HK1218888B - Use of microparticles and endothelial cells with decellularized organs and tissues - Google Patents

Description

Use of microparticles and endothelial cells and decellularized organs and tissues

Cross Reference to Related Applications

This application claims the benefit of U.S. application serial No. 61/790,118 filed on 2013, month 3and day 15, the disclosure of which is incorporated herein by reference.

Background

Tissue engineering is a rapidly developing field seeking to repair or regenerate damaged or diseased tissues and organs by implanting a combination of cells, scaffolds, and soluble mediators. Current stem cell differentiation and primary cell culture are typically achieved under 2-dimensional (2D) culture conditions. This system allows for the expansion of specific cell populations, but has limited ability to maintain functional cell phenotypes to maintain high density cell culture and long-term primary or differentiated cell function. For example, the number of stem cells can be expanded in large numbers while retaining the ability to differentiate into specific lineages, compared to the limited availability of large numbers of primary cells required for certain cell therapies. Control of stem cell fate (e.g., differentiation), whether in vivo or in vitro, is primarily due to genetic and molecular mediators (e.g., growth factors and transcription factors). While differentiation of stem and progenitor cells may result in cells with appropriate lineage-or tissue-specific gene expression, the differentiated cells may lack the functional properties required for in vitro or in vivo applications.

Summary of The Invention

The present invention provides methods of maintaining, reducing the decrease in, or increasing the diameter of a capillary vessel lumen in a re-endothelialized and/or re-cellularized (with cells other than endothelial cells) extracellular matrix of a mammalian organ, tissue, or portion thereof having an intact vascular bed, for example, ensuring a suitable capillary diameter after transplantation to maintain continuous blood flow. The method comprises providing or preparing a decellularized extracellular matrix of a mammalian organ, tissue or portion thereof having an intact vascular bed, and providing or preparing a population of endothelial cells or a population of stem or progenitor cells capable of differentiating into endothelial cells. Simultaneously introducing or sequentially introducing into the decellularized extracellular matrix an amount of cells and an amount of a first aqueous solution containing biocompatible nanoparticles or microparticles. The amount of cells is effective to re-endothelialize the vasculature of the decellularized extracellular matrix and, during or after re-endothelialize, the amount of nanoparticles or microparticles maintains, or decreases, e.g., decreases to about 4 microns, or increases, e.g., up to about 15 to about 20 microns, the capillary lumen diameter of the vasculature when circulating through the vasculature relative to a corresponding re-endothelialized decellularized extracellular matrix lacking the nanoparticles or microparticles. In one embodiment, the nanoparticle or microparticle is biodegradable. In one embodiment, the nanoparticles or microparticles are rapidly biodegradable after addition of an additive (addedagent) or energy source. In one embodiment, the nanoparticle or microparticle is not biodegradable. The nanoparticles may be formed of any biocompatible material and may be of any shape that allows them to pass through the vasculature. In one embodiment, the shape of the nanoparticle or microparticle is spherical or ellipsoidal. In one embodiment, the average diameter of the nanoparticles or microparticles is from about 0.5 μm to about 30 μm or from about 5 μm to about 20 μm. In one embodiment, the nanoparticles or microparticles are deformable. In one embodiment, the nanoparticles or microparticles are formed from polymers, including naturally occurring and synthetic (non-naturally occurring) polymers. In one embodiment, the nanoparticle or microparticle is formed from a protein and a non-protein polymer. In one embodiment, the nanoparticles or microparticles are modified to include carboxylates, esters, amines, aldehydes, alcohols, or halides and functional molecules, such as ligands for magnetic molecules. In one embodiment, an external energy source is added, such as, but not limited to: light, magnetic, mechanical force or ultrasound to degrade the particles. In one embodiment, the aqueous solution is added after re-endothelialization. In one embodiment, the nanoparticles or microparticles are removed by washing the re-endothelialized vasculature with another solution that is free of the particles and may contain a substance that degrades the particles. In one embodiment, the method comprises introducing a second aqueous solution comprising biocompatible nanoparticles or microparticles having an average diameter at least 10% greater than the average diameter of the nanoparticles or microparticles in the first solution. The concentration of the particles can vary and can be any amount that achieves the purpose. For example, the first solution may contain about 300 to about 50,000,000 particles per μ L.

The decellularized extracellular matrix can be from any organ or tissue, so long as the organ or tissue has an intact vascular (capillary) bed ("intact" vasculature) that allows solution to circulate into and out of its vascular ducts. In one embodiment, the decellularized organ is a decellularized heart, pancreas, liver, kidney, bone, or lung. Any cell type capable of re-endothelializing extracellular matrix vasculature of a decellularized organ, tissue or portion thereof having intact vasculature can be used. For example, the cells may be obtained from iPS cells. In one embodiment, the cells are introduced into the matrix by injection or perfusion or a combination thereof. In one embodiment, the cells are introduced into the vasculature by injection or perfusion, or a combination thereof. In one embodiment, the cells introduced into the decellularized extracellular matrix are primary cells. In one embodiment, the cells introduced into the decellularized extracellular matrix are a plurality of different cell types intended to fully or partially recellularize an organ, tissue, or portion thereof. In one embodiment, the cells introduced into the decellularized extracellular matrix are human embryonic stem cells. In one embodiment, the cells are allogeneic to the perfused decellularized organ, tissue, or portion thereof. In one embodiment, the cells are xenogeneic to the perfused decellularized organ, tissue or portion thereof.

Brief description of the drawings

FIG. 1A shows a photograph of perfusion decellularized pig liver.

FIGS. 1B-C show Scanning Electron Microscope (SEM) photographs of the vascular and parenchymal matrices, respectively, of perfusion decellularized pig liver.

Figure 2 provides a general view of a rat liver immersed in decellularization, in which matrix attrition can be observed at both low (left) and high (right) magnifications.

Figure 3 shows SEM photographs of immersion decellularized rat liver (a and B) and perfusion decellularized rat liver (C and D).

FIG. 4 provides the histological structure of immersed decellularized liver (A, H & E staining; B, trichrome staining) and perfusion decellularized liver (C, H & E staining; D, trichrome staining).

Fig. 5 shows a comparison of immersion decellularization (top row) and perfusion decellularization (bottom row) of rat hearts.

Figure 6 shows a comparison of immersion decellularization (top row) and perfusion decellularization (bottom row) using rat kidney.

Figure 7 shows SEM photographs of decellularized kidneys.

Fig. 8A shows an SEM photograph of a perfusion decellularized heart, while fig. 8B shows an SEM photograph of an immersion decellularized heart.

Detailed Description

The present invention provides engineered cells and ECMs that directly control cell behavior through physical as well as molecular interactions, via controlling the environment of those cells. In particular, the invention provides an engineered organ, tissue or bioreactor having perfusion decellularized ECM transplanted with a cell population comprising a combination of cells and subjected to culture conditions, e.g., comprising perfusion of soluble media, that produce substantially the same functional cells and capillary lumen diameters as the corresponding original organ, e.g., from about 5 μm to about 10 μm or from about 3 μm to about 20 μm. In particular, the present invention may provide improved regulation of cell differentiation, growth and phenotypic expression of adult and embryonic cells and partially differentiated progenitor cells, as well as improved maintenance of differentiated cell types, due to the maintenance of capillary lumen diameter. Also included are the growth and functional maintenance of primary cells, including cells of fetal origin, such as organ-specific cells obtained from fetal cells or neonatal cells (e.g., cells committed to a particular lineage but not terminally differentiated).

