HK1170662A - Decellularization and recellularization of organs and tissues - Google Patents

Description

Decellularization and recellularization of organs and tissues

Technical Field

The present invention relates to organs and tissues, and more particularly to methods and materials for decellularization and recellularization of organs and tissues.

Background

Biologically derived matrices have been developed for tissue engineering and regeneration. However, the matrices developed to date often have a compromised matrix structure and/or do not exhibit a vascular bed that is effective in reconstructing an organ or tissue. Methods of decellularization and recellularization of organs and tissues are described herein.

Summary of The Invention

Provided herein are methods and materials for decellularizing an organ or tissue, and methods and materials for recellularizing a decellularized tissue or organ.

In one aspect, a decellularized mammalian heart is provided. A decellularized mammalian heart includes a heart decellularized extracellular matrix having an outer surface. The extracellular matrix of the decellularized heart substantially maintains the shape of the extracellular matrix before decellularization, and the outer surface of the extracellular matrix is substantially intact.

Representative hearts include, but are not limited to: rodent heart, porcine heart, rabbit heart, bovine heart, ovine heart or canine heart. Another representative heart is the human heart. The decellularized heart can be a cadaveric heart. In some embodiments, the decellularized heart is a portion of an intact heart. For example, a portion of a complete heart includes, without limitation: myocardium, aortic valve, left atrioventricular valve, pulmonary valve, right atrioventricular valve, right atrium, left atrium, right ventricle, left ventricle, septum, coronary artery, pulmonary artery, or pulmonary vein.

In another aspect, the present invention provides a solid organ. The solid organ described herein includes the above decellularized heart and a regenerative cell population attached thereto. In some embodiments, the regenerative cells are pluripotent cells. In some embodiments, the regenerative cells are embryonic stem cells, umbilical cord cells, adult stem or progenitor cells, bone marrow-derived cells, blood-derived cells, Mesenchymal Stem Cells (MSCs), skeletal muscle-derived cells, Multipotent Adult Progenitor Cells (MAPCs), Cardiac Stem Cells (CSCs), or multipotent adult cardiac stem cells. In some embodiments, the regenerative cells are cardiac fibroblasts, cardiac microvascular cells or aortic endothelial cells. In some embodiments, the cell is a tissue-derived or skin-derived cell.

Generally, the number of regenerative cells attached to the decellularized heart is at least about 1,000. In some embodiments, the number of regenerative cells attached to the decellularized heart is about 1,000-10,000,000 cells/mg tissue (wet weight; i.e., weight before decellularization). In some embodiments, the regenerative cells are heterologous to the decellularized heart. In other embodiments, the solid organ is also transplanted into a patient, and the regenerative cells are autologous to the patient.

In yet another aspect, the present invention provides a method of preparing a solid organ. Such methods generally comprise providing a decellularized heart as described herein, and contacting the decellularized heart with a population of regenerative cells under conditions such that the regenerative cells are capable of engraftment and proliferation and/or differentiation in and on the decellularized heart. In one embodiment, the regenerative cells are injected or perfused into the decellularized heart.

In yet another aspect, the invention provides a method of decellularizing a heart. Such methods include providing a heart, cannulating the heart at one or more cavities, vessels, and/or ducts, and perfusing the cannulated heart with a first cell disrupting medium through one or more cannulas. For example, the perfusion may be multidirectional from the lumen, vessel and/or duct of each cannula. Generally, the cell disruption medium contains at least one detergent, such as SDS, PEG or Triton X.

The method may further comprise perfusing the cannulated heart through the plurality of cannulae with a second cell disruption medium.

Generally, the first cell disruption medium can be an anionic detergent, such as SDS, and the second cell disruption medium can be an ionic detergent, such as Triton X-100. In this method, the perfusion may last for about 2-12 hours per gram (wet weight) of cardiac tissue.

In one aspect, a solid organ is provided. Such solid organs include decellularized organs and regenerative cell populations attached thereto. Such decellularized organs comprise a decellularized extracellular matrix of said organ, wherein the extracellular matrix comprises an outer surface, wherein the outer surface, including the vascular tree, substantially retains the morphology of the decellularized extracellular matrix, and wherein the outer surface is substantially intact.

