellularization. Many of these studies achieved cell engraftment and function after cell seeding but did not pursue recellularization strategies to promote tissue function regeneration which would otherwise require long-term culturing, implementation of biophysical stimuli during recellularization, and considerations for tissue-specific mechanical properties. For example, measurements for shear stress may be considered for blood vessels post-recellularization given that endothelial cells change phenotypes when shear stress is induced. The importance of analyzing mechanical properties post-recellularization cannot be understated as the seeded cells sense and rely on the surrounding mechanical environment and thus, heavily impact composite tissue recellularization outcomes.

[0112] As shown in FIGS. 4A-4C, after decellularization, the bi-axial stress-strain analysis of the tissue can be performed. In some embodiments, the ultimate tensile stress (UTS) at circumferential or radial direction of a decellularized tissue (e.g., the decellularized heart valve described herein) using any of the decellularizing methods described herein is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to that of a fresh tissue. In some embodiments, the stress at failure in the circumferential direction of the decellularized heart valve is at least 500 kPa, at least 550 kPa, at least 600 kPa, at least 650 kPa, or at least 700 kPa. In some embodiments, the stress at failure in the radial direction of the decellularized heart valve is at least 350 kPa, at least 400 kPa, at least 450 kPa, at least 500 kPa, or at least 550 kPa. In some embodiments, decellularization using any of the methods described herein does not result in significant decrease of UTS.

[0113] In some embodiments, the strain to failure (E) at circumferential or radial direction of a decellularized tissue (e.g., the decellularized heart valve described herein) using any of the decellularizing methods described herein is at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% as compared to that of a fresh tissue. In some embodiments, the strain to failure in the circumferential direction of the decellularized heart valve is at least 0.5 8, at least 0.6 8, at least 0.7 8, at least 0.8 8, at least 0.9 8, or at least 1 8. In some embodiments, the strain to failure in the radial direction of the decellularized heart valve is at least 1 8, at least 1.1 8, at least 1.2 8, at least 1.3 8, at least 1.4 8, or at least 1.5 8. In some embodiments, decellularization using any of the methods described herein does not result in significant decrease of strain to failure.

[0114] In some embodiments, the stiffness at circumferential or radial direction of a decellularized tissue (e.g., the decellularized heart valve described herein) using any of the decellularizing methods described herein is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, or at least 120% as compared to that of a fresh tissue. In some embodiments, the stiffness in the circumferential direction of the decellularized heart valve is at least 600 kPa, at least 700 kPa, at least 800 kPa, at least 900 kPa, at least 1000 kPa, at least 1100 kPa, or at least 1200 kPa. In some embodiments, the stiffness in the radial direction of the decellularized heart valve is at least 150 kPa, at least 160 kPa, at least 170 kPa, at least 180 kPa, at least 190 kPa, at least 200 kPa, at least 210 kPa, at least 220 kPa, or at least 230 kPa. In some embodiments, decellularization using any of the methods described herein does not result in significant decrease of stiffness.

[0115] Delivery of cells or cell components and tissue regeneration by injectable hydrogels

[0116] The use of hydrogel -based biomaterials for the delivery and recruitment of cells to promote tissue regeneration in the body is of growing interest. Hydrogels are hydrophilic, water-swollen polymer networks formed from a variety of natural and synthetic polymeric building blocks. These building blocks have been engineered to enable crosslinking by chemical reaction or through physical interactions in the presence of cells and proteins that proceeds rapidly enough for injection and in situ hydrogel formation.

[0117] Injectable hydrogels are prepared using a wide range of materials. Cyto- and biocompatibility as well as reactive chemistries are critical factors for selecting base materials that can be used in hydrogels for cell delivery or recruitment in the body. Material crosslinking (formation and concentration of physical or covalent linkages), biodegradability, and biochemical properties are also important design criteria that can influence the structural, mechanical, and biological properties of the hydrogels initially and over time. In addition, the base polymeric materials must be stable in either solid or solution form for storage prior to use in translational or clinic studies.

[0118] The hydrophilic polymers used for hydrogel construction generally can be divided into two categories: natural polymers derived from tissues or other natural sources and synthetic polymers fabricated using organic chemistry and molecular engineering principles. Biocompatible natural polymers such as hyaluronic acid, chitosan, heparin, alginate, fibrin, collagen, chondroitin sulfate, and silk, mimic aspects of the native microenvironment, including its mechanical and biochemical properties for modulating cell adhesion, migration, and other key functions for tissue regeneration. These natural polymers have been used as building blocks for injectable hydrogel formation by physical (e.g., ionic, hydrogen bonding) or covalent crosslinking (e.g., reaction of functional groups on modified polymers). Synthetic polymers such as poly(ethylene glycol) [PEG], poly(vinyl alcohol) [PVA], poly(N- isopropyl acrylamide) [PNIPAAm], and polycaprolactone [PCL] have frequently been used for the design of injectable, cell -compatible hydrogels due to their commercial availability, low batch-to-batch variation, versatility for chemical modification, and consequently, the ease of tuning the mechanical properties of the resulting hydrogels. Since synthetic polymers lack the inherent biochemical cues for interaction with cells, they are used in combination with natural polymers or biomimetic peptides to facilitate cell adhesion, migration, and protein secretion.

[0119] To capture the favorable attributes of both traditional natural and synthetic polymers, synthetic peptides and recombinant proteins have been designed for injection and subsequent assembly in situ for cell delivery. Examples of such combinations include resilin-containing proteins, tryptophan- and proline-rich sequences in mixing-induced twocomponent hydrogels (MITCH), and peptide amphiphiles with hydrophilic peptide segments (lysine or glutamic acid repeats) conjugated to a hydrophobic fatty acid. These designer building blocks have been engineered with integrin-binding peptide sequences to promote cell adhesion and degradable sequences for cell-driven remodeling. Polymers commonly used in designing injectable hydrogels for cell delivery and tissue regeneration include hyaluronic acid (HA), Alginate, and PEG.

[0120] Crosslinking and degradation reactions can be used for controlling hydrogel formation and erosion. Injectable hydrogels can be formed using a variety of chemical or physical crosslinking strategies, which must be carefully selected to match the specific application of interest. For example, an appropriate crosslinking rate is essential for the proper formation of an injectable hydrogel in situ. If the gelation is too slow, precursors are likely to perfuse from the site of injection into surrounding tissues, leading to poor hydrogel properties and potentially to an inflammatory response to unreacted monomers. If the gelation is too rapid, shear thinning (and potentially premature gel formation) in the syringe may occur, introducing network defects that, in turn, affect gel mechanical properties and retention / release of cargo (i.e., cells). In addition, the ability to form the hydrogel in the presence of live cells and therapeutic proteins is the key for cell delivery and regenerative medicine applications.

[0121] For all cell or cell component delivery and tissue regeneration applications, hydrogels must degrade in a controlled manner after injection for the desired therapeutic use (e.g., cell release or infiltration, protein elaboration) without cytotoxic degradation byproducts. Typically, degradation can be achieved using cleavable polymeric backbones or dynamic / reversible crosslinks. As mentioned earlier, natural polymers such as hyaluronic acid and alginate undergo enzymatic hydrolysis in vivo, providing a mechanism for hydrogel degradation. For synthetic polymers, hydrogels have been traditionally engineered with ester linkages like poly(lactic acid) for degradation by ester hydrolysis in aqueous microenvironments preprogrammed based on the number of esters incorporated. In addition, several research groups have explored additional responsive or triggerable degradable chemistries, including photodegradation, retro Michael type, and retro Diels-Alder reactions, for precise control over material degradation in situ.

[0122] In some embodiments, the hydrogel described herein is a photocrosslinkable hydrogel, a shear-thinning hydrogel, a click-based hydrogel, or a self-assembling hydrogel. In some embodiments, the hydrogel is a gelatin methacrylate-hyaluronic acid methacrylate (GelMA-HAMA) hydrogel, an PNP shear-thinning hydrogel, or a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel.

[0123] In some embodiment, the hydrogel may be functionalized with one or more bioactive agents. For example, the bioactive agents (e.g. small molecules, polypeptides including cytokines and chemokines, differentiation factors, signaling pathway inhibitors etc.) may, for example, facilitate viability of the cells in the resultant engineered tissue and the further development or differentiation of cells. In one embodiment, one or more bioactive agents may be agents selected from the group consisting of: anti-proliferative agents, immunosuppressants, pro-angiogenic compounds, antibodies or fragments or portions thereof, antibiotics or antimicrobial compounds, antigens or epitopes, aptamers, biopolymers, carbohydrates, cell attachment mediators (such as RGD), cytokines, cytotoxic agents, drugs, enzymes, growth factors or recombinant growth factors and fragments and variants thereof, hormone antagonists, hormones, immunological agents, lipids, metals, nanoparticles, nucleic acid analogs, nucleic acids (e.g., DNA, RNA, siRNA, RNAi, and microRNA agents), nucleotides, nutraceutical agents, oligonucleotides, peptide nucleic acids (PNA), peptides, prodrugs, prophylactic agents, proteins, small molecules, therapeutic agents, or any combinations thereof.

[0124] In one embodiment, the cell suspension is mixed with the hydrogel such that the cell density is about 1 x 105, about 5 x io5, about 1 x 106, about 5 x 106, about 1 x io7, about 5 x

[0125] 107, or about 1 x 108cells / ml. ] For example, the cell density is from about 1 x 105to about 1 x

[0126] 108, about 1 x 105to about 5 x 107, about 1 x 105to about 1 x 107, about 1 x 105to about 5 x 106, about 1 x 105to about 1 x 106, about 1 x 105to about 5 x 105, about 5 x 105to about 1 x 108, about 5 x io5to about 5 x 107, about 5 x 105to about 1 x 107, about 5 x 105to about 5 x

[0127] 106, about 5 x io5to about 1 x 106, about 1 x 106to about 1 x 108, about 1 x 106to about 5 x

[0128] 107, about 1 x 106to about 1 x 107, about 1 x 106to about 5 x 106, about 5 x io6to about 1 x

[0129] 108, about 5 x io6to about 5 x 107, about 5 x 106to about 1 x 107, about 1 x 107to about 1 x 108, about 1 x 107to about 5 x 107, or about 5 x io7to about 1 x io8cells / ml. In some embodiments, the cell density i s prior to, at substantially the s; ime time, or after the time when the cells are encapsulated or the hydrogel is solidified.