The present invention provides the use of perfusion of decellularized organ or tissue derived extracellular matrix (ECM) and systems useful in the extracellular matrix vasculature of these matrices to support: these matrices with vascular (e.g. endothelial) cells are recellularized, or stem or progenitor cells are differentiated and/or matured into endothelial cells, or maintenance or differentiation of primary cells, or any combination thereof. Primary cells are cells obtained from an organism, which are then usually cultured in vitro, but those cells cannot be propagated indefinitely. Differentiated cells include primary cells and cells that have been differentiated in vitro, such as stem cells or progenitor cells in a perfusion decellularized matrix of the invention. In one embodiment, at least 5%, 10% or 20% or more of the differentiated cells have a functionally mature phenotype. Tissues are cell populations that share common structure and function, such as epithelial, connective, muscle (skeletal, cardiac, or smooth), and neural tissue, and include a soft layer that covers or lines or joins organs. An organ is a number of tissues (two or more) that serve a common function, joined together in structural units. Organs include, but are not limited to: brain, liver, pancreas, bone, heart, stomach, kidney, lung, whole body muscle, thymus, anus, and intestine. As used herein, an organ includes the whole organ that can be perfused or a portion of the organ or vascularized structures thereof, and a tissue includes any structure containing vascularized tissue, such as the trachea.

In one embodiment, the invention provides for the use of organ or tissue specific extracellular matrix (ECM) scaffolds for re-endothelialization, utilizing cells that may require differentiation or maturation, such as stem cells or progenitor cells. Differentiation is the process by which cells acquire a new phenotype different from the original cell population, for example, different cellular gene and/or protein expression and/or function. Maturation further elucidates the phenotype of the cell population as having the normal maturation functional capabilities of the cells in the cell population in vivo. In one embodiment, the scaffold is a perfusion decellularized ECM portion of the organ. In another embodiment, the scaffold is a perfusion decellularized ECM organ.

Perfusion decellularized ECM from organs or tissues retains more of the native microstructure, including intact blood vessels and/or microvasculature, than other decellularization techniques (e.g., immersion-based decellularization). For example, perfusion decellularized ECM from organs or tissues retains collagen content as well as other binding and signaling factors and vasculature structure, thus providing a niche environment with natural cues for functional differentiation or maintenance of cell function of the introduced cells. In one embodiment, perfusion decellularized ECM from an organ or tissue is perfused with cells and/or media under suitable conditions (including suitable pressure and flow) using the vasculature perfused with decellularized ECM to mimic the conditions normally present in an organism. The normal pressure of a human-sized organ is about 40 to about 200mm Hg, with the flow rate produced depending on the incoming perfused vessel diameter. For a normal human heart, the resulting perfusion rate is about 20 to about 200mL/min/100 g. With this system, seeded cells can achieve seeding concentrations from about 5x up to about 1000x higher than those achieved under 2D cell culture conditions and unlike 2D culture systems, the ECM environment allows for further functional differentiation of the cells, e.g., differentiation of progenitor cells into cells exhibiting an organ or tissue specific phenotype with sustained function. In one embodiment, the combination of culture conditions and ECM origin allows for functional differentiation of cells introduced into the ECM.

In one embodiment, the method comprises selecting a perfusion decellularized matrix of an organ or tissue and a cell population comprising endothelial cells or progenitor cells capable of differentiating into endothelial cells. Contacting the selected perfusion decellularized matrix with a population of cells under conditions and for a time to provide recellularization of the perfusion decellularized matrix and to differentiate progenitor cells in the population of cells into functional cells. In one embodiment, nanoparticles and microparticles are introduced and circulated through the vasculature with the cells. In one embodiment, after re-endothelialization, nanoparticles and microparticles are introduced and circulated through the vasculature. In one embodiment, the organ is a heart. In another embodiment, the organ is a liver. In another embodiment, the organ is a pancreas. In another embodiment, the organ is a lung.

In one embodiment, the invention provides a method of maintaining capillary lumen diameter in a re-endothelialized perfusion decellularized matrix. The method comprises selecting a perfusion decellularized matrix of an organ or tissue and a population of stem cells capable of differentiating into endothelial cells or endothelial cells. Contacting the selected perfusion decellularized matrix with cells under conditions and for a time to provide re-endothelialization of the perfusion decellularized matrix and provide endothelial cells or differentiate stem cells in the population into functional endothelial cells. In one embodiment, the stem cell is an Induced Pluripotent Stem (iPS) cell. In one embodiment, the stem cell is an Embryonic Stem (ES) cell, e.g., a human ES cell. In one embodiment, the stem cell is an adult stem cell.

In one embodiment, the methods of the invention use a portion of the organ or tissue ECM, such as the atria or ventricles of the heart, or the internal structure of the pancreas, including the islets. In one embodiment, the thickness of the portion is from about 5 to about 10 mm. In one embodiment, the thickness of the portion is from about 70 to about 100 mm.

The ECM organ or tissue matrix can be obtained from any source including, but not limited to, heart, liver, lung, skeletal muscle, brain, pancreas, spleen, kidney, uterus, eye, spinal cord, systemic muscle, or bladder, or any portion thereof (e.g., aortic valve, mitral valve, pulmonary valve, tricuspid valve, pulmonary vein, pulmonary artery, coronary vasculature, septum, right atrium, left atrium, right ventricle, or left ventricle). A solid organ refers to an organ having "substantially closed" vasculature. By "substantially closed" vasculature with respect to an organ is meant that, assuming that the main vessel is cannulated, ligated, or otherwise restricted, most of the fluid is contained within the solid organ or flows out of the native vascular structure after perfusion with the fluid, and does not leak out of the solid organ. Despite having a "substantially closed" vasculature, many of the organs listed above have defined "inlet" or "outlet" vessels that can be used to introduce and move fluid throughout the organ during perfusion. In addition, other types of vascularized organs or tissues may be perfusion decellularized, such as, for example, all or part of a joint (e.g., knee, shoulder or hip), anus, trachea or spinal cord. Further, avascular tissue, e.g., cartilage or cornea, may be decellularized when part of a larger vascularized structure (e.g., the entire leg).

Perfusion decellularization of organs with substantially closed vasculature is particularly useful as it preserves the intact matrix and microenvironment, including intact vasculature and microvasculature, which can be used for cell delivery and for delivering nutrients and/or differentiation or maintenance factors to cells in vitro. The cells and nutrients and/or other factors may be delivered by other methods, such as, for example, injection or passive methods, or a combination thereof. In one embodiment, the target cell population is perfused into a perfusion decellularized organ ECM that allows seeding into the interstitial space or matrix outside the vascular conduit. This includes active migration and/or return of the cells to their native microstructure, e.g., return of endothelial cells to the vasculature. In one embodiment, the target cell population is perfused into the perfusion decellularized ECM, followed by introduction of a second cell population, e.g., a beta cell population, followed by introduction of a population of endothelial cells that reside in the vascular conduit as in its native microenvironment. In one embodiment, the target cell population is perfused into the perfusion decellularized ECM, followed by introduction of a second cell population, e.g., an endothelial cell population, which is left in the vascular conduit as in its native microenvironment, followed by introduction of a cell population comprising beta cells. In another embodiment, two or more cell populations are combined and perfused together. In another embodiment, the two or more different cell populations are introduced sequentially by perfusion, direct injection, or a combination of both. Introduction of the particles of the invention into the ex vivo re-endothelialized vasculature maintains capillary lumen diameter, decreases capillary lumen diameter reduction or increases capillary lumen diameter.

Cells may be introduced into a medium that supports cell proliferation, metabolism, and/or differentiation. Alternatively, after the cells have colonized the ECM, the medium is changed to a medium that supports cell proliferation, metabolism and differentiation. The cultured cells may be present in the ECM at physiological cell densities and allow for maintenance and/or functional differentiation of the cells in the presence of suitable microenvironments in the ECM and/or culture media that support cell proliferation, metabolism and/or differentiation.

The cells and ECM may be xenogeneic or allogeneic. In one embodiment, partially or fully differentiated human cells may be combined with perfusion decellularized organs or tissues from small animals (e.g., non-human mammals). In one example, endothelial cells and partially differentiated human ES-derived hepatocytes are seeded into perfusion decellularized liver matrices from humans, thereby providing allogeneic or xenogeneic cell-seeded matrices, respectively.