Representative solid organs include heart, kidney, liver or lung. In one embodiment, the solid organ is a liver or a part of a liver. In another embodiment, the solid organ is a heart (e.g., a rodent heart, a porcine heart, a rabbit heart, a bovine heart, a ovine heart, or a canine heart; e.g., a heart exhibiting contractile activity). A representative heart is a human heart. The heart may be a portion of an intact heart (e.g., an aortic valve, a left atrioventricular valve, a pulmonary valve, a right atrioventricular valve, a right atrium, a left atrium, a right ventricle, a left ventricle, a myocardium, a septum, a coronary artery, a pulmonary artery, and a pulmonary vein). In another embodiment, the solid organ is a kidney. The solid organs described herein generally include a variety of histological structures, including blood vessels.

In some embodiments, the number of regenerative cells attached to the decellularized heart is at least about 1,000. In some embodiments, the number of regenerative cells attached to the decellularized heart is about 1,000-10,000,000 cells/mg tissue. The regenerative cells may be pluripotent cells. In addition, the regenerative cells may be embryonic stem cells or subsets thereof, umbilical cord cells or subsets thereof, bone marrow cells or subsets thereof, peripheral blood cells or subsets thereof, adult-derived stem or progenitor cells or subsets thereof, tissue-derived stem or progenitor cells or subsets thereof, Mesenchymal Stem Cells (MSC) or subsets thereof, skeletal muscle-derived stem or progenitor cells or subsets thereof, Multipotent Adult Progenitor Cells (MAPC) or subsets thereof, Cardiac Stem Cells (CSC) or subsets thereof, or multipotent adult heart-derived stem cells or subsets thereof. Examples of regenerative cells include cardiac fibroblasts, cardiac microtubule endothelial cells, aortic endothelial cells or hepatocytes. In some embodiments, the regenerative cells are heterologous or xenogeneic to the decellularized organ.

In some embodiments, the solid organ is transplanted into a patient and the regenerative cells are autologous to the patient. In other embodiments, the solid organ is transplanted into a patient, and the decellularized organ is allogeneic or xenogeneic to the patient.

In another aspect, a method of preparing an organ is provided. Such methods generally include providing a decellularized organ, wherein said decellularized organ comprises a decellularized extracellular matrix of the organ, wherein said extracellular matrix comprises an outer surface, wherein said extracellular matrix comprising the vascular tree substantially maintains the morphology of said extracellular matrix prior to decellularization, wherein said outer surface is substantially intact; contacting said decellularized organ with a population of regenerative cells under conditions wherein said regenerative cells are capable of engrafting, proliferating and/or differentiating in and on said decellularized organ. In one embodiment, the regenerative cells are injected into the decellularized organ. Representative decellularized organs include heart, kidney, liver, spleen, pancreas, or lung.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings, detailed description, and claims.

Brief description of the drawings

FIG. 1 illustrates the preliminary preparation of cardiac decellularization. The aorta, pulmonary artery and superior vena cava were cannulated (A, B and C, respectively), and the inferior vena cava, brachiocephalic artery, left common carotid artery and left subclavian artery were ligated. Arrows indicate antegrade and retrograde perfusion directions.

FIG. 2 illustrates one embodiment of a decellularization/recellularization apparatus.

Fig. 3A is a photograph of decellularized liver and kidney, and fig. 3B is a photograph of decellularized heart and lung. The photograph on the left shows the histological staining of the tissue, depicting the quantification of the nucleic acid remaining in the cadaveric organ, while the photograph on the right shows the histological staining of the acellular matrix and the quantification of the nucleic acid remaining in the perfusion acellular organ.

FIG. 4 shows photographs of perfusion decellularized pig kidney (left) and rat kidney (center; panels show perfusion with Evans blue dye) and EM photographs of tubule-enclosed glomeruli and perfusion decellularized collection tubes.