[0130] In some embodiments, the hydrogel is a photocrosslinkable hydrogel, e.g., a GelMA- HAMA hydrogel, and cells are encapsulated by crosslinking the gel with UV light for about 10 seconds to about 5 minutes (e.g., about 30 seconds) at a UV intensity of about 10 mW / cm2to about 200 mW / cm2. The exact crosslinking time and UV intensity can be adjusted by a skilled person in the art. For example, the crosslinking time can range from 10 seconds to about 5 minutes, from 10 seconds to about 3 minutes, from 15 seconds to about 1 minute, from 20 seconds to about 45 seconds, or from 25 seconds to about 40 seconds. The UV intensity can range from about 10-200 mW / cm2, about 10-150 mW / cm2, 10-120 mW / cm2, 10-100 mW / cm2, 30-200 mW / cm2, 30-150 mW / cm2, 30-120 mW / cm2, 30-100 mW / cm2, 50- 200 mW / cm2, 50-150 mW / cm2, 50-120 mW / cm2, or 50-100 mW / cm2. In some embodiments, the UV intensity is about 80 mW / cm2or about 90 mW / cm2.

[0131] In some embodiments, the hydrogel described herein is a GelMA-HAMA hydrogel, which includes about l%-50% GelMA (gelatin methacrylate), about 0.01%-10% HAMA (hyaluronic acid methacrylate), and about 0.01%-10% PI (2-hydroxy-4’ -(2 -hydroxy ethoxy)- 2-methylpropiophenone), by weight per volume (w / v). For example, the concentration of GelMA can be about 1% to about 50%, about 1% to about 40%, about 1% to about 30%, about 1% to about 20%, about 1% to about 10%, about 1% to about 5%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 10%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, or about 12% to about 18%; the concentration of HAMA can be about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 2%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 2%, or about 0.7% to about 1.5%; and the concentration of PI can be about 0.01% to about 10%, about 0.01% to about 5%, about 0.01% to about 2%, about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 2%, about 0.5% to about 10%, about 0.5% to about 5%, about 0.5% to about 2%, or about 0.7% to about 1.5%. In a particular embodiments, GelMA is about 10% to about 20%, HAMA is about 0.5% to about 2%, and PI is about 0.5% to about 2% in the GelMA-HAMA hydrogel.

[0132] In some embodiments, the hydrogel described herein is a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel. The hydrogel may have a concentration of about 1% to about 20% P-aminoacrylate synthetic polyethylene glycol (PEG). For example, the concentration can be about 1% to about 20%, about 1% to about 15%, about 1% to about 12%, about 5% to about 20%, about 5% to about 15%, about 5% to about 12%, or about 8% to about 12%. In some embodiments, the hydrogel described herein is an PNP shear-thinning hydrogel.

[0133] Depending on the viscosity of the hydrogel used, a skilled person in the art can determine the location to inject the cell -gel mixture to the heart valve, e.g., the arterial wall (e.g., the pulmonary artery wall at the leaflet insertion site) or nodulus of the decellularized heart valve. For example, if the hydrogel has a relatively low viscosity, it is easier to achieve homogeneous distribution within the sub-endothelial layer (e.g., spongiosa) of the heart valve tissue. In some embodiments, the viscosity of the hydrogel is from at least about 0.001 Pascal-second (Pa s) to about 100 Pa s or more, when measured at 25°C. For example, the viscosity can be about 0.001 Pa s to about 0.01 Pa s, about 0.01 Pa s to about 0.1 Pa s, about 0.1 Pa s to about 1 Pa s, about 1 Pa s to about 10 Pa s, or about 10 Pa s to about 100 Pa s.

[0134] The hydrogels described herein may also be sued to deliver a therapeutic agent into a sub-surface layer of a tissue, e.g., a sub -endothelial layer (e.g., spongiosa) of the heart valve tissue. For example, the therapeutic agent can be a peptide, a protein, a growth factor, a cytokine, and / or a chemokine.

[0135] In some embodiments, the cell components described herein can be any molecules that are produced by living cells or organisms, e.g., a protein (e.g., a growth factor or a cytokine), a nucleic acid, a carbohydrate, a lipid, and / or an extracellular vesicle. In some embodiments, the cell components described herein include extracellular vesicles. Details of extracellular vesicles can be found, e.g., in Liu, Y.J., et al. "A review of the regulatory mechanisms of extracellular vesicles-mediated intercellular communication." Cell Communication and Signaling 21.1 (2023): 77, which is incorporated herein by reference in its entirety.

[0136] The hydrogels described herein may be prepared using any of the following methods: thermogelling interactions (hydrophonic or free radicals); ionic interactions; shear-thinning; host-guest interactions / self assembling; enzymatic interactions; photopolyermization, and / or click chemistry.

[0137] Cell culturing and engineered tissues

[0138] The engineered tissue (e.g., the engineered heart valve described herein) can be created by using autogenic cell populations derived from the subject's own tissue. The engineered tissue can also be allogenic or xenogenic, where cell populations are derived from the same species or a mammalian species that are different from the subject. For example, tissue cells can be derived from mammals such as monkeys, dogs, cats, mice, rats, cows, horses, pigs, goats and sheep.

[0139] As described herein, the engineered tissue can be a valve, e.g., a heart valve or a venous valve. The heart has four main valves that ensure blood flows in the correct direction through the heart and to the rest of the body. The four heart valves include a pulmonary valve, an aortic valve, a mitral valve, and a tricuspid valve.

[0140] The isolated cells are preferably cells obtained by a swab or biopsy, from the subject's own tissue. A biopsy can be obtained by using a biopsy needle under a local anesthetic, which makes the procedure quick and simple. The small biopsy core of the isolated tissue can then be expanded and cultured to obtain the tissue cells. Cells from relatives or other donors of the same species can also be used with appropriate immunosuppression.

[0141] Methods for the isolation and culture of cells are discussed by Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107-126. Cells may be isolated using techniques known to those skilled in the art. For example, the tissue can be cut into pieces, disaggregated mechanically and / or treated with digestive enzymes and / or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage. If necessary, enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination. These include but are not limited to trypsin, chymotrypsin, collagenase, elastase, and / or hyaluronidase, DNase, pronase, and dispase. Mechanical disruption can also be accomplished by a number of methods including, but not limited to, scraping the surface of the tissue, the use of grinders, blenders, sieves, homogenizers, pressure cells, or insonators to name but a few.

[0142] Cell types include, but are not limited to, endothelial cells such as human endothelial cells, progenitor cells isolated from the peripheral blood bone that can be induced to differentiate into different cells, stem cells, committed stem cells, and / or differentiated cells may be used. Also, depending on the type of tissue or organ being made, specific types of committed stem cells can be used. For instance, myoblast cells can be used to build various muscle structures. Other types of committed stem cells can be used to make organs or organlike tissue such as heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. Other cells include, but are not limited to, endothelial cells, muscle cells, smooth muscle cells, fibroblasts, osteoblasts, myoblasts, neuroblasts, fibroblasts, glioblasts; germ cells, hepatocytes, chondrocytes, keratinocytes, cardiac muscle cells, connective tissue cells, epithelial cells, endothelial cells, hormone-secreting cells, cells of the immune system, neurons, cells from the heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra, and the like. In some embodiments it is unnecessary to pre-select the type of stem cell that is to be used, because many types of stem cells can be induced to differentiate in an organ specific pattern once delivered to a given organ. For example, a stem cell delivered to the liver can be induced to become a liver cell simply by placing the stem cell within the biochemical environment of the liver.

[0143] Examples also include cells that have been genetically engineered, transformed cells, and immortalized cells. One example of genetically engineered cells useful is a genetically engineered cell that makes and secretes one or more desired molecules. When matrices comprising genetically engineered cells are implanted in an organism, the molecules produced can produce a local effect or a systemic effect, and can include the molecules identified above as possible substances. Cells may produce substances that inhibit or stimulate inflammation; facilitate healing; resist immuno-rejection; provide hormone replacement; replace neurotransmitters; inhibit or destroy cancer cells; promote cell growth; inhibit or stimulate formation of blood vessels; augment tissue; and to supplement or replace the following tissue, neurons, skin, synovial fluid, tendons, cartilage, ligaments, bone, muscle, organs, dura, blood vessels, bone marrow, and extracellular matrix.

[0144] The shape of the extracellular matrix may help send signals to the cells to grow and reproduce in a specific type of desired way. Other factors and differentiation inducers may be added to the matrix to promote specific types of cell growth. Once the tissue has been reduced to a suspension of individual cells, the suspension can be fractionated into subpopulations from which the cells elements can be obtained. This also may be accomplished using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection), separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counterstreaming centrifugation), unit gravity separation, counter-current distribution, electrophoresis and fluorescence-activated cell sorting {see e.g. Freshney, (1987) Culture of Animal Cells. A Manual of Basic Techniques, 2d Ed., A. R. Liss, Inc., New York, Ch. 11 and 12, pp. 137- 168). For example, salivary cells may be enriched by fluorescence-activated cell sorting. Magnetic sorting may also be used.

[0145] Cell fractionation may also be desirable, for example, when the donor has diseases such as cancer or tumor. A cell population may be sorted to separate the cancer or tumor cells from normal noncancerous cells. The normal noncancerous cells, isolated from one or more sorting techniques, may then be used for tissue reconstruction.