Perfusion of decellularized ECM

Studies have shown that connective tissue cells behave very differently in 3D culture compared to 2D culture (cukiermannet et al,Science,294:1708(2001)). For example, culturing fibroblasts on flat substrates induces polarity, which does not occur in vivo. Further, when fibroblasts and other cell types are cultured in a 3D tissue-derived matrix, they countMature integrin-containing adhesive plaque complexes were produced in minutes, similar to those found in vivo, whereas in 2D cultures or even simple collagen type 3D I gels or matrigel, only the original adhesive complexes were produced. These adhesion complexes are required for proper growth factor activation receptor signaling and rapid (within 5 minutes) priming of synthesis of its own ECM components as well as factors that alter the ECM (Cukierman et al, 2001; Abbott,Nature,424:870(2003)). In addition, cells in ECM culture deposit autocrine growth factors in a tissue-derived matrix, a process required for proper presentation of growth factors to target cells. In 2D culture, such factors are mainly secreted into the culture medium.

As mentioned above, physical interactions with the ECM, in addition to chemical substances, molecules (e.g., soluble mediators) or genetic (cell type) factors, can modulate cell fate. For example, ECM-based cell control can occur through a variety of physical mechanisms, such as microscale and nanoscale ECM geometry, ECM elasticity, or mechanical signals transmitted from the ECM to the cell.

The invention includes the use of engineered perfusion decellularized ECM that allows for better control of cell behavior through physical as well as molecular interactions, such as from adult stem cells or embryonic stem cells. The perfusion decellularized matrix of the invention mimics the complex and highly ordered nature of native ECM, as well as the possible interactions between cells and ECM. In particular, the ECM can provide a tissue-specific predisposition to stem or progenitor cells. In particular, different matrix proteins may be important for the specificity of the ECM due to their contribution to the ECM structure or due to their ability to interact with growth factors and/or resident cells themselves.

Perfusion decellularization of tissue or organ ECM provides intact ECM with structural, biochemical, and mechanical properties that provide functional cells to be able to differentiate and maintain. Thus, perfusion decellularization of an organ allows the organ to function as a tissue/organ specific bioreactor for stem or progenitor cell differentiation. Furthermore, perfusion decellularization of organ or tissue ECM is preferred over immersion decellularization in terms of preservation of intact matrix (including intact vasculature) with structural and biochemical cues. Furthermore, perfusion decellularization offers advantages over immersion decellularization when the tissue or organ thickness exceeds about 2 mm.

Decellularization of organs or tissues

Decellularization typically involves the following steps: stabilizing a solid organ (e.g., its vascularized structure) or tissue, decellularizing the solid organ or tissue, renaturing and/or neutralizing the solid organ or tissue, washing the solid organ, degrading any DNA remaining in the organ, sterilizing the organ or tissue, and allowing the organ to achieve homeostasis.

The initial step in decellularizing the vascularized structure or tissue of an organ is the insertion of a cannula into the organ or tissue. A cannula may be inserted into a vessel, duct, and/or lumen of an organ or tissue using methods and materials known in the art. The cannulated organ vascularized structure or tissue is then perfused with a cell disruption medium. Perfusion through the organ may be multidirectional (e.g., antegrade and retrograde).

In the art, Langendorff perfusion of the heart is conventional physiological perfusion (also known as four-chamber mode of operation perfusion). See, for example, Dehnert, The Isolated Perfused wave-blood Heart accumulating to Langendorff, In Methods In Experimental Physiology and Pharmacology, biologicals measurements Techniques V.biomesstechnik-Verlag March GmbH, West Germany, 1988.

Briefly, for Langendorff perfusion, the aorta is cannulated and connected to a reservoir containing physiological solution to allow the heart to run outside the body for a specified duration. To achieve perfusion decellularization, the protocol has been modified to perfuse a cell disruption medium delivered in a retrograde direction downstream of the aorta, delivered at a constant flow rate, for example, by an infusion or roller pump or by a constant hydrostatic pressure pump. In both cases, the aortic valve is forced to close and the perfusion fluid flows to the coronary ostia (thus perfusing the entire ventricular mass of the heart by antegrade), and then into the right atrium through the coronary sinus. For working mode perfusion, a second cannula is connected to the left atrium, and perfusion may be reversed.

In one embodiment, the physiological solution comprises Phosphate Buffered Saline (PBS). In one embodiment, the physiological solution is a physiologically compatible buffer supplemented with, for example, a nutritional supplement (e.g., glucose). For example, for the heart, the physiological solution may be a Modified Krebs-Henseleit buffer (Modified Krebs-Henseleit buffer) containing 118mM NaCl, 4.7mM KCl, 1.2mM MgSO₄、1.2mM KH₂PO₄、25mM NaHCO₃11mM glucose, 1.75mM CaCl₂2.0mM pyruvate and 5U/L insulin; or a Krebs buffer (Krebs buffer) containing 118mM NaCl, 4.7mM KCl, 25mM NaHCO₃、1.2mM MgSO₄、1.2mM KH₂PO₄、2mM CaCl₂Filled with 95% O₂、5％CO₂. The heart may be perfused with glucose (e.g., about 11mM) as the sole substrate or in combination with about 1 or 1.2mM palmitate. For the kidney, the physiological solution may beThe kidney is perfused with solution. For the liver, the physiological solution may be a Klebsiella-Henshelet buffer containing 118mM NaCl, 4.7mM KCl, 1.2mM MgSO₄、1.2mM KH₂PO₄、26mMNaHCO₃8mM glucose and 1.25mM CaCl₂Supplemented with 2% BSA.

Methods of perfusing other organs or tissues are known in the art. For example, the following references describe perfusion of the lung, liver, kidney, brain, and extremities. Van Putte et al,Ann.Thorac.Surg.,74(3):893(2002)；den Butter et al.,Transpl.Int.,8:466(1995)；Firth et al.,Clin.Sci.(Lond.),77(6):657(1989)；Mazzetti et al.,Brain Res.,999(1):81(2004)；Wagner et al.,J.Artif.Organs,6(3):183(2003)。

one or more cell disruption media may be used to decellularize an organ or tissue. The cell disruption medium typically comprises at least one detergent such as, but not limited to, SDS, PEG, CHAPS, or Triton X. The cell disruption medium may comprise water to render the medium osmotically incompatible with the cells. Alternatively, the cell disruption medium may comprise a buffer compatible with cell permeation (e.g., PBS). The cell disruption medium may also comprise enzymes such as, without limitation, one or more collagenases, one or more dispases, one or more dnases, or proteases such as trypsin. In certain instances, the cell disruption medium can further or alternatively comprise an inhibitor of one or more enzymes (e.g., a protease inhibitor, a nuclease inhibitor, and/or a collagenase inhibitor).

In certain embodiments, the cannulated organ or tissue may be perfused sequentially with two different cell disruption media. For example, the first cell disruption medium can comprise an anionic detergent, such as SDS, and the second cell disruption medium can comprise an ionic detergent, such as Triton X. Following perfusion with at least one cell disruption medium, the cannulated organ or tissue may be perfused with, for example, a wash solution and/or a solution containing one or more enzymes (such as those disclosed herein).