FIG. 5 is a photograph of a whole rat decellularized from the lower abdomen to the head.

FIG. 6 is a photograph showing liver recellularization. FIG. 6A shows perfusion of decellularized rat liver; fig. 6B shows injection of primary hepatocytes into individual decellularized rat liver lobes via portal vein catheter.

FIG. 7 shows that recellularization can be directed. FIG. 7A shows primary rat hepatocytes delivered into decellularized liver tail lobes; fig. 7B shows primary rat hepatocytes delivered into the right lateral inferior/superior lobe of decellularized rat liver.

FIG. 8 is an SEM photograph showing recellularization of decellularized rat liver. Delivery of 40x10 through the portal vein⁶Primary rat hepatocytes, cultured for 1 week (A-D).

FIG. 9 shows recellularized rat liver staining 1 week after injection of primary rat hepatocytes into the caudate knob. FIG. 9A is a Mashimura trichrome stain (10X) and FIG. 9B is an H & E stain (10X).

Figure 10 shows the liver TUNEL analysis 1 week after recellularisation in the caudate process with primary rat hepatocytes. Fig. 10A is TUNEL staining showing a mixture of live and apoptotic cells (10X) and fig. 10B shows mahalanobis trichrome staining (10X).

FIG. 11 shows the Mayer's trichrome staining of human HepG2 cells 1 week after in vitro perfusion of decellularized rat liver. Fig. 11A shows the caudate process and fig. 11B shows the superior/inferior right lateral lobe. Both are 10 times larger; v ═ a container in the matrix.

FIG. 12 is cell retention efficiency. The figure shows the primary rat hepatocytes (1-6) or HepG2(7 and 8) cells retained after injection. Cells were counted before and after injection. Percent retention was calculated based on the original number minus retained cells.

FIG. 13 shows that HepG2 cells remained viable in the cell organelle removal function. Metabolism of the Alamalan showed HepG2 cells (about 30X10 days of injection)⁶) Survived and proliferated to a limit after injection of caudate process (diamonds) and right lateral superior/inferior lobe of liver (squares).

FIG. 14 shows a further detailTime course of urea production by primary rat hepatocytes after cellularization (about 35X 10)⁶One cell, 7 days).

FIG. 15 shows the time course of albumin production by primary rat hepatocytes per day after recellularization (about 35X 10)⁶One cell, 7 days).

FIG. 16 shows the time course of ethoxyisophenazone-O-deethylase (EROD) activity of primary rat hepatocytes injected in the tail lobes of the liver (23X 10)⁶One cell, 8 days).

Figure 17 shows embryonic and adult-derived stem/progenitor cells propagated on decellularized heart, lung, liver and kidney for at least 3 weeks.

FIG. 18 shows the survival of mouse embryonic stem cells (mESC) and proliferative adult stem cells (skeletal myoblasts; SKMB) on decellularized heart, lung, liver and kidney.

Fig. 19 is SEM photographs of cadaveric (left) and decellularized (right) hearts. LV, left ventricle; RV, right ventricle.

Fig. 20 is a histological (top) and SEM (bottom) comparison of cadaver (left) and recellularized rat liver (right).

Fig. 21 is a photograph showing (a) fully decellularized pig liver matrix, and SEM of perfusion decellularized pig liver showing (B) vascular and (C) mesenchymal matrix integrity.

FIG. 22 is a photograph showing a full view of an infiltrating decellularized liver. In spite of the appearance of an intact liver, matrix flaking and loss of the bursa can be seen in the enlarged views below (A) and above (B).

The SEM photographs of fig. 23 show that after infiltration decellularization (a and B), the organ lacks the grignard capsule, whereas after 1% SDS perfusion decellularization (C and D), the organ retains the capsule.

FIG. 24 is a photograph showing the histology of the liver of an infiltrated decellularized rat (A, H & E; B, trichrome Ma) and the histology after perfusion decellularization of 1% SDS (C, H & E; D, trichrome Ma).