[0146] Isolated cells can be cultured in vitro to increase the number of cells available for seeding into the biocompatible substrate (e.g., a decellularized porcine heart valve). To prevent an immunological response after implantation of the artificial tissue construct, the subject may be treated with immunosuppressive agents such as, cyclosporin or FK506. Isolated cells may be transfected with a nucleic acid sequence. Useful nucleic acid sequences may be, for example, genetic sequences which reduce or eliminate an immune response in the host. For example, the expression of cell surface antigens such as class I and class II histocompatibility antigens may be suppressed. In addition, transfection could also be used for gene delivery. Cells may be transfected with specific genes prior to seeding onto the biocompatible substitute. Thus, the cultured cells can be engineered to express gene products that would produce a desired protein that helps ameliorate a particular disorder.

[0147] The tissue cells grown on the engineered tissue (e.g., any of the engineered heart valves described herein) may be genetically engineered to produce gene products beneficial to implantation, e.g., anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1, and anti-IL-2. Alternatively, the tissue cells may be genetically engineered to "knock out" expression of native gene products that promote inflammation, e.g. , GM-CSF, TNF, IL-I, IL- 2, or "knock out" expression of MHC in order to lower the risk of rejection.

[0148] Methods for genetically engineering cells for example with retroviral vectors, adenoviral vectors, adeno-associated viral vectors, polyethylene glycol, or other methods known to those skilled in the art can be used. These include using expression vectors which transport and express nucleic acid molecules in the cells. (See Geoddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Vector DNA is introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989), and other laboratory textbooks.

[0149] Once seeded onto the matrix during recellularization, the cells can proliferate and develop on the matrix to form a tissue layer. Importantly, because the matrix has an infrastructure that permits culture medium to reach the tissue layer, the cell population continues to grow, divide, and remain functionally active to develop into a tissue that has a morphology which resembles the analogous structure in vivo.

[0150] It is important to recreate, in culture, the cellular microenvironment found in vivo for the particular tissue being engineered. By using a matrix that retains an infrastructure that is similar or the same as an in vivo tissue structure, the optimum environment for cell-cell interactions, development and differentiation of cell populations, is created.

[0151] Growth factors and regulatory factors can be added to the media to enhance, alter or modulate proliferation and cell maturation and differentiation in the cultures.

[0152] The growth and activity of cells in culture can be affected by a variety of growth factors such as growth hormone, somatomedins, colony stimulating factors, erythropoietin, epidermal growth factor, hepatic erythropoietic factor (hepatopoietin), and like. Other factors which regulate proliferation and / or differentiation include prostaglandins, interleukins, and naturally-occurring chalones.

[0153] The engineered tissue (e.g., any of the engineered heart valves described herein) can be used in a variety of applications. For example, the engineered tissue can be implanted into a subject to replace or augment existing tissue. The subject can be monitored after implantation of the engineered tissue or organ, for amelioration of the disorder. The engineered tissue can be used in vitro to screen a wide variety of compounds, for effectiveness and cytotoxicity of pharmaceutical agents, chemical agents, growth / regulatory factors. The cultures can be maintained in vitro and exposed to the compound to be tested. The activity of a cytotoxic compound can be measured by its ability to damage or kill cells in culture. This may readily be assessed by vital staining techniques. The effect of growth / regulatory factors may be assessed by analyzing the cellular content of the matrix, e.g., by total cell counts, and differential cell counts. This may be accomplished using standard cytological and / or histological techniques including the use of immunological and / or chemical techniques employing antibodies that define type-specific cellular antigens. The effect of various drugs on normal cells cultured in the engineered tissue may be assessed.

[0154] Method of treatment

[0155] Heart valve diseases are treated by surgical repair of valves, although often the valves are too diseased to repair and must be replaced. Replacement heart valves can be categorized as either artificial mechanical valves, transplanted valves, or tissue valves. Replacement heart valves are designed to optimize hemodynamic performance, thrombogenicity and durability. Another factor taken into consideration is the relative ease of surgical implantation. Mechanical valves are typically constructed from nonbiological materials such as plastics, metals and other artificial materials which, while durable, are expensive and prone to blood clotting which increases the risk of an embolism. Anticoagulants taken to help against blood clotting can further complicate the patient's health due to increased risks for hemorrhages. Transplanted valves are natural valves taken from cadavers. These valves are typically removed and frozen in liquid nitrogen, and are stored for later use. They are typically fixed in glutaraldehyde to eliminate antigenicity and are sutured in place, typically with a stent. Artificial tissue valves are valves constructed from animal tissue, such as bovine or porcine tissue. Efforts have also been made at using tissue from the patient for which the valve will be constructed. Bioprosthetic and homograft valves face limited durability and are more prone to degeneration, especially in pediatric patients as a consequence of their higher metabolic rates and active immune system.

[0156] Provided herein are methods of treating heart valve dysfunction by replacing a diseased heart valve with an engineered heart valve (e.g., any of the engineered heart valves described herein). In some embodiments, the methods of treating heart valve dysfunction disclosed herein include replacing the diseased heart valve by implanting the engineer heart valve (e.g., any of the decellularized or recellularized heart valves described herein). In some embodiments, the engineered heart valve may be colonized by a subject's own cells, providing a matrix or scaffold for growth of the cells. In some embodiments, the engineered heart valve described herein includes cells (e.g., any of the stem cells, interstitial cells, and / or endothelial cells described herein) isolated from a subject (e.g., a human subject) who is the recipient of the engineered heart valve. The use of autologous cells from the subject can reduce excessive immune response and / or rejection of the engineered heart valve.

[0157] As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human, to whom treatment according to the methods of the present invention is provided. Veterinary and non-veterinary applications are contemplated by the present invention. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

[0158] In some embodiments, the methods described in this disclosure involves identifying a subject as having, being at risk of developing, or suspected of having a disorder associated with heart valve dysfunction. Diseases or conditions that cause heart valve dysfunction include, but are not limited to, rheumatic fever, infective endocarditis, congenital heart defects, degenerative valve disease, heart attack, high blood pressure, Marfan syndrome, and radiation therapy.

[0159] Heart valve dysfunction can develop with heart valves stenosis, in which a valve does not open properly, and / or insufficiency, also called regurgitation, in which a valve does not close properly. In addition to stenosis and insufficiency of heart valves, heart valves may need to be surgically repaired or replaced due to certain types of bacterial or fungal infections in which the valve may continue to function normally, but nevertheless harbors an overgrowth of bacteria (vegetation) on the leaflets of the valve that may embolize and lodge downstream in a vital artery. If such vegetations are on the valves of the left side (i.e., the systemic circulation side) of the heart, embolization may occur, resulting in sudden loss of the blood supply to the affected body organ and immediate malfunction of that organ. The organ most commonly affected by such embolization is the brain, in which case the patient suffers a stroke. Thus, surgical replacement of either the mitral or aortic valve (left-sided heart valves) may be necessary for this problem even though neither stenosis nor insufficiency of either valve is present. Likewise, bacterial or fungal vegetations on the tricuspid valve may embolize to the lungs resulting in a lung abscess and therefore, may require replacement of the tricuspid valve even though no tricuspid valve stenosis or insufficiency is present.

[0160] EXAMPLES

[0161] The materials and methods described here have been used to generate the examples described herein.

[0162] Materials and Methods

[0163] / . Cell culture a. Isolation and culture of sheep EPC (shEPC)

[0164] Peripheral blood was drawn from the carotid artery and diluted with heparin to a final concentration of 20 U / mL during the collection procedure to prevent clotting. Next, one volume of isolation buffer (IB) was added to the heparizined blood; IB including 50 mL ACD buffer (22.3 g / L glucose (G6152, Sigma-Aldrich), 22.0 g / L sodium citrate (1613859 USP, Sigma-Aldrich) and 8.0 g / L citric acid (C2404-1006, Sigma-Aldrich) in distilled water), 2.5 g bovine serum albumin (A7906, Sigma-Aldrich) and 450 mL l x phosphate buffered saline (PBS, MT21040CV, Thermo Fisher Scientific). shEPC were isolated from blood using a density gradient-based centrifuge protocol and Ficoll-Paque™ PLUS (17-1440-02, GE Healthcare) as the density gradient medium. In brief, Ficoll-Paque™ PLUS was added to the chambers beneath SepMate™-50 filter inserts (CLS3991, Sigma-Aldrich). On top of each filter insert, 35 mL diluted blood was added and the mononuclear cells were separated by centrifuging tubes for 15 minutes at 1400 rpm with the brake disabled. Afterwards, the plasma layer was aspirated and the lymphocyte layer was collected. For every 5 mL of collected lymphocytes, three volumes of IB were added, and then cells were spun down at 700 rpm for 10 minutes. The supernatant was discarded, and the pellets were resuspended a final time in 10 mL IB. Cells were spun down again at 700 rpm for 10 minutes and the resulting pellet was plated on fibronectin-coated (1 : 1000, FC010, EMD Millipore) 6-well plates. Cells were cultured in 2 mL shEPC-culture medium (shEPC-CM) including: epithelial basal medium (EBM-2, CC-3156, Lonza) with 20% fetal bovine serum (FBS, 10438026, Thermo Fisher Scientific), 1% penicillin / streptomycin / glutamine 100* (PSG, 10378016, Thermo Fisher Scientific) and endothelial cell GM2 supplements (C-39211, PromoCell). The cells were kept in a culture incubator at 21% O2, 5% CO2 and 37°C, henceforth indicated as standard culture conditions. Plates were not disturbed for 48 hours to allow attachment of shEPCs to the well bottoms. On Day 3 of culture, unattached cells were removed through washing with PBS and the remaining cells were supplied with shEPC-CM. Thereafter, the culture medium was changed every two days and colony formation was documented each day. The cells of a colony were passaged 1 :3 after reaching 80-90% confluency by aspirating the medium and washing once with PBS. Then, 0.25% Trypsin-EDTA (25200056, Thermo Fisher Scientific) was used to detach shEPCs from the plates. If detaching took more than 5 minutes, a cell scraper was used. Trypsin was neutralized with shEPC-CM and cells were spun down for 5 minutes at 1200 rpm before being re-plated on fibronectin-coated culture plates. b. Isolation and culture of sheep MSC (shMSC)