Alternating perfusion directions (e.g., antegrade and retrograde) can help decellularize an entire organ or tissue. Decellularization generally decellularizes the organ from inside to outside, resulting in less damage to the ECM. Organs or tissues can be decellularized at a suitable temperature between 4-40 ℃. Depending on the size and weight of the organ or tissue and the particular detergent and the concentration of detergent in the cell disruption medium, the organ or tissue is typically perfused with the cell disruption medium for about 0.05 hours to about 5 hours per gram of solid organ or tissue (typically >50 grams), or for about 2 hours to about 12 hours per gram of solid organ or tissue (typically <50 grams). Organs can be perfused up to about 0.75 hours to about 10 hours per gram of solid organ or tissue (typically >50 grams), or about 12 hours to about 72 hours per gram of tissue (typically <50 grams), including washing. Decellularization time depends on the vascular and cellular density of the organ or tissue, which has a limited scaling to the total mass. Thus, the above time ranges and qualities are provided as a general guideline. Perfusion is typically adjusted to accommodate physiological conditions, including pulsatile flow, velocity, and pressure.

Decellularized organs or tissues have extracellular matrix (ECM) components of all or most regions of the organ or tissue, including ECM components of the vascular tree. The ECM component can include any or all of the following components: fibronectin, fibrillin, laminin, elastin, collagen family members (e.g., collagen I, III and IV), ECM-associated growth proteins (including growth factors and cytokines), glycosaminoglycans, matrices, reticular fibers, and thrombospondin, which can maintain the organization into a well-defined structure, such as a substrate. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells and nuclei in tissue sections or the removal of more than 97% of detectable DNA as measured by fluorescence analysis using standard histological staining procedures. The remaining cell debris may be removed from the decellularized organ or tissue.

During and after decellularization, the morphology and structure of the ECM is maintained. As used herein, "morphology" refers to the overall shape of an organ, tissue or ECM, while "structure" as used herein refers to the outer surface, inner surface and ECM therebetween.

The morphology and structure of the ECM can be detected visually and/or histologically. For example, the substrate on the exterior surface of a solid organ, or within the vasculature of an organ or tissue, should not be removed or significantly damaged by perfusion decellularization. In addition, the fibrils of the ECM should be similar to or not significantly altered from those of organs or tissues that are not decellularized.

One or more compounds may be applied to a decellularized organ or tissue, for example, to preserve the decellularized organ, or to prepare the decellularized organ or tissue for recellularization, and/or to assist or stimulate cells during the recellularization process. Such compounds include, but are not limited to: one or more growth factors (e.g., VEGF, DKK-1, FGF, BMP-1, BMP-4, SDF-1, IGF, and HGF), immunomodulators (e.g., cytokines, glucocorticoids, IL2R antagonists, leukotriene antagonists), and/or factors that modulate the coagulation cascade (e.g., aspirin, heparin-binding protein, and heparin). In addition, the decellularized organ or tissue can be further treated with, for example, radiation (e.g., UV, gamma) to reduce or eliminate any type of microorganism remaining in the decellularized organ or tissue.

Exemplary perfusion decellularization of the heart

PEG decellularization protocol

Hearts were washed without recirculation in 200mL PBS containing 100U/mL penicillin, 0.1mg/mL streptomycin, 0.25. mu.g/mL amphotericin B, 1000U glycogen, and 2mg adenosine. The heart was then decellularized with 35mL polyethylene glycol (PEG; 1g/mL) using manual recirculation for up to 30 minutes. The organs were then washed with 500mL PBS using a recirculating pump for up to 24 hours. The washing step is repeated at least twice for at least 24 hours each time. The heart was exposed to 35mL of DNase I (70U/mL) for at least 1 hour using manual recirculation. The organs were washed again with 500ml PBS for at least 24 hours.

Triton X and trypsin decellularization protocol

Hearts were washed without recirculation for at least about 20 minutes in 200mL PBS containing 100U/mL penicillin, 0.1mg/mL streptomycin, 0.25. mu.g/mL amphotericin B, 1000U glycogen, and 2mg adenosine. The heart was then decellularized with 0.05% trypsin for 30 minutes, followed by perfusion with 500mL PBS containing 5% Triton-X and 0.1% ammonium hydroxide for about 6 hours. The heart was perfused with deionized water for about 1 hour, then with PBS for 12 hours. The hearts were then washed 3 times with 500mL PBS using a recirculating pump, each for 24 hours. Using manual recirculation, the heart was perfused with 35mL of DNase I (70U/mL) for 1 hour, and washed twice with recirculation pump in 500mL PBS for at least about 24 hours each.

1% SDS Decellularization protocol

Hearts were washed without recirculation for at least about 20 minutes in 200mL PBS containing 100U/mL penicillin, 0.1mg/mL streptomycin, 0.25 μ g/mL amphotericin B, 1000U glycogen, and 2mg adenosine. The heart was decellularized with 500mL of water containing 1% SDS for at least about 6 hours using a recirculating pump. The heart was then washed with deionized water for about 1 hour and PBS for about 12 hours. Using a recirculating pump, the heart was washed three times with 500mL PBS for at least about 24 hours each. The heart was then perfused with 35mL of DNase I (70U/mL) for about 1 hour using manual recirculation, and washed three times with 500mL of PBS using a recirculation pump for at least about 24 hours each.

Triton X decellularization protocol

The heart was washed with 200mL PBS containing 100U/mL penicillin, 0.1mg/mL streptomycin, 0.25. mu.g/mL amphotericin B, 1000U glycogen, and 2mg adenosine (adenosine) for at least about 20 minutes without recirculation. The heart was then decellularized with 500mL of water containing 5% Triton X and 0.1% ammonium hydroxide using a recirculating pump for at least 6 hours. The heart was then perfused with deionized water for about 1 hour, followed by perfusion with PBS for about 12 hours. The hearts were washed with a recirculating pump by perfusing the hearts with 500mL PBS three times for at least 24 hours each. The heart was then perfused with 35mL of DNase I (70U/mL) for about 1 hour using manual recirculation and washed three times in 500mL PBS for about 24 hours each.

Can be at 60cm H₂The coronary perfusion pressure of O perfuses the heart. Although not required, the heart can be placed in a decellularization chamber, completely submerged, and perfused with PBS containing antibiotics for 72 hours in a recirculation mode at 5 mL/min continuous flow, to wash as much of the cellular components and detergent as possible.

Detection of cardiac decellularization

Can be cut through tissueThe absence of myofilaments and nuclei in the disc was used to gauge successful decellularisation. Successful preservation of vascular structures can be assessed by perfusion with 2% infantrine prior to embedding the tissue sections. When first perfused with a constant coronary artery pressure, it is dissolved in deionized H₂Efficient decellularization is observed when the heart is perfused anterogradely with an ionic detergent of O (1% Sodium Dodecyl Sulfate (SDS), approximately 0.03M) and then with a non-ionic detergent (1% Triton X-100) to remove SDS and possibly renature extracellular matrix (ECM) proteins. Intermittently, the heart may be perfused retrograde with phosphate buffered solution to clear blocked capillaries and small blood vessels.

To visualize the decellularized intact vasculature, the decellularized heart was stained with evans blue via Langendorff perfusion to stain the vascular basement membrane and quantify macrovascular and microvascular density. Further, polystyrene particles can be perfused into and through the heart to quantify coronary artery volume, the level of vascular leakage, and the distribution of perfusion assessed by analyzing coronary effluent and tissue sections. A combination of three criteria was evaluated and compared to isolated non-decellularized hearts: 1) average distribution of polystyrene particles, 2) significant change in leakage at a certain level, 3) microvascular density.

Fiber orientation can be assessed by polarized light microscopy of Tower et al (Ann Biomed Eng.,30(10):1221(2002), which can be applied in real time to samples subjected to uniaxial or biaxial stress.

Exemplary perfusion decellularization of liver

For liver isolation, the vena cava was exposed by a middle laparotomy, dissected and cannulated with a mouse aortic cannula (RadnotiGlass, monprovia, Calif.). The hepatic artery and vein and bile duct were transected, and the liver was carefully removed from the abdomen and submerged in sterile PBS (Hyclone, Logan, Utah) to minimize traction on the portal vein. Heparinized PBS was perfused for 15 minutes, followed by 2-12 hours with deionized water containing 1% SDS (Invitrogen, Carlsbad, Calif.) and 15 minutes with deionized water containing 1% Triton-X (Sigma, st. The liver was then perfused continuously for 124 hours with antibiotic-containing PBS (100U/ml penicillin-G (Gibco, Carlsbad, Calif.), 100U/ml streptomycin (Gibco, Carlsbad, Calif.), 0.25. mu.g/ml amphotericin B (Sigma, St. Louis, Mo.).