Figure 25 is a photograph showing a comparison between rat cardiac infiltration decellularization (top panel) and perfusion decellularization (bottom panel). Left column, whole organ; middle panel, H & E histological staining; right column, SEM.

Figure 26 is a photograph showing a comparison between rat kidney infiltration decellularization (top panel) and perfusion decellularization (bottom panel). Left column, whole organ; middle panel, H & E histological staining; right column, SEM.

Fig. 27 is SEM photographs of perfusion decellularized kidneys (fig. 27A) and infiltrated decellularized kidneys (fig. 27B).

Fig. 28 is SEM photographs of a perfusion decellularized heart (fig. 28A) and an infiltrating decellularized heart (fig. 28B).

FIG. 29 is an SEM photograph of infiltrated decellularized liver.

Like reference symbols in the various drawings indicate like elements.

Detailed Description

Solid organs are generally composed of 3 major components, namely the extracellular matrix (ECM), cells embedded in the extracellular matrix, and vascular beds. Decellularizing a solid organ as described herein can remove most or all of the cellular components while substantially retaining the extracellular matrix (ECM) and vascular bed. The decellularized solid organ can then be used as a scaffold for recellularization. Mammals from which solid organs can be obtained include, but are not limited to: rodents, pigs, rabbits, cattle, sheep, dogs and humans. The organs and tissues used in the methods described herein may be from a cadaver, or may be a fetus, a neonate, or an adult.

Solid organs as referred to herein include, but are not limited to: heart, liver, lung, skeletal muscle, brain, pancreas, spleen, kidney, stomach, uterus, and bladder. A solid organ as used herein refers to an organ having a "substantially closed" vascular system. A "substantially closed" vascular system in relation to an organ means that the solid organ can contain most of the fluid when perfused with fluid, provided the main vessel is cannulated, ligated or otherwise restricted, without the fluid leaking out of the solid organ. Despite having a "substantially closed" vascular system, many of the solid organs listed above still define "inlet" and "outlet" vessels that can be used to introduce and remove fluids that pass through the organ during perfusion.

In addition to the solid organs described above, other types of vascular organs or tissues, such as all or part of a joint (e.g., knee, shoulder, hip, or spinal cord), trachea, skin, mesentery or intestinal tract, small and large intestine, esophagus, ovary, penis, testis, spinal cord, or single or branched vessel, may also be decellularized using the methods disclosed herein. Moreover, the methods disclosed herein can also be used to decellularize avascular (or relatively avascular) tissue, such as cartilage or cornea.

An decellularized organ or tissue (e.g., heart or liver) or any portion thereof (e.g., aortic valve, left atrioventricular valve, pulmonary valve, right atrioventricular valve, pulmonary vein, pulmonary artery, coronary artery, septum, right atrium, left atrium, right ventricle, left ventricle, or lobe) described herein, with or without recellularization, can be transplanted into a patient. Alternatively, the recellularized organ or tissue described herein can be used to detect, for example, cells undergoing differentiation and/or cellular composition of the organ or tissue.

Decellularization of organs or tissues

The present invention provides methods and materials for decellularizing a mammalian organ or tissue. If possible, the first step in decellularizing an organ or tissue is cannulating the organ or tissue. Vessels, conduits and/or cavities of organs or tissues may be cannulated using methods and materials known in the art. The next step in decellularizing an organ or tissue is to perfuse the cannulated organ or tissue with a cell disruption medium. Organ perfusion may be multidirectional (e.g., antegrade and retrograde).

Langendorff perfusion, i.e., physiological perfusion (also known as four-chamber mode of operation perfusion), is routinely used in the art for the heart. See, e.g., Methods in Experimental Physiology and pharmacology: dehnert in Biological Measurement Techniques V (Experimental physiological and pharmacological methods: bioassay technology V edition), The Isolated Perfused Warm-blood Heart According to Langendorff (perfusing Warm-blooded animals hearts According to Landaff separation), Biomesstechnik-Verlag March GmbH, West Germany, 1988. Briefly, Landaff perfusion cannulates the aorta and connects to a reservoir containing a cell disruption medium. The cell disruption medium can be delivered retrograde down the aorta at a constant flow rate, for example by infusion or roller pumps or by constant hydrostatic pressure. In both modes, the aortic valve is rigidly closed and the perfusate enters the coronary ostia directly (thereby filling the entire ventricle of the heart) and then drains through the coronary sinus into the right atrium. To perform working mode perfusion, a second cannula is connected to the left atrium, which may be changed from retrograde to antegrade.