[0165] Bone marrow was collected from sheep and diluted to a final concentration of 25 U / mL of heparin during the procedure. For every 25 mL heparinized bone marrow, 10 mL IB was added and shMSC were isolated based on a density gradient. SepMate™-50 tubes were filled with 13-15 mL Ficoll-Paque™ PLUS and 35 mL bone marrow-IB was pipetted on top of the filter inserts. The tubes were centrifuged for 35 minutes at 2700 rpm with the brake disabled. Afterwards, plasma was aspirated, and the mononuclear cell layer was collected. Next, two volumes of IB were added to every 5 mL of collected cells and the cells were spun down for 5 minutes at 2700 rpm. The supernatant was discarded and the pellet was resuspended in 10 mL IB. Cells were pelleted again by centrifugation for 10 minutes at 1200 rpm. The supernatant was removed, and the cells were resuspended in 2 mL IB before 10 mL red blood cell (RBC) lysis buffer (00-4333-57, Invitrogen) was added. Red blood cells were lysed by keeping the mixture on ice for 10 minutes. The buffer was removed by adding another 5 mL IB and centrifuging for 5 minutes at 1200 rpm. The supernatant was aspirated and the pellet was washed with 10 mL IB. Cells were spun down a final time for 5 minutes at 1200 rpm and then plated on 6-well plates with 2 mL mesenchymal stem cell basal medium (MSCBM, PT-3238, Lonza) supplemented with MSCGM® SingleQuots® (PT-4105, Lonza) in each well. The cells were kept in standard culture conditions and plates were not disturbed for 24 hours to accommodate attaching of the MSCs. On Day 3, cells were washed once with PBS before supplying the new MSC-medium. The medium was replaced every two days and the MSCs were passaged 1 :5 after formed colonies reached 80-90% confluency. After two passages, medium was changed from MSCGM® to normal shMSC-culture medium (shMSC- CM), which included: Dulbecco’s modified eagle medium (DMEM, Thermo Fisher Scientific, 31053028, Waltham MA USA) supplemented with 10% FBS and 1% PSG. To passage MSCs, medium was aspirated and cells were washed once with PBS. Next, the MSCs were detached from the culture plates using 0.25% Trypsin-EDTA. Trypsin was neutralized with shMSC-CM and cells were spun down at 1200 rpm for 5 minutes.

[0166] 2. Decellularization and recellularization of porcine pulmonary valves a. Decellularization of porcine pulmonary valves

[0167] Porcine hearts from pigs weighing 20-25 kg were obtained from a certified vendor. The pulmonary valve was excised from the surrounding tissue and the valve was cut open carefully at one commissure to keep all three leaflets intact. The valves were washed once in PBS and one fresh leaflet was removed for either biological or mechanical analysis. Then, the remaining valve leaflets were transferred to 50 mL tubes containing a decellularization solution. The tissue was decellularized at 4°C on a rocking plate (VWR® 100 Rocking Platform Shaker, speed level 7) during several cycles: 3 cycles of 12 hours in demineralized water (demi-ELO) with 0.5% sodium deoxycholate (SDC, D6750, Sigma-Aldrich) and 0.5% sodium dodecyl sulfate (SDS, 71725, Sigma-Aldrich), followed by 3 cycles of 24 hours in demi-ELO supplemented with 1% PSG and 0.05% sodium azide (s8032, Sigma-Aldrich). Each time the solution was replaced, the valves were washed in H2O by thoroughly shaking the tubes. After the decellularization protocol was completed, the decellularized valves were washed for 24 hours in PBS at 4°C on the rocking plate (speed level 7). Then, a cardiac surgical fellow trimmed the pulmonary artery wall before sterilization to leave only a small rim adjacent to the leaflet insertion line. The valve leaflets were washed in H2O to remove the decellularization solution and sterilized for 5 min in 70% ethanol, followed high UV-light for 3 minutes on each side at 90 mW / cm2and overnight in 30% PSG at 4°C. After sterilization the valves were washed thoroughly in H2O to remove traces of ethanol and antibiotics, and one decellularized leaflet was removed for the biological or mechanical analysis. The sterile valves were kept in PBS at 4°C for several days before recellularization. b. Recellularization of porcine pulmonary valves

[0168] To promote attachment of EPC on the leaflet surface, the valves were incubated in PBS with 1 : 1000 fibronectin for 1 hour at 37°C. Before seeding, the valves were washed three times in H2O. Lyophilized gelatin methacrylate (GelMA) and hyaluronic acid methacrylate (HAMA) were dissolved in PBS to a final concentration of 5% GelMA, 1% HAMA and 1% PI (2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone, 410896, Sigma-Aldrich). Both the GelMA and HAMA had been produced by at the Massachusetts Institute of Technology (MIT). The GelMA was mixed with shMSCs to a final concentration of 6-8 x 106cells / mL. Cells were encapsulated by crosslinking the gel with UV-light for 30 seconds at 80 mW / cm2. The GelMA-HAMA-shMSC gel was transferred to an insulin syringe (29G, 8881600350, MedontheGo.com) and injected into the spongiosa of the decellularized leaflet via the pulmonary artery wall at the leaflet insertion line. After injection, the leaflet was moved to a 6-well plate and seeded with shEPC. This seeding was achieved by resuspending the cell pellet containing 3-4 x 106cells in 200 pl shEPC-CM without PSG and placing small droplets onto the surface of the leaflet. The reseeded valves were placed on an orbital shaker platform (VWR® Standard orbital shaker, speed level 2) inside a cell culture incubator for two hours, under standard culture conditions, after which 5 mL shEPC-CM without PSG was added to each well. The next day, medium was aspirated and the valve was flipped over to seed the other surface with shEPC. Again, the pellet was resuspended in shEPC-CM without PSG and the leaflet surface was covered. Cells were allowed to attach for 2 hours before the leaflets received 5 mL shEPC-CM without PSG. The reseeded valves were kept on the orbital shaker (speed level 2) under standard culture conditions for up to 4-7 days before use. The medium was checked daily for signs of infection, in which case the experiments was terminated.

[0169] 3. Biological assays

[0170] Leaflets were collected at various time points. A fresh leaflet was removed on Day 0 of decellularization and a decellularized leaflet was collected after sterilization. Each leaflet was cut in half and its wet weight was documented. One half was used to determine the collagen concentration whereas the other half for determining the glycosaminoglycan (GAG) content. The halves were transferred to separate Eppendorf® tubes and were snap frozen immediately. Samples were stored at -20°C. a. Soluble and insoluble collagen assay

[0171] The collagen concentration in fresh and decellularized porcine heart valves was determined using the Sircol™ Soluble Collagen Assay (Biocolor) and the Sircol™ Insoluble collagen assay (Biocolor). Assays were performed according to the manufacture’s instructions, with minor adjustments. Soluble collagen was obtained from snap-frozen leaflet halves by adding 1 mL extraction solution: 0.1 mg / mL pepsin (P7000, Sigma- Aldrich) and 0.5 M acetic acid (A9967, Sigma-Aldrich) in diEEO. The samples were incubated overnight at 4°C. The solution was collected and stored at 4°C while the remaining valve tissue was incubated with 1 mL fragmentation reagent for 2 hours at 65°C and vortexed regularly to extract insoluble collagen. The extraction solution was neutralized by adding cold 100 pl acid neutralization reagent to every sample. Next, 200 pl isolation and concentration reagent was added. Samples were incubated at room temperature for 30 minutes and vortexed regularly. 100 pl of the soluble and insoluble samples were collected, and the remaining samples were stored at -20°C. To visualize the soluble and insoluble collagens, 1 mL Sircol™ dye was added to every sample. The reactions were incubated for 30 minutes at room temperature and vortexed regularly. Afterwards, dyed collagen was spun down for 10 minutes at 12000 rpm. The supernatant was removed carefully through aspiration and the pellet was dried using a super-absorbent tissue. To each pellet, 500 pl Sircol™ alkali reagent was added and the samples were vortexed until the pellets dissolved. Finally, 200 pl duplicates of the samples and standard line were pipetted into a clear 96-well plate and absorbance was measured at 550nm. b. Glycosaminoglycan assay

[0172] The glycosaminoglycan (GAG) concentrations of fresh and decellularized porcine pulmonary valves were determined using the Blyscan™ Glycosaminoglycan Assay according to the manufacturer’s instructions with minor modifications. In short, GAG was extracted from snap-frozen tissues by incubating the samples overnight at 60°C in 1 mL extraction solution, which included 10 mM L-cysteine (168149, Sigma-Aldrich) and 25 mg / mL papain (P3375, Sigma-Aldrich) in PBE buffer (14.21 mg / mL Na2HPO4 (255793, Sigma-Aldrich) and 2.29 mg / mL edetic acid (1233508, Sigma-Aldrich) in distilled H2O, pH 6.5). For each sample, 100 pl was removed and used in the assay, and remaining samples were stored at - 20°C. To color GAG, 1 mL dye reagent was added to every sample. The reactions were incubated for 30 minutes and vortexed regularly, and dyed GAG was spun down at 12000 rpm for 10 minutes. The supernatant was discarded, and the pellets were carefully dried using a super-absorbent tissue. Then, pellets were dissolved in 500 pl dissociation reagent and vortexed. 200 pl duplicates of each sample were pipetted into a standard 96-well plate and the absorbance was measured at 656 nm.