After 120 minutes of SDS perfusion, it was perfused with Triton-X100, which was sufficient to generate fully decellularized liver. Movat's five-color chromatically staining of decellularized liver confirmed that typical liver tissue was retained with a central vein and a portal area containing the hepatic artery, bile duct, and portal vein.

Recellularization of organs or tissues

The decellularized organ or tissue is contacted with a population of cells, either differentiated (mature or primary), stem, or partially differentiated, which include different cell types. Thus, the cell may be a totipotent, pluripotent or multipotent cell, and may be an uncommitted or committed cell, and may be a single lineage cell. The cells may be undifferentiated cells, partially differentiated cells, or fully differentiated cells, including cells of fetal origin. The cells may comprise progenitor, precursor, or "adult" derived stem cells, including umbilical cord cells and fetal stem cells. Cells useful in the matrices of the invention include embryonic stem cells (as defined by the National Institute of Health (NIH); see, e.g., the glossary of the world Wide Web site stemcells.

Examples of cells that may be used to recellularize an organ or tissue include, without limitation: embryonic stem cells, umbilical cord blood cells, tissue-derived stem or progenitor cells, bone marrow-derived stem or progenitor cells, blood-derived stem or progenitor cells, Mesenchymal Stem Cells (MSCs), skeletal muscle-derived cells, Multipotent Adult Progenitor Cells (MAPCs), or iPS cells. Additional cells that may be used include Cardiac Stem Cells (CSCs), multipotent adult heart-derived stem cells, cardiac fibroblasts, cardiac microvasculature endothelial cells, aortic endothelial cells, coronary endothelial cells, microvascular endothelial cells, venous endothelial cells, arterial endothelial cells, smooth muscle cells, cardiac muscle cells, hepatocytes, beta cells, keratinocytes, purkinji (purkinji) fibers, neurons, cholangioepithelial cells, pancreatic islet cells, alveolar cells, clara cells, brush border cells, or podocytes. Bone marrow-derived stem cells such as bone marrow mononuclear cells (BM-MNCs), endothelial or vascular stem or progenitor cells, and peripheral blood-derived stem cells such as Endothelial Progenitor Cells (EPCs) may also be used.

The number of cells introduced by perfusion of a decellularized scaffold may depend on the organ (e.g., what organ, size and weight of the organ) or tissue, as well as the type and developmental stage of the regenerative cells. Different types of cells may have different tropisms with respect to the population density that those cells will reach. Similarly, different organs or tissues may be cellularized at different densities. For example, a decellularized organ or tissue can be "seeded" with at least about 1,000 (e.g., at least 10,000, 100,000, 1,000,000, 10,000,000, or 100,000,000) cells; or there may be from about 1,000 cells/mg tissue (wet weight, e.g., prior to decellularization) to about 10,000,000 cells/mg tissue (wet weight) attached thereto.

Cells can be introduced ("seeded") into decellularized organs or tissues by injecting them at one or more sites. In addition, various types of cells can be introduced into decellularized organs or tissues. For example, a population of differentiated cell types may be injected at multiple sites of a decellularized organ or tissue, or different cell types may be injected into different portions of a decellularized organ or tissue. Alternatively, or in addition to injection, the cells or cell mixtures may be introduced into the cannulated decellularized organ or tissue by perfusion. For example, cells may be perfused to a decellularized organ using a perfusion medium, which may then be replaced with an expansion medium and/or a differentiation medium to induce growth and/or differentiation of the cells. Site-specific differentiation can be achieved by placing cells at different sites within the organ, for example in areas of the heart such as the atria, ventricles or nodules.

During recellularization, the organ or tissue is maintained under conditions under which at least some cells within or on the decellularized organ or tissue can proliferate and/or differentiate. Those conditions include, without limitation: suitable temperature and/or pressure, electrically and/or mechanically active, force, suitable O₂And/or CO₂The amount, the appropriate amount of moisture, and the sterile or near-sterile conditions. During recellularization, the decellularized organ or tissue and the regenerative cells attached thereto are maintained in a suitable environment. For example, the cells may require a nutritional supplement (e.g., nutrients and/or a carbon source such as glucose), an exogenous hormone or growth factor, and/or a specific pH.

The cells and decellularized organ or tissue may be allogeneic (e.g., human decellularized organ or tissue seeded with human cells), or the cells and decellularized organ or tissue may be xenogeneic (e.g., porcine decellularized organ or tissue seeded with human cells). As used herein, "allogeneic" refers to cells obtained from the same species as the organ or tissue source (e.g., related or unrelated individuals), while "xenogeneic" as used herein refers to cells obtained from a different species than the organ or tissue source.

The stem cell or progenitor cell culture Medium may contain a variety of components, including, for example, KODMEM Medium (knock-out Dulbecco's Modified Eagle's Medium), DMEM, Ham's F12 Medium, FBS (fetal bovine serum), FGF2 (fibroblast growth factor 2), KSR, or hLIF (human leukemia inhibitory factor). The cell differentiation medium may also contain supplements such as L-glutamine, NEAA (non-essential amino acids), P/S (penicillin/streptomycin), N2, B27, and beta-mercaptoethanol. It is contemplated that additional factors may be added to the cell differentiation medium, including, but not limited to: fibronectin, laminin, heparin sulfate, retinoic acid, members of the epidermal growth factor family (EGF), members of the fibroblast growth factor family (FGF) including FGF2, FGF7, FGF8, and/or FGF10, members of the platelet derived growth factor family (PDGF), Transforming Growth Factor (TGF)/Bone Morphogenetic Protein (BMP)/Growth Differentiation Factor (GDF) factor family antagonists including, but not limited to, noggin, follistatin, tenascin, gremlin, cerberus/DAN family proteins, ventropin, high dose activin, and amnionless protein or variants or functional fragments thereof. TGF/BMP/GDF antagonists in the form of TGF/BMP/GDF receptor-Fc chimeras may also be added. Other factors that may be added include molecules that can activate or inactivate signals through the Notch receptor family, including but not limited to like proteins and Jagged family proteins as well as inhibitors of Notch processing or cleavage, or variants or functional fragments thereof. Other growth factors may include insulin-like growth factor family members (IGFs), insulin, wingless gene-related (WNT) factor family, and hedgehog (hedgehog) family or variants or functional fragments thereof. Additional factors may be added to promote proliferation and survival of mesendoderm, endoderm, mesoderm or definitive endoderm stem or progenitor cells, as well as survival and differentiation of these progenitor cell derivatives.

In one embodiment, the perfusion decellularized matrix is combined with iPS or ES cells differentiated using an Embryoid Body (EB) method. For example, human iPS cell lines that are reprogrammed by transduction, e.g., lentivirus-mediated transduction of transcription factors (OCT4, SOX2, NANOG and LIN 28; Oct3/4, Sox2, Klf4 and c-Myc; or Oct3/4, Sox2 and Klf4) are used. iPS clones of fetal origin or neonatal origin may be used. Human ES cell lines may also be used. Can be cultured in a 6-well plate (Nunc) containing DMEM/F12 medium at 19,500 cells/cm²In (d), iPS cells and ES cells were maintained in irradiated Mouse Embryonic Fibroblasts (MEF), supplemented with 20% KnockOut serum replacement (KnockOut serum replacer) (Invitrogen), 0.1mmol/L non-essential amino acids, 1mmol/L L-glutamine, and 0.1mmol/L β -mercaptoethanol (Sigma).In addition, for iPS cells, the medium can be supplemented with 100ng/mL zebrafish basic fibroblast growth factor, for hES cells with 4ng/mL human recombinant basic fibroblast growth factor (Invitrogen.) and iPS and ES cell lines can be maintained in gelled 100-mm dishes in DMEM (Sigma-Aldrich) containing 15% fetal calf serum (FCS; Sigma-Aldrich), 0.1. mu. mol/L2-mercaptoethanol (2ME) and 1,000 units/mL LIF (Chemicon International). for differentiation, these cells can be treated with 0.25% trypsin/ethylenediaminetetraacetic acid (GIBCO) and treated with 3 × 10⁴Concentration of individual cells/well they were transferred to a gelled 6-well plate containing α -minimal essential medium (GIBCO) supplemented with 10% FCS and 0.05. mu. mol/L2 ME.