Methods of perfusing other organs or tissues are known in the art. For example, the following references describe methods of perfusion of the lung, liver, kidney, brain and extremities. Van Putte et al, 2002, journal of cardiothoracic surgery (ann. thorac. surg.), 74 (3): 893-8; den button et al, 1995, journal of international transplantation (Transpl. int. }, 8: 466-71; Firth et al, 1989, clinical sciences (Clin. Sci.) (Lond.), 77 (6): 657-61; Mazzetti et al, 2004, Brain research (Brain Res.), 999 (1): 81-90; Wagner et al, 2003, journal of artificial organs (J.Artif. Organs.), 6 (3): 183-91).

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 SDS, PEG or Triton X. The cell disruption medium may comprise water, making the medium incompatible with cell permeability. Alternatively, the cell disruption medium may include a buffer (e.g., PBS) to make it compatible with cell permeability. Cell disruption media may also include enzymes such as, but not limited to: one or more collagenases, one or more dispases, one or more dnases, or a protease such as trypsin. In some cases, the cell disruption medium also (or alternatively) includes 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 continuously 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-100. Following perfusion with the at least one cell disruption medium, the cannulated organ or tissue may be perfused, for example, with a wash solution and/or a solution containing one or more enzymes as disclosed herein.

Changing the direction of perfusion (e.g., antegrade and retrograde) can facilitate efficient decellularization of the entire organ or tissue. Decellularization as described herein is essentially an organ decellularization from inside to outside with very little damage to the ECM. Organs or tissues can be decellularized at a suitable temperature of 4-40 ℃. Depending on the size and weight of the organ or tissue, and the particular detergent and detergent concentrations in the cell disruption medium, the organ or tissue is typically perfused with the cell disruption medium for about 2-12 hours per gram of solid organ or tissue. Including washing, organs can be perfused for up to about 12-72 hours per gram of tissue. Perfusion is typically adjusted to accommodate physiological conditions, including pulsatile blood flow, velocity, and pressure.

As described herein, a decellularized organ or tissue is composed primarily of extracellular matrix (ECM) components of all or most of the organ or tissue, including ECM components of the vascular network. The ECM component can include any or all of: fibronectin, fibrillin, laminin, elastin, collagen family members (e.g., collagen I, III and IV), aminodextran, matrices, reticular fibers, and thrombospondin, which may be organized into a defined structure such as a basal layer. Successful decellularization is defined as the absence of detectable myofilaments, endothelial cells, smooth muscle cells, and the absence of nuclei from the histological sections using standard histological staining methods. It is also preferred, but not necessary, to remove residual cellular debris from the decellularized organ or tissue.

Maintaining the morphology and architecture of the ECM during and after decellularization (i.e., remaining substantially intact) is very important for efficient recellularization and production of organs or tissues. As used herein, "morphology" refers to the overall shape of an organ or tissue or ECM, while "texture" as used herein refers to the outer surface, inner surface and ECM therebetween.

The morphology and architecture of the ECM can be detected visually and/or by histological methods. For example, it is not desirable to remove or severely disrupt the basal layer on the outer surface of a solid organ or within the vasculature of an organ or tissue during decellularization. Furthermore, the fibrils of the ECM should be similar to or not significantly altered by organs or tissues that are not decellularized. As used herein, unless otherwise indicated, decellularized refers to perfusion decellularized, and, unless otherwise indicated, decellularized organs or matrices described herein are obtained using perfusion decellularization methods described herein. Perfusion decellularization described herein can be compared to infiltration decellularization described in U.S. patent nos. 6,753,181 and 6,376,244, among others.