[0173] 4. Mechanical analysis: bi-axial stress-strain

[0174] To obtain the ultimate tensile strength (UTS), ultimate tensile strain (E, or strain to failure) and the elasticity (O) of the heart valves a biaxial stress-strain analysis was performed. The data were collected using a Biaxial Test System (BioTester, CellScale) and the accompanying LabJoy software. The machine was equipped with 5N Load Cells and specimens were mounted using 0.5 mm BioRakes. Before mounting, the thickness of each sample was measured in pm using a thickness gauge tester. The circumferential direction of the valve fibers aligned along the X-axis, and the radial fiber direction was aligned along the Y-axis of the machine. The distance between the actuators was set to 5000 pm to accommodate the length of each sample. The tissues were stretched for 20 seconds followed by a 10 second recovery period, and the collection frequency of data was set to 10 Hz. For each direction, the displacement (pm) and accompanying force (mN) were measured. The raw data were transferred to Microsoft Excel for further analysis, and the stress and strain for both fiber directions were calculated according to the following formulas:

[0175] ( vIII) radial (y) stress = - ’J JPorcey

[0176] . . , Displacementy radial (y) strain = - -

[0177] Lengthy

[0178] The data were graphed as a stress-strain curve in Excel, and the UTS and E were extracted. The stiffness was determined by calculating the slope of the stress-strain curve. 5. OCT embedding of tissue samples

[0179] Tissues were fixed overnight in 10% neutral buffered formalin (NBF-4-G, Azer Scientific) and then transferred to 30% sucrose (21600, Electron Microscopy Sciences) for 24 hours at 4°C. Next, the tissues were kept in a 1 :1 mixture of 30% sucrose and Tissue-Tek® embedding solution (OCT compound, 4583, Sakura) for 2 hours at 4°C. Finally, the samples were transferred to disposable plastic molds (22-363-555, Fisherbrand) and fixed in 100% Tissue-Tek®. Samples were processed by the Brigham and Women’s Hospital core facility pathology department. Sections were cut with a cryostat at 15 pm thickness and stained with haematoxylin and eosin (HE). Slides were imaged with brightfield microscopy.

[0180] 6. Implanting of a reseeded decellularized porcine pulmonary leaflet in an ovine model

[0181] The in vivo study was performed at the Animal Research Children’s Hospital (ARCH). All procedures were approved of and carried out under ARCH protocols by the groups’ research staff and cardiac surgical fellows. Welfare of the animals was documented by the ARCH Large Animals surgical technicians and medication was prescribed by the ARCH veterinarian. a. Implantation

[0182] The animals (n=3) underwent a single pulmonary artery leaflet replacement via leftthoracotomy (Table 1). Echocardiography was performed directly after implantation, before closing of the chest, to confirm valve leaflet motion. An additional transthoracic echo was performed on the animal ten days after the surgery to document the pulmonary valve function.

[0183] Table 1. Overview of animals for in vivo experiments b. Explant

[0184] After four to six weeks of survival, the animal was anesthetized and the leftthoracotomy was reopened. The pulmonary valve was again visualized by echocardiography before the animal was heparinised and sacrificed. The pulmonary valve was removed and visually inspected for the presence of thrombus. Then, the xenograft leaflet and root as well as the native leaflet and root were removed from the surrounding tissue and embedded for histological analysis.

[0185] 7. Data analysis

[0186] The data were analyzed and graphed in GraphPad Prism 7.0. Results are displayed as the mean ± standard deviation (SD). Normal distribution of the data was confirmed using the Shapiro-Wilkinson test for paired data. Difference between groups were tested using a paired t-test: P-values below 0.5 were assumed significant.

[0187] Example 1. Isolation of shEPC and shMSC

[0188] After isolation of the buffy coat from peripheral blood using a density gradient, the cell suspension was plated on fibronectin-coated 6-well plates. Within 48 hours, cells had attached to bottoms of the wells and the remaining cells were washed away. Attached cells formed colonies in one to six weeks. The attached cells were recognized as EPC, due to their characteristic cobblestone-like morphology. After allowing colonies to reach 80-90% confluency, they were passaged to expand in preparation for the seeding procedures. The isolation procedure succeeded for sheep 744 and sheep 714. However, the cells isolated from sheep 475 developed into non-shEPC-like cells within the colonies, therefore the leaflet intended for this sheep was not seeded with shEPC. Mononuclear cells were isolated from bone marrow using a density gradient and plated on standard 6-well plates without additional coating. Selection of shMSC from the cell suspension occurred based on attachment of cells within 48 hours, after which unattached cells were removed through washing. Early colonies formed within one week, and cell morphology indicated the attached cells were shMSC. The colonies were grown in MSCGM® for the first two passages, which increased the pace of colony formation. After the third passage, medium was switched to normal shMSC-CM for the remaining expansion period. For all three animals, shMSC were successfully isolated and expanded.

[0189] Example 2. Decellularization and trimming of porcine pulmonary valves

[0190] The pulmonary valves from pigs weighing 20-25 kg were excised, cut open and washed in PBS. FIG. 1A shows a representative image of a fresh porcine pulmonary valve. The pulmonary root containing the valve measured 40-60 mm in circumference at the level of the sino-tubular junction, or 14-18 mm in diameter. During the decellularization process, the tissue lost its red color, although the ventricular muscle attached to the valve remained slightly brown in color. On the final day of the decellularization process, the valves increased in size slightly due to swelling (FIG. IB). Before sterilization, the valve was trimmed to remove all non-leaflet tissue except the rim of pulmonary artery wall at the leaflet insertion site by the cardiac surgical fellow (FIG. 1C). Generally, one of the three leaflets was sacrificed to obtain two completely intact decellularized leaflets. The surrounding tissue was cut away, leaving only the base of the pulmonary artery wall at the valve insertion site, which was necessary for suturing the leaflet into the recipient animal’s pulmonary root. The trimmed leaflets were then sterilized as described in the Materials and Methods section.

[0191] 1. Biological composition of fresh and decellularized porcine pulmonary valves

[0192] To ascertain whether the decellularization procedure removed key ECM components, the collagen and GAG content of fresh and decellularized porcine valves was determined. A total of five pulmonary valves (N=5) was used, which each yielded three leaflets. One leaflet was collected immediately as the fresh sample, the other two were decellularized. After sterilization a second leaflet was harvested as the decellularized sample. Each leaflet was halved: one half for collagen extraction and the other half for GAG extraction.

[0193] 2. Soluble and insoluble collagen concentration

[0194] The soluble and insoluble collagen content was measured using the Sircol™ soluble collagen assay and the Sircol™ insoluble collagen assay (Biocolor, UK) (FIG. 2). The amount of collagen was normalized to the wet weight of samples. There was no difference observed in the insoluble collagen content of fresh leaflets compared to decellularized leaflets (P > 0.05). The concentration of insoluble collagen was 107.20 ± 27.84 pg / mg in fresh leaflets and 96.91 ± 18.86 pg / mg in decellularized leaflets. Additionally, no difference was observed between the soluble collagen content of the three groups (P > 0.05). Fresh leaflets contained 0.61 ± 0.18 pg / mg soluble collagen and decellularized leaflets measured at 0.36 ± 0.16 pg / mg.

[0195] 3. GAG concentration

[0196] The amount of glycosaminoglycan (GAG) in fresh, decellularized and recellularized leaflets was determined using the Blyscan™ Glycosaminoglycan assay (Biocolor, UK). During decellularization, a significant amount of GAG were lost (P < 0.05). Specifically, fresh leaflets contained 0.71 ± 0.08 pg / mg GAG compared to 0.44 ± 0.08 pg / mg in decellularized leaflets (FIG. 3).

[0197] Example 3. Bi-axial stress-strain analysis of fresh and decellularized pulmonary valves

[0198] The mechanical characteristics of fresh and decellularized pulmonary leaflets were measured using a Biaxial Test System from CellScale (FIGS. 4A-4C). A stress-strain curve was plotted for both the circumferential and radial fiber direction, from which the UTS, strain and elasticity were deduced. Fresh leaflets failed in the circumferential direction at UTS = 703 ± 226 kPa, which was slightly higher than that of decellularized leaflets at UTS = 631 ± 129 kPa (P > 0.05). The stress at failure in the radial direction was comparable for both conditions (P > 0.05), fresh leaflets failed at UTS = 512 ± 230 kPa compared to decellularized leaflets at UTS = 459 ± 120 kPa. Accompanying the UTS is the strain (E) at this point, which did not significantly change during decellularization for both fiber directions (P > 0.05). The circumferential fibers failed at E = 1.46 ± 0.48 for fresh leaflets and at E = 0.85 ± 0.17 for decellularized leaflets. Radial fibers of fresh tissue failed at E = 1.53 ± 0.47 and decellularized ones at E = 1.35 ± 0.36. The modus of elasticity (E), also the stiffness of the leaflets, was deduced from the slope of the stress-strain curve. The stiffness of the circumferential fibers increased slightly during decellularization: E = 790 ± 367 kPa in fresh samples compared to E = 933 ± 301 kPa in decellularized sample (P > 0.05). This increase was not seen in the radial direction, where fresh material was slightly more elastic (E = 220 ± 90 kPa) compared to decellularized samples (E = 190 ± 38 kPa).

[0199] Example 4. Histology of fresh and decellularized leaflets

[0200] To confirm the removal of cells by the decellularization protocol, and to assess the collagen structure, fresh and decellularized leaflets were stained with HE. Samples fixed in 10% formalin were subsequently embedded in OCT compound according to the protocol described herein. Tissue sections were cut at 15 pm thickness using a cryostat and stained with HE by the Brigham and Women’s Hospital histology department. The slides were imaged using brightfield microscopy, and representative images captured of the pulmonary artery wall and leaflet are shown in FIG. 5. Cell nuclei were stained in dark blue and collagen fibers were stained in pink. The tightly packed collagen fibers of the ventricularis were visible as a thin layer (arrow 1), followed by the GAG-rich spongiosa (arrow 2) and the fibrosa layers, which was also rich in collagen (arrow 3). Fresh leaflets were evenly populated by cells in the spongiosa and both surfaces were covered with an endothelial layer. Decellularized leaflets showed the same structure compared to fresh leaflets, albeit there were no cells present. This indicates that the decellularization process was successful at removing cells whilst keeping the general ECM structure intact.