Clones can be isolated from the plates by the following method: the clones are incubated with 1mg/mL dispase (Gibco) solution for 8 to 15 minutes at 37 ℃ and placed in suspension-cultured ultra-low adsorption plates, for example for 4 days. During suspension culture, the medium can be changed on day 1, and then cultured for another 3 days without changing the medium. The EBs are then placed on 0.1% gelatin-coated culture plates, for example at a density of 50 to 100 EBs per well, or in perfusion decellularized ECM, and cultured in differentiation media (e.g., changed daily).

In certain instances, an organ or tissue produced by the methods described herein is transplanted into a patient. In those cases, the cells used to recellularize the decellularized organ or tissue can be obtained from the patient such that the cells are "autologous" to the patient. Cells from a patient can be obtained, for example, from blood, bone marrow, tissue or organ at various stages of life (e.g., prenatal, neonatal or perinatal, adolescent or as an adult) using methods known in the art. Alternatively, the cells used to recellularize the decellularized organ or tissue may be syngeneic with the patient (i.e., from the homozygotic twins), the cells may be Human Lymphocyte Antigen (HLA) -matched cells from, for example, a patient's relatives or HLA-matched individuals unrelated to the patient, or the cells may be allogeneic to the patient from, for example, a non-HLA-matched donor.

The decellularized solid organ can be autologous, allogeneic or xenogeneic to the patient, regardless of the source of the cells (e.g., autologous or non-autologous).

During recellularization, the progression of the cells can be monitored. For example, during recellularization, the number of cells on or within an organ or tissue can be assessed by biopsy tissue sections obtained at one or more time points. In addition, the amount of cells that have undergone differentiation can be monitored by determining whether a different marker is present in the cell or population of cells. Markers associated with different cell types and different differentiation stages of those cell types are known in the art and can be readily determined using antibodies and standard immunoassays. See, for example, Current Protocols in Immunology,2005, Coligan et al, eds., John Wiley & Sons, Chapters 3and 11. Nucleic acid analysis as well as morphological and/or histological evaluation can be used to monitor recellularization.

The recellularized graft is continuously perfused. During the culture period, cell viability is maintained, and quantification of TUNEL positive cells can be performed, for example to determine apoptotic cells.

Controlled system for decellularising and/or recellularising an organ or tissue

Systems (e.g., bioreactors) for decellularizing and/or recellularizing an organ or tissue typically include: at least one intubation device for inserting a cannula into an organ or tissue, perfusion apparatus for perfusing an organ or tissue through the cannula, and apparatus (e.g., a sealing system) for maintaining a sterile environment of the organ or tissue. Intubation and perfusion are well known techniques in the art. Intubation devices typically include a suitably sized hollow tube for introduction into a vessel, duct, and/or lumen of an organ or tissue. Typically, one or more vessels, conduits and/or lumens in an organ are cannulated. The perfusion apparatus may comprise a holding container for a liquid (e.g. cell disruption medium) and a mechanism (e.g. pump, pneumatic pressure, gravity) for flowing the liquid through the organ via one or more cannulae. A variety of techniques known in the art can be used to maintain sterility of the organ or tissue during decellularization and/or recellularization, such as controlling and filtering the gas flow and/or perfusion with, for example, antibiotics, antifungal agents, or other antimicrobial agents, to prevent the growth of unwanted microorganisms.

A system for decellularizing and recellularizing an organ or tissue as described herein may have the ability to monitor certain perfusion characteristics (e.g., pressure, volume, flow patterns, temperature, gas, pH), mechanical forces (e.g., ventricular wall motion and stress), and electrical stimulation (e.g., pacing). Since the coronary vascular bed varies with the process of decellularisation and recellularisation (e.g. vascular resistance, volume), perfusion apparatus that regulate pressure are advantageous in order to avoid large fluctuations. The effectiveness of perfusion can be assessed in the outflow and tissue sections. Perfusion volume, flow pattern, temperature, local O can be monitored using standard methods₂And CO₂Pressure and pH.

Sensors may be utilized to monitor the system (e.g., bioreactor) and/or the organ or tissue. Sonar micrometering, micropressure and/or conductometry may be used to obtain pressure-volume or preload-supplemented stroke work information relative to myocardial wall motion and performance. For example, a sensor may be used to monitor the pressure of fluid flowing through an organ or tissue in which the cannula is inserted; the ambient temperature of the system and/or the temperature of the organ or tissue; the pH and/or flow rate of the fluid through the intubated organ or tissue; and/or the biological activity of the recellularized organ or tissue. In addition to being equipped with sensors to monitor such characteristics, systems for decellularizing and/or recellularizing an organ or tissue may also include devices for maintaining or regulating such characteristics. Apparatus for maintaining or adjusting such features may include components such as: a thermometer, a thermostat, an electrode, a pressure sensor, an overflow valve, a valve for changing the liquid flow rate, a valve for opening or closing a liquid connection with a solution for changing the pH of the solution, a balloon, an extracorporeal pacemaker and/or a compliance lumen. To help ensure stable conditions (e.g., temperature), the chambers, reservoirs, and tubes may be water-jacketed.

During recellularization, it is advantageous to place a mechanical load on the organ and cells attached thereto. As an example, a balloon inserted into the left ventricle via the left atrium may be used to apply mechanical stress to the heart. A piston pump, which allows for volume and velocity adjustment, can be connected to the balloon, simulating left ventricular wall motion and stress. To monitor wall motion and stress, left ventricular wall motion and stress may be measured using micro tonometry and/or sonar micro-measurement. In some embodiments, an extracorporeal pacemaker may be connected to a piston pump to provide stimulation synchronized with each deflation of the ventricular balloon (which corresponds to a contraction of the heart). Peripheral ECGs from the surface of the heart can be recorded, allowing pacing voltages to be adjusted, monitoring depolarization and repolarization, and providing a simplified surface map of the heart that recellularizes or recellularizes the heart.

Mechanical ventricular dilation can also be achieved by attaching a peristaltic pump to a cannula that is inserted through the left atrium into the left ventricle. Similar to the procedure described above involving balloons, ventricular dilation by periodic fluid flow (e.g., pulsatile flow) through the cannula may be synchronized with electrical stimulation.

Using the methods and materials disclosed herein, a mammalian heart can be decellularized and recellularized, and when maintained under suitable conditions, a functional heart can be generated that undergoes contractile function as well as responding to pacing stimuli and/or pharmacological agents.

A system for generating an organ or tissue may be controlled by a computer readable storage medium in combination with a programmable processor (e.g., a computer readable storage medium as used herein having stored thereon instructions that cause a programmable processor to perform particular steps). For example, such a storage medium, in combination with a programmable processor, may receive and process information from one or more sensors. Such storage media in connection with the programmable processor may also transmit information and instructions back to the bioreactor and/or organ or tissue.