One or more compounds may be used in or on the surface of an decellularized organ or tissue, for example, to preserve the decellularized organ, or to provide for recellularization of the decellularized organ or tissue and/or to aid 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, for example, with radiation (e.g., UV, gamma) to reduce or eliminate any type of microorganisms remaining within or on the surface of the decellularized organ or tissue.

Recellularization of organs or tissues

The present invention provides materials and methods for producing an organ or tissue. An organ or tissue may be produced by contacting a decellularized organ or tissue with a population of regenerative cells as described herein. As used herein, a regenerative cell is any cell used to recellularize an decellularized organ or tissue. The regenerative cells may be totipotent cells (totipotent cells), pluripotent cells (pluripotent cells) or multipotent cells (multipotent cells), and may be either uncommitted or committed. The regenerative cells may also be single lineage cells. In addition, the regenerative cells may be undifferentiated cells, partially differentiated cells, or fully differentiated cells. Regenerative cells, as used herein, include embryonic stem cells (as defined by the National Institute of Health (NIH); see, e.g., the glossary on http:// www.stemcells.nih.gov). Regenerative cells also include progenitor cells, precursor cells, and "adult" stem cells including umbilical cord cells and fetal stem cells.

Examples of regenerative cells that can be used to recellularize an organ or tissue include, but are not limited to: embryonic stem cells, umbilical cord blood cells, tissue stem or progenitor cells, bone marrow stem or progenitor cells, blood stem or progenitor cells, adipose tissue stem or progenitor cells, Mesenchymal Stem Cells (MSCs), skeletal muscle cells, or Multipotent Adult Progenitor Cells (MAPCs). Other regenerative cells that may be employed include Cardiac Stem Cells (CSCs), multipotent adult cardiac stem cells, cardiac fibroblasts, cardiac microvascular endothelial cells or aortic endothelial cells. Bone marrow stem cells such as bone marrow mononuclear cells (BM-MNC), endothelial or vascular stem or progenitor cells, and peripheral blood stem cells such as Endothelial Progenitor Cells (EPC) may also be used as regenerative cells.

The number of regenerative cells introduced into the interior and surface of a decellularized organ to produce an organ or tissue depends on the type and developmental stage of the organ (e.g., which organ, size and weight of the organ) or tissue and regenerative cells. The colony density that different cell types may tend to achieve also varies. Similarly, different organs or tissues may be recellularized 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) regenerative cells; or may have about 1,000-10,000,000 cells/mg tissue (wet weight, i.e., before decellularization) attached thereto. Regenerative cells can be introduced ("seeded") into the interior of a decellularized organ or tissue by injection into one or more locations. In addition, more than one cell type (i.e., mixed cells) can be introduced inside the decellularized organ or tissue. For example, a mixture of cells may be injected into multiple sites of a decellularized organ or tissue, or different types of cells may be injected into different sites of a decellularized organ or tissue. Alternatively, or in addition to injection, the regenerative cells or mixed cells may be introduced into the interior of the cannulated decellularized organ or tissue by perfusion. For example, the regenerative cells may be perfused into the interior of the decellularized organ using a perfusion medium, followed by the induction of growth and/or differentiation of the regenerative cells using an expansion and/or differentiation medium.

During recellularization, the organ or tissue is maintained under conditions in which at least some of the regenerative cells are capable of proliferating and/or differentiating within or on the surface of the decellularized organ or tissue. These conditions include, but are not limited to: suitable temperature and/or pressure, electrical and/or mechanical action, external force, suitable O₂And/or CO₂Amount, suitable humidity, and 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, regenerative cells may require nutritional supplements (e.g., nutrients and/or carbon sources such as glucose), exogenous hormones or growth factors, and/or a particular pH.

The regenerative cells may be allogeneic with respect to the decellularized organ or tissue (e.g., a human decellularized organ or tissue seeded with human regenerative cells), or the regenerative cells may be xenogeneic with respect to the decellularized organ or tissue (e.g., a porcine decellularized organ or tissue seeded with human regenerative cells). As used herein, "allogeneic" refers to cells from the same species from which the organ or tissue is derived (e.g., autologous (i.e., autologous) or unrelated individuals), while "xenogeneic" as used herein refers to cells from a different species from which the organ or tissue is derived.