[0201] Example 5. Animal studies for implantation of reseeded decellularized valves

[0202] In order to observe the performance of the decellularized valves, a single pulmonary valve leaflet replacement was performed in three sheep. The leaflets to be implanted were injected with shMSC encapsulated in GelMA-HAMA, which had been isolated from each animals’ bone marrow. Additionally, the surface of the leaflets intended for sheep 744 and sheep 714 were seeded with their respectively isolated shEPC. For all animals, the echocardiography performed at implant showed normal opening and closing of the pulmonary valves with little to no regurgitation of the blood flow. This was also the observation at explant for all animals. FIG. 6 shows the pulmonary valves of the animals at explant. The explanted pulmonary valves showed the implanted leaflet in the middle, with the two remaining native leaflets on both sides. The implants showed no signs of fibrosis and the leaflets were supple. The pulmonary valve of sheep 475 was explanted after 6 weeks in vivo, and the implanted leaflet showed a thrombus. Upon closer inspection, the cardiac surgical fellow present at the explant determined this thrombus to be fresh, possibly having formed during the explant procedure or even post-mortem prior to explant. A similar observation was made at the valve explant of sheep 744 after 4 weeks in vivo, whose implanted leaflet also showed a fresh thrombus. A similar observation was made at the valve explant of sheep 714 after 4 weeks in vivo, whose implanted leaflet did not show thrombus.

[0203] The histology of the implanted leaflets was inspected by HE staining. A pre-implant leaflet was included for reference material, as well as a native leaflet from the explanted valve. The histology results for sheep 475 are presented in FIG. 7. No images for the preimplant leaflet were available, due to problems in specimen processing. The collagen fibers, stained pink, was more organized in the native leaflet of sheep 475 compared to the explant. However, the three-layered structure of the explant was relatively similar to the native tissue. The explanted leaflet was colonized by cells, as indicated by the blue-stained nuclei. At the base of the leaflet, partial epithelization and the presence of cells in the spongiosa was observed. At the more distal parts of the leaflet, the endothelization was more sporadic and few cells were observed in the spongiosa layer. This same pattern of cell distribution was observed in the explant of sheep 744, where the spongiosa near the base of the leaflet was more evenly populated with cells compared to the leaflet (FIG. 8). However, the endothelization was continuous in this animal over the entire valve leaflet and the deposition of collagen was highly organized. Interestingly, no cells were observed in the pre-implant leaflet in either the spongiosa or on the leaflet surface. Although the pre-implant for sheep 714 did show some cells in the spongiosa layer, these were only located near the base of the leaflet (FIG. 9). This pre-implant leaflet did not appear to contain cells in either the spongiosa or on the leaflet surface. In neither explanted leaflet was there any evidence of inflammatory cells or immune reaction.

[0204] As a summary, decellularization of porcine pulmonary valves was investigated using a sodium dodecyl sulphate (SDS)-based protocol. Meanwhile, sheep-derived EPC (shEPC) and sheep-derived MSC (shMSC) were isolated from blood and bone marrow, respectively. The effects of the decellularization protocol on the leaflets were investigated by performing a bi-axial stress-strain analysis. Additionally, the biological composition of the leaflets was studied by measuring the GAG and collagen content of the leaflets. For the in vivo studies, a photo-crosslinkable hydrogel of gelatin methacrylate (GelMA) enriched with hyaluronic acid methacrylate (HAMA) was used to encapsulate shMSC GelMA contains cell binding motifs and target sequences for ECM-remodel enzymes, and it is biocompatible and biodegradable. Presence of hyaluronic acid aids in recapitulating the native tissue ECM, as it is the most prominent GAG in the native valve leaflet, and has proven beneficial to MSC functionality. shMSC were encapsulated in GelMA-HAMA and injected into the spongiosa of the decellularized leaflets via the arterial wall at the site of leaflet attachment and shEPC were seeded on the leaflet surface. The reseeded leaflets were characterized on their in vivo functionality by performing a single pulmonary leaflet replacement in an ovine model. The performance of the valves was documented by echocardiography at implant and explant. Additionally, the histological organization of the tissues was investigated through HE staining.

[0205] Example 6. Methods for cell delivery in decellularized heart valves using injectable hydrogels

[0206] To address the growth and remodeling of decellularized valves, we have developed a novel recellularization method to uniformly repopulate decellularized xenograft leaflets as well as control the cell type seeded inside the decellularized tissue by using injectable hydrogels. A schematic workflow is shown in FIG. 10.

[0207] / . Study design a. To investigate the feasibility of this method

[0208] Sheep valvular interstitial cells (VICs) were isolated and cultured in vitro. The VICs were combined with a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel with a click-like polymerization and injected into decellularized valves. These TEHVs were cultured for 0, 5, or 7 days in the following groups and fixed for histology processing:

[0209] 1. Aortic valve recellularized (n=l), Day 0

[0210] 2. Aortic valve recellularized (n=4), Day 5

[0211] 3. Aortic valve recellularized (n=2), Day 7

[0212] 4. Pulmonary valve recellularized (n=4), Day 7 b. To investigate the translation of this method using more readily available cell sources

[0213] Three sheep underwent a single pulmonary valve leaflet replacement via leftthoracotomy. Autologous mesenchymal stem cells (MSCs) and endothelial colony forming cells (ECFCs) were isolated and cultured in vitro. The decellularized valve scaffolds were injected with the MSCs encapsulated in a GelMA-HAMA hydrogel and for two sheep the outside of the leaflets were additionally seeded with ECFCs. The TEHVs were cultured in vitro and implanted in the donor matched sheep for 4-6 weeks. c. Generation of decellularized leaflets

[0214] Decellularization of aortic and pulmonary heart valves was performed using a detergent-based protocol. Briefly, porcine pulmonary and aortic valved conduits were treated by 4 cycles of 12 hours each with 0.5% sodium dodecyl sulfate and 0.5% deoxycholate, followed by a 24 hour washing step. Subsequently the leaflets were treated with 3 cycles of 24 hour with 0.05% of sodium azide and 1% penicillin / streptomycin. The valves were washed in sterile water between each step. Throughout the entire decellularization process, valves were kept at 4°C on a rocking plate (VWR® 100 Rocking Platform Shaker). The decellularized valves were trimmed and sterilized in 70% ethanol under high UV-light for 3 minutes on each side at 90 mW / cm2followed by Dulbecco's Modified Eagle Medium (DMEM) supplemented with 30% Pen Strep Glutamine (PSG) overnight. Before seeding, the valves were treated with Human Plasma Fibronectin. d. Cell isolation All cells were obtained from female juvenile Dorset sheep that were housed in the Animal Research Children’s Hospital Boston (ARCH). Valvular interstitial cells were obtained as described in Gould, R.A., and Butcher, J.T. "Isolation of valvular endothelial cells." Journal of Visualized Experiments'. JoVE 46 (2010): 2158. Briefly, ovine leaflets were collected and valvular endothelial cells were removed by enzymatic digestion and physical disruption. Next, the leaflets were incubated overnight in a 600 U / mL collagenase solution and the isolated cells were cultured in DMEM low glucose supplemented with 1% fetal bovine serum (FBS; Gibco™, 10082-147) and 1% PSG.

[0215] The isolation of ECFCs and MSCs was performed as described herein. In short, blood samples were obtained from the right femoral vein, and heparin was added to a final concentration of 20 U / ml. For the isolation of MSCs, bone marrow samples were extracted from sheep femurs and heparin was added to a final concentration of 28 U / mL. Isolation buffer was prepared to include: 90% phosphate buffered saline (Gibco™, 10010-023), 10% distilled water, glucose (Sigma Aldrich, G8270) (0.223% (w / v)), sodium citrate (Sigma- Aldrich, S4641) (0.22% (w / v)), citric acid (Sigma-Aldrich, C2404) (0.08% (w / v)), and bovine serum albumin (BSA, Sigma-Aldrich, A7906) (5% (w / v). The samples above were combined, and the ECFCs and MSCs were isolated by density gradient centrifugation using Ficoll-Paque™ Plus as the medium according to the manufacturer’s protocol. Cells were cultured up till passage 3- 4 before being used to seed the scaffolds. e. Gel formation and cell encapsulation

[0216] Lyophilized GelMA and HAMA were dissolved in PBS to a final concentration of 5% GelMA, 1% HAMA and 1% PI (2-hydroxy-4’-(2-hydroxyethoxy)-2-methylpropiophenone, 410896, Sigma- Aldrich). The GelMA was mixed with shMSCs to a final concentration of 6-8 x 106cells / mL. Cells were encapsulated by crosslinking the gel with UV-light for 30 seconds at 80 mW / cm2. The P-aminoacrylate synthetic polyethylene glycol (PEG) hydrogel with a click-like polymerization was created by mixing with VICs to a final concentration of 5%- 10%. The cell concentration was about 0.5 x 106to about 1 x io6cells per mL.

[0217] Using an insulin syringe (28G x 12.7 MM (1 / 2”) BD, 329461), 500 pL of the cell- loaded hydrogel was injected into the spongiosa layer of each valve. The surface of the leaflet was seeded with isolated ECFCs at a density of 2.5-3 x io5cells / cm2. Next, the leaflet was placed in an incubator to allow cell attachment. After 2 hours, EBM-2 medium without PSG was added. The next day, the leaflet was turned, and the other side of the construct was seeded with isolated ECFCs at a density of 2.5-3 x io5cells / cm2. The valves were cultured in EBM-2 medium without PSG until further use. f In vivo implantation

[0218] The study was approved by the local Animal Experimentation Unit Ethical Committee. Female juvenile Dorset sheep (n=3) underwent a single pulmonary artery leaflet replacement via left-thoracotomy using cardiopulmonary bypass. Echocardiography was performed directly after implantation, before closing of the chest, to confirm valve leaflet motion. An additional transthoracic echo was performed on the animal 10 days after the surgery to document the pulmonary valve function. After four to six weeks of survival, the animals were anesthetized and the left-thoracotomy was reopened. The pulmonary valve was again visualized by epicardial echocardiography before the animal was heparinised and sacrificed. The pulmonary valve was removed and visually inspected for the presence of thrombus. Then, the xenograft leaflet and root as well as the native leaflet and root were removed from the surrounding tissue and embedded for histological analysis. g. Valve evaluation

[0219] To visualize the movement of the leaflet, an intraoperative echocardiogram (Philips, iE33TMmachine) was performed post implant after the animal was taken off bypass and again before sacrifice, respectively after 4 (n=2) and 6 (n=l) weeks, to visualize the movement and regurgitation of the leaflet. All leaflets were macroscopically examined, fixed, and stained with haematoxylin and eosin (HE). On the explanted leaflets, additional immunohistochemistry staining was performed by the following antibodies: CD31 (PECAM- 1) (Novus Biologicals, NB100-65900) at a dilution of 1 : 100 in combination with VE- Cadherin (Abeam, ab33168) at a dilution of 1 :200; anti smooth muscle actin (Sigma, A2547- 100) at a dilution of 1 :200 in combination with Vimentin at a dilution of 1 :300 (Cell Signaling, 5741); CD64 (Abeam, abl40779) at a dilution of 1 :200 in combination with Von Willebrand Factor (Abeam, ab6994) at a dilution of 1 : 1200; CD163 (Biorad, MCA1853) at a dilution of 1:250 in combination with iNOS (Abeam, ab3523) at a dilution of 1 :400.