The biological activity of an organ or tissue undergoing recellularization can be monitored. The biological activity may be the biological activity of the organ or tissue itself, such as electrical activity, mechanical stress, contractility, and/or stress of the organ or tissue wall in relation to the heart tissue. In addition, cells attached to an organ or tissue may be monitored for biological activity, e.g., ion transport/exchange activity, cell division, and/or cell viability. See, for example, Laboratory Textbook of atom and physiology (2001, Wood, Presence Hall) and Current Protocols in Cell Biology (2001, Bonifacino et al, Eds, John Wiley & Sons). As discussed above, it may be useful to simulate the payload of an organ during recellularization. The computer readable storage medium of the present invention in combination with a programmable processor may be used for components necessary to coordinate the monitoring and maintaining of a payload on an organ or tissue.

In one embodiment, the weight of an organ or tissue can be recorded into a computer readable storage medium described herein, which in combination with a programmable processor, is capable of calculating the exposure time and perfusion pressure for a particular organ or tissue. Such storage media may record the front and rear loads (pressure before and after priming, respectively) and the flow velocity. In this embodiment, for example, a computer readable storage medium in combination with a programmable processor controls the adjustable perfusion pressure, perfusion direction, and/or perfusion solution type via one or more pumps and/or valves.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1

Comparison of perfusion and immersion

Fig. 1A shows a photograph of perfusion decellularized pig liver, and fig. 1B and 1C show SEM of vascular and parenchymal matrix of perfusion decellularized pig liver, respectively. These photographs show the integrity of vascular ducts and matrix perfusing decellularized organs. On the other hand, fig. 2 shows a general view of a rat liver immersed in decellularization, in which the attrition of the matrix is visible at both low magnification (left) and high magnification (right).

FIG. 3 shows SEM of immersion in decellularized rat liver (A and B) and SEM of perfusion decellularized rat liver (C and D). These results clearly indicate that immersion decellularization significantly damaged organ capsules (Glisson's capsule), whereas perfusion decellularization retained organ capsules. Furthermore, FIG. 4 shows the histological structure of the immersed decellularized liver (A, H & E staining; B, trichrome staining) and the perfusion decellularized liver (C, H & E staining; D, trichrome staining). Upon injection, immersion into decellularized rat liver did not retain cells or dye.

Fig. 5 shows a comparison between immersion decellularization (top row) and perfusion decellularization (bottom row) of rat hearts. The left column of photographs shows the entire organ. From these two photographs, it can be seen that the perfusion decellularized organ (bottom left) is more transparent than the immersion decellularized organ (top left), which retains the iron-rich "red-brown" color of the cadaver muscle tissue and appears to still contain cells. The photographs in the middle column show the H & E staining pattern of decellularized tissue. Staining showed that many cells within the parenchyma and within the walls of the vasculature were preserved after immersion decellularization (above-mid), whereas after perfusion decellularization (below-mid), almost every cell and cell debris was removed, but the vascular catheter was evident. Furthermore, the scanning electron micrographs in the right column show a clear difference in the ultrastructure of the matrix after immersion decellularization (upper right) compared to after perfusion decellularization (lower right). Again, intact retention of cellular components throughout the myocardial cross section was observed in the entire wall immersed decellularized heart, but in perfusion decellularized heart, almost complete loss of these cellular components was observed, as well as retention of spatial and structural features of the intact myocardium, including vascular ducts. For example, perfusion of decellularized matrices retains structural features within the matrix, including weaves (w), curls (coils (c), and struts (s)), despite complete loss of cells.

Figure 6 shows the same comparison of immersion decellularization (top row) and perfusion decellularization (bottom row) using rat kidney. Unlike the heart, it looks very similar because immersion into the decellularized whole kidney (upper left) is quite transparent to perfusion decellularized whole kidney (lower left). However, in perfusion decellularized kidneys, the vascular conduit network within the perfusion decellularized organ is more pronounced and a higher degree of branching is observed than in immersion decellularized constructs. In addition, the perfusion decellularized kidney surrounded by mesentery retains the intact organ capsule, and as shown, it can be decellularized with the attached adrenal gland. The photographs in the middle column show the H & E staining pattern of both tissues. Staining showed that cellular components and/or debris and possibly even intact nuclei were retained after immersion decellularization (upper-middle), whereas almost every cell and/or all cellular debris was removed after perfusion decellularization (lower-middle). Likewise, SEM photographs show that immersion into decellularized kidney matrix (upper right) suffered more damage than perfusion of decellularized kidney matrix (lower right). In immersion into decellularized kidneys, organ capsules are lost or damaged to make "holes" or abrasion of the matrix surface more evident, whereas in perfusion decellularized organs, the capsules are intact.

Figure 7 shows SEM photographs of decellularized kidneys. Fig. 7A shows perfusion of a decellularized kidney, while fig. 7B shows immersion of a decellularized kidney. Fig. 8A shows an SEM photograph of a perfusion decellularized heart, while fig. 8B shows an SEM photograph of an immersion decellularized heart. These images further show the damage of the organ ultrastructure caused by immersion decellularization and the viability of the matrix after perfusion decellularization.

Example 2

Exemplary particles for use in the methods of the invention

The particles used in the method of the invention include nanoparticles or microparticles, such as nanospheres or microspheres, which may be formed from a variety of different biocompatible materials, such as degradable or non-degradable synthetic materials, biological (natural) materials or modified biological materials. Examples of materials from which nanoparticles or microparticles can be formed include, but are not limited to: alginate, polysaccharide, collagen, dextran, hyaluronic acid, glass, ceramic, metal (including titanium), particles with a ferrite core, PLA, PGA, PLA/PGA, monodisperse melamine resin particles, polystyrene, nylon, PMMA, and the like. By way of example, and not limitation, suitable polymeric materials may include the following polymers: polyoxides such as poly (ethylene oxide) and poly (propylene oxide); polyesters, such as poly (ethylene terephthalate); a polyurethane; a polysulfonate salt; polysiloxanes, such as poly (dimethylsiloxane); a polysulfide; polyacetylene; polysulfones; polysulfonamides; polyamides, such as polycaprolactam and poly (hexamethylene adipamide); a polyimide; a polyurea; heterocyclic polymers such as polyvinylpyridine and polyvinylpyrrolidone; naturally occurring polymers such as natural rubber, gelatin, cellulose; a polycarbonate; a polyanhydride; and polyolefins such as polyethylene, polypropylene, and ethylene-propylene copolymers. The polymeric material may also contain functional groups such as carboxylates, esters, amines, aldehydes, alcohols, or halides to provide, for example, sites for attachment of chemical or biological moieties that are desired to enhance the utility of the particles in chemical or biological analytes.

Methods for preparing nanoparticles or microparticles from polymers are well known in the art. For example, proteins may be combined with non-protein polymers to form composite nanospheres or microspheres. In one embodiment, the particles are bioerodible synthetic or natural polymers. The term "bioerodible" or "biodegradable" as used herein refers to a material that degrades enzymatically, thermally, electrically, ionically, pH, mechanically or chemically, or dissociates into simpler chemical species. An example of a natural polymer is a polysaccharide. Synthetic polymers that degrade in vivo to innocuous products include poly (lactic acid) (PLA), poly (glycolic acid) (PGA), and copolymers of PLA and PGA, polyorthoesters, polyanhydrides, polyphosphazenes, polycaprolactone, polyhydroxybutyrate, mixtures thereof, and copolymers thereof. PLA, PGA, and PLA/PGA copolymers are particularly useful for forming prolamin composite microspheres. PLA polymers are typically prepared from cyclic esters of lactic acid. Non-optically active DL-lactic acid mixtures of L (+) and D (-) types of lactic acid and mixtures of D (-) and L (+) lactic acid can be used to prepare PLA polymers. The process for the preparation of polylactic acid is clearly documented in the patent literature. Suitable polylactic acids, their properties and preparation are described in detail in the following U.S. patents, the teachings of which are incorporated herein by reference: dorough, 1,995,970; 2,703,316 to Schneider; 2,758,987 to Salzberg; 2,951,828 to Zeile; 2,676,945 to Higgins; and 2,683,136; 3,531,561-Trehu. Temperature sensitive polymers include, but are not limited to: poly (N-isopropylacrylamide), hydroxypropylcellulose, poly (vinylcaprolactam), polyvinylmethyl ether, and polyhydroxyethylmethylmethacrylate.