In some cases, it is desirable to transplant an organ or tissue produced by the methods described herein into a patient. In those cases, the regenerative cells used to recellularize the decellularized organ or tissue can be from the patient such that the regenerative cells are "autologous" to the patient. The patient's regenerative cells can be obtained, for example, from blood, bone marrow, tissue or organs at various stages of life (e.g., prenatal, neonatal or perinatal, adolescent or adult) using methods known in the art. Alternatively, the regenerative cells used to recellularize the decellularized organ or tissue can be syngeneic with the patient (i.e., from the sibling twin), the regenerative cells can be Human Lymphocyte Antigen (HLA) matched cells from, for example, a patient's relatives or HLA matched individuals not related to the patient, or the regenerative cells can be from, for example, a donor of the same species as the patient that is not HLA matched.

Regardless of the source of the regenerative cells (e.g., autologous or non-autologous), the decellularized solid organ can be an autologous, allogeneic or xenogeneic organ of the patient.

In some cases, decellularized organs can be recellularized with cells in vivo (e.g., after transplantation of the organ or tissue into an individual). In vivo recellularization can be performed as described above (e.g., injection and/or perfusion) with, for example, any of the regenerative cells described herein. In addition, in vivo seeding of decellularized organs or tissues with endogenous cells may occur naturally or be mediated by factors delivered into the recellularized tissue.

The development of regenerative cells can be monitored during recellularization. For example, the number of cells on the surface or within an organ or tissue can be assessed by taking a biopsy at one or more time points during recellularization. In addition, the number of differentiations undergone by the regenerating cells can be monitored by determining the presence or absence of various markers in the cells or cell populations. Markers associated with different types of cells and different differentiation stages of these cell types are known in the art and can be readily detected using antibodies and standard immunoassays. See, for example, Current protocols Immunology (recent methods of Immunity), 2005, ed.p. Coligan et al, John Wiley & Sons, chapters 3 and 11. Nucleic acid assays as well as morphological and/or histological evaluation can be used to monitor the recellularization process. Functional analysis of the recellularized organ can also be assessed. For example, contraction and vascular pressure can be assessed in a recellularized heart; albumin production, urea production and cytochrome p450 activity can be assessed in recellularized liver; blood or media filtration and urea production can be assessed in the recellularized kidney; blood, glucose and insulin can be evaluated in the recellularized pancreas; force production or response to stimulation can be assessed in the recellularized muscle; thrombogenicity can also be assessed in the recellularized vessel.

Control system for decellularization and/or recellularization of organs or tissues

The invention also provides a system (e.g., a bioreactor) for decellularizing and/or recellularizing an organ or tissue. Such systems typically include at least one intubation device for intubation of the organ or tissue, a perfusion apparatus for perfusing the organ or tissue through the cannula, and means for maintaining the organ or tissue in a sterile environment (e.g., a sealed system). Both intubation and perfusion are well known in the art. Intubation devices typically include hollow tubes of a suitable size for insertion into a vessel, duct, and/or cavity of an organ or tissue. Typically, one or more vessels, catheters and/or cavities within the organ are inserted. The perfusion apparatus may include a handheld container of liquid (e.g., cell disruption medium) and a mechanism (e.g., pump, air pressure, gravity) to perfuse the liquid through one or more cannulas into the organ. Various techniques known in the art may be employed to maintain sterility of the organ or tissue during decellularization and/or recellularization, such as controlling and filtering the gas flow and/or co-infusing, for example, antibiotics, antifungals, or other antimicrobials to prevent growth of harmful microorganisms.