[0220] 2. Results a. Assessment of decellularization Aortic and pulmonary porcine leaflets were decellularized using an in-house used protocol as described herein. The removal of cells inside the leaflets was confirmed by HE and DAPI stainings (FIG. 11). To evaluate the extracellular matrix proteins, collagen, elastin and GAG after decellularization, the decellularized leaflets were compared to native leaflets. Collagen and elastin did not significantly decrease and the GAG content slightly decreased, while the overall leaflet ECM structure was found to be intact (data not shown). b. Selection of hydrogel for cell distribution inside the decellularized scaffolds To deliver cells inside the spongiosa layer of the decellularized leaflet, the most important parameter for the hydrogel is that it needs be injectable and has a favourable solgel transition. To investigate a novel delivery method, a widely used GelMA-HAMA hydrogel, an PNP shear-thinning hydrogel, and a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel with a click-like polymerization developed at MIT were tested. All gels were able to be injected as a liquid a to solidify into the dECM, in theory making them all suitable for this injection method. However, the PNP shear-thinning hydrogel showed complications including sterilization challenges, batch-to-batch variation, and damage of the ECM at the injection site (FIG. 12).

[0221] Using the GelMA-HAMA hydrogel, the results showed the precursor solutions could not be injected as a liquid and polymerize once inside the leaflet through the outside of the leaflet. Therefore, a very low-viscous hydrogel was mixed with cells and polymerized before injection. This hydrogel-cell mixture was injected into the interstitial space, but due to the relative high viscosity, no homogeneous distribution throughout the leaflet was obtained. In particular, no gel and cells were observed in the tip region of the leaflet. After 1 month of culture in vitro, the cells were still viable and inside the scaffold (FIG. 13A). As shown in FIG. 13B, an endothelial layer was observed on all explants. In particular, the endothelial cells were observed in the tip region of the leaflet. As shown in FIGS. 13C-13D, MSCs were observed in the tip and the belly regions of all leaflets. As shown in FIG. 13E, Ml (iNOS) and M2 (CD 163) macrophages were observed in the anulus of the leaflet.

[0222] The p-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel with a click-like polymerization can be injected as a liquid and polymerize inside the scaffold. In addition, the gelation timeframe is tuneable. Here, a gelation timeframe of 1 minute was used, which resulted in a homogenous gel-cell distribution throughout the entire scaffold, including the tip region (FIGS. 14A-14C). A homogeneous distribution was observed for both pulmonary and aortic leaflets including cell survival up to 7 days in vitro. In addition, DAPI staining (FIGS. 14D-14E) and HE staining (FIGS. 14F-14G) of the transverse cross sections showed that the cells were observed throughout the entire leaflet (not just one plane). c. Optimization of entry site to ensure homogeneous cell distribution

[0223] The injection site is of great importance to homogeneously distribution and to allow cells to move inside the scaffold with minimal effects on the leaflet architecture. To minimalize the surface damage of the leaflet, and the activation of the anticoagulant cascade or the attraction of immune cells, the injection was conducted via the wall of the leaflet inside the annulus area, leaving the outer valve leaflet intact. However, this did not result in a homogeneous distribution of the gel.

[0224] Therefore, two primary entry routes were chosen, injection from the wall and injection from the nodulus as indicated in FIGS. 15-17. Depending on the type of gel that is used, the best site to inject should be determined. If the gel has a higher viscosity upon injection, the wall is the most suitable area. However, if the gel has a low viscosity or the gel has click chemistry, a single injection via the nodulus can allow the injected material reach the entire scaffold. In addition, based on the histology, it was not possible to determine which side the leaflet was injected. d. In vivo performance

[0225] To examine the tissue engineered valves in vivo, juvenile Dorset sheep (n=3) underwent a single pulmonary artery leaflet replacement via left-thoracotomy, comprising one leaflet replacement with MSCs in the spongiosa layer using a GelMA-HAMA hydrogel and two leaflets were additionally seeded with ECFCs on the outside. Although the click gel showed a better cell distribution, hydrogel GelMA-HAMA was used as it had been widely used and biocompatible. During the study, the biocompatibility of the newly developed gel was under research, therefore the well-known GelMA-HAMA was used to rule out any gel- related issues. Histological examination (FIGS. 18 and 19A-19H) showed that all explanted leaflets presented a nonconfluent endothelial cell layer (CD31, VE-Cadherin). The root area, where the MSCs were injected, showed cells expressing mesenchymal stem cell markers (aSMA, Vimentin). The presence of macrophages (CD64) of both subtype Ml (iNOS) and M2 (CD 163) indicates cellular ingrowth. Example 7. Methods for cell delivery in decellularized heart valves using injectable hydrogels

[0226] Decellularized xenograft valve tissues are attractive scaffolds for tissue engineered heart valves (TEHVs) by removing cells and other bioactive components while preserving the extracellular matrix. Great progress has been made in decellularization strategies, however, the repopulation of the tissues by host-cells in vivo remains marginal. Surface repopulation is achieved but little cellular ingrowth is observed. Current seeding methods have not yet overcome these challenges while the success of TEHVs depend on the scaffold transformation into living tissue through the interactions between scaffold properties and the regenerative capacity of the host. In this proof-of-concept study, a novel method was developed to introduce cells inside the spongiosa layer of decellularized valves using injectable hydrogels.

[0227] To investigate the feasibility, sheep valvular interstitial cells (VICs) were encapsulated in a gelatin methacrylate hyaluronic acid methacrylate (GelMA-HAMA) hydrogel or a P-aminoacrylate polyethylene glycol (PEG)-based hydrogel, and combined with decellularized porcine valves. These TEHVs were cultured in vitro and analyzed. To investigate the translation, three sheep underwent single pulmonary valve replacements. Sheep mesenchymal stem cells (MSCs) and endothelial colony forming cells (ECFCs) were isolated. Decellularized porcine valves were combined with MSCs encapsulated in the GelMA-HAMA hydrogel or the P-aminoacrylate synthetic PEG-based hydrogel, and the outside of two leaflets were seeded with ECFCs. The TEHVs were implanted in the donor matched sheep for up to six weeks. Results showed that a homogeneous cell distribution throughout the entire decellularized scaffold was achieved in vitro. In addition, the leaflets functioned well without stenosis, regurgitation, or valve thickening in vivo. Post-explant histology showed persistence of the seeded cells inside the scaffold and cellular infiltration by different macrophage subtypes.

[0228] The results indicate that cells were successfully delivered into the spongiosa layer of valve leaflets with good spatial cell distribution using hydrogels. In addition, single leaflet implantations showed that the leaflet functioned well for up to six weeks in vivo with persistence of the seeded cells inside the leaflet.

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[0273] OTHER EMBODIMENTS

[0274] Embodiment 1. A method of delivering a plurality of cells or cell components into a tissue, comprising: a. encapsulating a plurality of cells into a hydrogel; and b. contacting the hydrogel encapsulating the cells with a tissue, thereby delivering the plurality of cells into the tissue.

[0275] Embodiment 2. The method of embodiment 1, wherein the plurality of cells comprise stem cells and / or endothelial cells.

[0276] Embodiment 3. The method of embodiment 2, wherein the stem cells are mesenchymal stem cells (MSCs).

[0277] Embodiment 4. The method of embodiment 2, wherein the endothelial cells are endothelial colony forming cells (ECFCs).

[0278] Embodiment 5. The method of embodiment 1, wherein the cells are valvular interstitial cells (VICs).

[0279] Embodiment 6. The method of any one of embodiments 1-5, wherein the cell components are extracellular vesicles (EVs).

[0280] Embodiment 7. A method of delivering a therapeutic agent into the spongiosa layer of a decellularized tissue, comprising: a. encapsulating a therapeutic agent into a hydrogel; and b. contacting the hydrogel encapsulating the therapeutic agent with a decellularized tissue, thereby delivering the therapeutic agent into the tissue.

[0281] Embodiment 8. The method of embodiment 7, wherein the therapeutic agent is a peptide, a protein, a growth factor, a cytokine, and / or a chemokine.

[0282] Embodiment 9. The method of any one of embodiments 1-8, wherein the hydrogel is a self-assembling hydrogel.

[0283] Embodiment 10. The method of any one of embodiments 1-8, wherein the hydrogel is a click-based hydrogel.

[0284] Embodiment 11. The method of embodiment 9 or 10, wherein the hydrogel is a [3- aminoacrylate polyethylene glycol -based hydrogel.

[0285] Embodiment 12. The method of any one of embodiments 1-8, wherein the hydrogel is a photocrosslinkable hydrogel. Embodiment 13. The method of embodiment 12, wherein the photocrosslinkable hydrogel is a gelatin methacrylate hyaluronic acid methacrylate (GelMA-HAMA) hydrogel.

[0286] Embodiment 14. The method of any one of embodiments 1-13, wherein the tissue is an engineered tissue, optionally decellularized tissue.

[0287] Embodiment 15. The method of any one of embodiments 1-14, wherein the tissue a heart valve tissue.

[0288] Embodiment 16. The method of embodiment 15, wherein the heart tissue is a decellularized heart valve tissue.