In one embodiment, the granules are formed from a polysaccharide that is readily degradable by an enzyme (e.g., amylase). This would allow for rapid removal of the particles prior to implantation.

The particles may be of a diameter similar to that of red blood cells, so that a single particle passes through the capillary bed. The particles can be, for example, from about 0.01 μm to about 30 μm, from about 0.5 μm to about 20 μm, or from about 5 μm to about 10 μm.

The particles may be of any shape suitable for passage through the vasculature, including but not limited to: spheres, ovals, discs, donuts or stars, the particles having a concave or convex surface and may have a smooth to matte surface.

The particles may have or be modified to have properties including, but not limited to: hydrophilic surface to ensure easy passage; the ability of the surface to undergo shrinkage under pressure, such as a hydrogel or protein coating; can be removed from the matrix by degradation, mechanical (e.g., magnetic agglomeration), or energy to break it down into smaller pieces that can be successfully expelled from the matrix.

To ensure that the capillaries formed are of sufficient diameter so that red blood cells are not trapped after transplantation, the particles can be added at the time of endothelial cell seeding or about 12 to 96 hours or up to several weeks after cell seeding. For example, to increase the diameter of re-endothelialized blood vessels in an otherwise decellularized graft or a graft re-cellated with cells other than endothelial cells, the capillaries can be forced open by: starting with a small particle size and then slowly increasing the particle size with hours/day until the desired size can be infused through the matrix.

In one embodiment, the particles are formed from a temperature sensitive polymer that can be easily degraded or dissociated by a change in temperature. This would allow for rapid removal of the particles prior to implantation.

In one embodiment, the particles are formed from a magnetic polymer, and the addition of a magnetic source to the circulating solution enables removal of the circulating particles prior to implantation.

In one embodiment, the particles are removed from the re-endothelialized tissue or organ prior to transplantation. For biodegradable particles, specific reagents or conditions will be added to degrade, dissolve or digest the particles, followed by washing. Removal of the particles may occur weeks/days before transplantation, or just before transplantation.

The normal concentration of red blood cells is about 3 million to about 5 million/μ L. Since the vasculature represents about 10% of the total tissue or organ void volume, the concentration of particles can be about 300-.

To determine the efficacy of the particles in maintaining, increasing, or decreasing the decrease in capillary lumen diameter, the percentage of particles recovered after perfusion of the particles through the re-endothelialized matrix at physiological pressure was determined. For example, particles for use in the methods are those, e.g., > 50%, 60%, 70% or more of particles having a size of approximately blood cell diameter or having an average diameter of about 5 to 8 μm are recovered after perfusion through a tissue or organ, or blood capable of perfusion through a tissue or organ at physiological pressure is > 50%, 60%, 70% returned. In non-recellularized organs, tissues or parts thereof, most of the particles (5 to 8 μm) reside in the interstitial spaces and do not show significant return. In a non-particle treated re-endothelialized organ, tissue, or portion thereof, a majority of the particles (e.g., those of about 5 μm to about 8 μm) reside in the vasculature and do not pass through the capillary bed.

All publications, patents and patent applications are herein incorporated by reference. While in the foregoing specification this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein can be varied considerably without departing from the basic principles of the invention.

Claims (35)

1. A method of maintaining, reducing a decrease in, or increasing capillary vessel lumen diameter in a re-endothelialized extracellular matrix of a mammalian organ, tissue or portion thereof having an intact vascular bed, comprising:

providing a decellularized extracellular matrix of a mammalian organ, tissue or portion thereof having an intact vascular bed, and a population of endothelial cells or a population of stem or progenitor cells capable of differentiating into endothelial cells; and

introducing into the decellularized extracellular matrix an amount of the cells and a first aqueous solution containing an amount of biocompatible microparticles, wherein the amount of the cells is effective to re-endothelialize vasculature of the decellularized extracellular matrix, and wherein during or after re-endothelialization the amount of the microparticles maintains, reduces a decrease in, or increases a capillary lumen diameter within the vasculature as the microparticles circulate through the vasculature relative to a corresponding re-endothelialized decellularized extracellular matrix lacking the microparticles.

2. The method of claim 1, wherein the microparticles maintain flow through the capillary bed.

3. The method of claim 1 or 2, wherein the microparticle is biodegradable.

4. The method of claim 1 or 2, wherein the microparticle is not biodegradable.

5. The method of claim 1, wherein the microparticles are spherical or ellipsoidal.

6. The method of claim 1, wherein the microparticles are deformable.

7. The method of claim 1, wherein the microparticles are formed from a polymer.

8. The method of claim 7, wherein the polymer is a naturally occurring polymer.

9. The method of claim 1, wherein the microparticles comprise alginate, polysaccharide, collagen, dextran, hyaluronic acid, glass, ceramic, metal, polylactic acid (PLA), polyglutamic acid (PGA), or a copolymer of PLA and PGA (PLA/PGA).

10. The method of claim 7, wherein the polymer is a non-naturally occurring polymer.

11. The method of claim 1, wherein the microparticles are modified to comprise a carboxylate, ester, amine, aldehyde, alcohol, or halide.

12. The method of claim 1, wherein the microparticles are formed from a proteinaceous polymer and a non-proteinaceous polymer.

13. The method of claim 1, wherein the microparticles have an average diameter of 0.5 μ ι η to 20 μ ι η.

14. The method of claim 1, wherein the microparticles comprise a hydrophilic surface.

15. The method of claim 1, wherein the microparticles are magnetic.

16. The method of claim 1, wherein the microparticle comprises a surface modification capable of binding to a ligand.

17. The method of claim 1 or 6, wherein the microparticle comprises a hydrogel.

18. The method of claim 1, wherein the solution is added after re-endothelialization.

19. The method of claim 1, further comprising introducing a second aqueous solution comprising biocompatible microparticles having an average diameter at least 10% greater than the average diameter of the microparticles in the first aqueous solution.

20. The method of any one of claims 19, further comprising introducing a third aqueous solution comprising biocompatible microparticles having an average diameter at least 10% greater than the average diameter of the microparticles in the second aqueous solution.

21. The method of claim 1, wherein the first aqueous solution comprises 300 to 500,000 microparticles per μ L.

22. The method of claim 1, further comprising washing the vasculature with a solution free of the microparticles.

23. The method of claim 22, wherein the solution free of nanoparticles or microparticles comprises an agent capable of degrading the microparticles.

24. The method of claim 22, wherein the solution free of nanoparticles or microparticles is applied concurrently with an external factor capable of degrading or removing the microparticles, the external factor comprising temperature, pH, ultrasound, light, or electrical energy.

25. The method of claim 1, wherein the microparticles comprise polystyrene.

26. The method of claim 1, wherein the microparticles comprise a polysaccharide.

27. The method of claim 25 or 26, wherein the microparticles have an average diameter of 5 to 20 microns.

28. The method of claim 1, wherein the organ is a heart, pancreas, bone, liver, kidney, or lung.

29. The method of claim 1, wherein the cells are obtained from iPS cells.

30. The method of claim 1, wherein the population is introduced into the matrix by injection or perfusion, or a combination thereof.

31. The method of claim 1, wherein the population comprises primary cells.

32. The method of claim 1, wherein the population comprises a plurality of different cell types.

33. The method of claim 1, wherein the population comprises human embryonic stem cells.

34. The method of claim 1, wherein the cells are allogeneic to the perfusion decellularized organ or tissue.

35. The method of claim 1, wherein the cells are xenogeneic to the perfusion decellularized organ or tissue.