[0289] Embodiment 17. The method of embodiment 16, wherein the hydrogel encapsulating the cells is delivered to a subendothelial layer (e.g., spongiosa layer) of the heart valve tissue.

[0290] Embodiment 18. The method of any one of embodiments 1-17, wherein the hydrogel has a concentration of about 1% to about 20% (w / v).

[0291] Embodiment 19. The method of any one of embodiments 1-18, wherein the plurality of cells or cell components are encapsulated into the hydrogel at a concentration of about 0.1 million to about 10 million cells per milliliter (mL).

[0292] Embodiment 20. A method of implanting an engineered tissue in a subject, comprising: a. encapsulating a plurality of cells into a hydrogel; b. contacting the hydrogel encapsulating the cells with an engineered tissue, thereby delivering the one or more cell into the engineered tissue; and c. implanting the engineered tissue into the subject.

[0293] Embodiment 21. The method of embodiment 20, wherein the subject is a mammal.

[0294] Embodiment 22. The method of embodiment 20 or 21, wherein the subject is a human subject.

[0295] Embodiment 23. The method of embodiment 1-22, wherein the engineered tissue is a biologically engineered tissue.

[0296] Embodiment 24. The method of any one of embodiments 20-23, wherein the engineered tissue is a heart valve.

[0297] Embodiment 25. The method of embodiment 24, wherein the engineered heart valve comprises the plurality of cells in subsurface of the heart valve.

[0298] Embodiment 26. The method of embodiment 24, wherein the engineered heart valve comprises the plurality of cells in the spongiosa layer of the heart valve.

[0299] Embodiment 27. An engineered heart valve comprising a plurality of exogenous cells in the spongiosa layer of the heart valve. Embodiment 28. A composition comprising a P-aminoacrylate polyethylene glycol- based hydrogel encapsulating a plurality of cells.

[0300] Embodiment 29. Use of a P-aminoacrylate polyethylene glycol-based hydrogel for delivering a plurality of cells into a tissue.

Claims

WHAT IS CLAIMED IS:

1. A method of delivering a plurality of cells or cell components into a sub-surface layer of a tissue, comprising:(a) encapsulating a plurality of cells into a hydrogel; and(b) delivering the hydrogel encapsulating the plurality of cells to the sub-surface layer of the tissue, thereby delivering the plurality of cells into the sub-surface layer of the tissue.

2. The method of claim 1, wherein the tissue is a heart valve tissue, and the sub-surface layer of the issue is a sub -endothelial layer (e.g., spongiosa) of the heart valve tissue.

3. The method of claim 1 or 2, wherein the tissue is a decellularized tissue.

4. The method of any one of claims 1-3, wherein the plurality of cells comprise primary cells or cell lines, wherein the plurality of cell components comprise a protein (e.g., a growth factor or a cytokine), a nucleic acid, a carbohydrate, and / or a lipid, optionally the plurality of cell components comprise extracellular vesicles.

5. The method of any one of claims 1-4, wherein the plurality of cells comprise stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells).

6. A method of delivering a therapeutic agent into a sub-endothelial layer (e.g., spongiosa) of the heart valve tissue, comprising:(a) encapsulating the therapeutic agent into a hydrogel; and(b) delivering the hydrogel encapsulating the therapeutic agent to the subendothelial layer (e.g., spongiosa) of the heart valve tissue, thereby delivering the therapeutic agent into the sub-endothelial layer (e.g., spongiosa) of the heart valve tissue.

7. The method of claim 6, wherein the therapeutic agent comprises a peptide, a protein, a growth factor, a cytokine, and / or a chemokine.

8. The method of any one of claims 1-7, wherein the hydrogel is a photocrosslinkable hydrogel, a shear-thinning hydrogel, a click-based hydrogel, or a self-assembling hydrogel.

9. The method of any one of claims 1-8, wherein the hydrogel is a gelatin methacrylatehyaluronic acid methacrylate (GelMA-HAMA) hydrogel, an PNP shear-thinning hydrogel, or a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel.

10. A method of recellularizing a decellularized heart valve, comprising:(a) delivering a hydrogel encapsulating a plurality of stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells) to a subendothelial layer (e.g., spongiosa) of the decellularized heart valve; and / or(b) contacting a plurality of endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells) with a surface of the decellularized heart valve, thereby recellularizing the decellularized heart valve.

11. The method of claim 10, wherein the hydrogel is a gelatin methacrylate-hyaluronic acid methacrylate (GelMA-HAMA) hydrogel.

12. The method of claim 11, wherein the hydrogel comprises about l%-50% GelMA (gelatin methacrylate), about 0.01%-10% HAMA (hyaluronic acid methacrylate), and about0.01%- 10% PI (2-hydroxy-4’ -(2-hydroxyethoxy)-2-m ethylpropiophenone).

13. The method of claim 10, wherein the hydrogel is a P-aminoacrylate synthetic polyethylene glycol (PEG)-based hydrogel.

14. The method of claim 13, wherein the hydrogel has a concentration of about 1% to about 20% P-aminoacrylate synthetic polyethylene glycol (PEG).

15. The method of any one of claims 10-14, wherein the plurality of stem cells or interstitial cells are encapsulated into the hydrogel at a concentration of about 0.1 million to about 100 million cells per milliliter (mL), e.g., by crosslinking under UV light; and whereinthe plurality of endothelial cells are contacted with the surface of the decellularized heart valve at a density of about 0.01 million to about 10 million per cm2.

16. The method of any one of claims 10-15, wherein the hydrogel encapsulating the plurality of stem cells or interstitial cells is injected to the spongiosa of the decellularized heart valve via the arterial wall (e.g., the pulmonary artery wall at the leaflet insertion site) and / or nodulus of the decellularized heart valve.

17. The method of any one of claims 10-16, wherein the surface of the decellularized heart valve is incubated with fibronectin before contacting with the plurality of endothelial cells.

18. The method of any one of claims 10-17, further comprising culturing the plurality of stem cells or interstitial cells after step (a), and / or culturing the plurality of endothelial cells after step (b).

19. A method of decellularizing a tissue, comprising:(a) contacting the tissue with a first solution comprising about 0.001%-5% SDC (sodium deoxycholate) and about 0.001%-5% SDS (sodium dodecyl sulfate) for about 1- 24 hours (e.g., 12 hours);(b) optionally repeating step (a) for 1-5 cycles with a fresh first solution for each cycle;(c) contacting the tissue with a second solution comprising about 0.001%-5% sodium azide for about 1-24 hours (e.g., 12 hours); and(d) optionally repeating step (c) for 1-5 cycles with a fresh second solution for each cycle, thereby decellularizing the tissue.

20. The method of claim 19, wherein the second solution further comprises about 0.1%- 10% PSG (penicillin / streptomycin / glutamine).

21. The method of claim 19 or 20, further comprising washing the tissue to remove the first and / or second solutions.

22. The method of any one of claims 19-21, wherein the decellularizing substantially maintains the structural and / or biochemical integrity of the tissue.

23. The method of any one of claims 19-22, wherein no cells are present in the tissue after the decellularizing, e.g., as determined by haematoxylin and eosin (HE) staining.

24. The method of any one of claims 19-23, wherein the tissue is a heart valve tissue.

25. A decellularized tissue obtained using the method of any one of claims 19-24.

26. A method of implanting an engineered heart valve in a subject, comprising:(a) obtaining a decellularized heart valve;(b) recellularizing the decellularized heart valve, thereby obtaining the engineered heart valve; and(c) implanting the engineered heart valve into the subject.

27. A method of implanting an engineered heart valve in a subject, comprising:(a) decellularizing a heart valve isolated from a mammal;(b) recellularizing the decellularized heart valve, thereby obtaining the engineered heart valve; and(c) implanting the engineered heart valve into the subject.

28. The method of claim 27, wherein the mammal is a pig and the heart valve is a porcine pulmonary valve.

29. The method of any one of claims 26-28, wherein the subject is a human subject (e.g., a pediatric patient) or a model animal (e.g., sheep).

30. The method of any one of claims 26-29, wherein the decellularized heart valve is recellularized with cells isolated from the subject.

31. The method of any one of claims 26-30, wherein the implanted engineered heart valve exhibits a normal function, e.g., normal opening and closing with no observable regurgitation of the blood flow as determined by echocardiography, at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5years, 6 years, 7 years, 8 years, 9 years, 10 years, 15 years, 20 years, 30 years, 40 years, or 50 years after implantation.

32. The method of any one of claims 26-31, wherein the implanted engineered heart valve does not induce inflammation or rejection.

33. An engineered heart valve comprising a first population of exogenous cells in a subendothelial layer (e.g., spongiosa) of a heart valve tissue.

34. The engineered heart valve of claim 33, wherein the heart valve tissue is a pulmonary valve or an aortic valve.

35. The engineered heart valve of claim 33 or 34, wherein the plurality of exogenous cells comprises stem cells (e.g., mesenchymal stem cells) or interstitial cells (e.g., valvular interstitial cells).

36. The engineered heart valve of any one of claims 33-35, further comprising a second population of exogenous cells on a surface of the heart valve tissue, wherein the second population of exogenous cells comprise endothelial cells (e.g., valvular endothelial cells, endothelial progenitor cells, or endothelial colony forming cells).

37. The engineered heart valve of any one of claims 33-36, wherein the first and / or second populations of exogenous cells are isolated from a human subject (e.g., a pediatric patient) or a model animal (e.g., sheep).

38. The engineered heart valve of any one of claims 33-37, wherein the heart valve tissue is a porcine pulmonary valve.

39. A composition comprising a gelatin methacrylate-hyaluronic acid methacrylate (GelMA- HAMA) hydrogel, an PNP shear-thinning hydrogel, or a P-aminoacrylate polyethylene glycol (PEG)-based hydrogel encapsulating a plurality of cells.

0. Use of a gelatin methacrylate-hyaluronic acid methacrylate (GelMA-HAMA) hydrogel, an PNP shear-thinning hydrogel, or a p-aminoacrylate polyethylene glycol (PEG)-based hydrogel for delivering a plurality of cells into a tissue.

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