The system for decellularizing and recellularizing an organ or tissue described herein has the ability to monitor certain perfusion properties (e.g., pressure, volume, gas flow pattern, temperature, gas, pH), mechanical forces (e.g., wall motion and pressure), and electrical stimulation (e.g., pacing). Because the coronary vascular bed can be decellularized and recellularizedWhile variable (e.g., vascular resistance, volume), pressure-adjustable perfusion apparatus can advantageously avoid large pressure fluctuations. Effusions and histological sections can be used to assess perfusion effectiveness. The perfusion volume, airflow pattern, temperature, local O can be monitored by standard methods₂And CO₂Pressure and pH.

Sensors may be used to monitor the system (e.g., bioreactor) and/or the organ or tissue. Ultrasound microassay, micropressometry and/or conductometry may be used to obtain pressure-volume or preload associated with myocardial wall motion and performance may restore stroke work information. For example, a sensor may be used to monitor the pressure of the fluid as it flows through the cannulated organ or tissue; ambient temperature of the system and/or temperature of the organ or tissue; the pH and/or flow rate of the fluid as it flows through the cannulated organ or tissue; and/or the biological activity of the recellularized organ or tissue. In addition to having sensors capable of monitoring such properties, systems for decellularizing and/or recellularizing an organ or tissue may also include means for maintaining or modulating such properties. Means for maintaining or adjusting such characteristics may include components such as thermometers, thermostats, electrodes, pressure sensors, relief valves, valves to change the flow rate of a liquid, valves to open and close the connection of a fluid to a solution to change the pH of the solution, gas chambers, extracorporeal pacemakers, and/or flexible cavities. To help ensure stable conditions (e.g., temperature), the cavity, reservoir, and conduit may have a water jacket.

It is beneficial to apply mechanical load to the organ and cells attached thereto during recellularization. For example, a chamber inserted into the left ventricle via the left atrium may be used to apply mechanical pressure to the heart. A piston pump capable of adjusting volume and rate can be connected to the air chamber to stimulate left ventricular wall motion and pressure. To monitor wall motion and pressure, left ventricular wall motion and pressure may be measured using micro tonometry, ultrasound micrometry, pressure volume changes, or echocardiography. In some embodiments, an external pacemaker may be connected to a piston pump to provide stimulation synchronized with each ventricular chamber deflation (equivalent to systole). Peripheral ECGs can be recorded from the surface of the heart to regulate pacing voltages, monitor depolarization and repolarization, and provide simplified surface maps of the heart both at and after recellularization.

Mechanical ventricular dilation can also be achieved by connecting a peristaltic pump to a cannula inserted into the left ventricle via the left atrium. Similar to the method described above with respect to the air chamber, ventricular dilation, which is achieved by periodic fluid movement (e.g., pulsatile blood flow) through the cannula, may be synchronized with electrical stimulation.

Using the methods and materials disclosed herein, mammalian hearts can be decellularized and recellularized, and when maintained in appropriate conditions, can produce a functional heart with contractile function and response to pacing stimuli and/or agents. The recellularized functioning heart can be transplanted into a mammalian body to function for a period of time.

FIG. 2 shows an embodiment of a system (e.g., a bioreactor) for decellularizing and/or recellularizing an organ or tissue. The illustrated embodiment is a bioreactor for decellularizing and recellularizing a heart. This embodiment has a peristaltic pump (a) of adjustable rate and volume; a variable rate and volume piston pump (B) connected to the intraventricular air chamber; an external pacemaker (C) of adjustable voltage, frequency and amplitude; an ECG recorder (D); a pressure sensor in the 'arterial line' (which is equal to the coronary pressure) (E); a pressure sensor in the 'venous' line (which is equal to the coronary sinus pressure) (F); and a synchronizer (G) between the pacemaker and the piston pump.

A computer-readable storage medium, in conjunction with a programmable processor (e.g., as used herein, a computer-readable storage medium having instructions stored thereon which enable the programmable processor to perform specific steps), may control a system for creating an organ or tissue. For example, such a storage medium, in conjunction with a programmable processor, may receive and process information from one or more sensors. Such a storage medium, in conjunction with the programmable processor, may also transmit information and instructions back to the bioreactor and/or the organ or tissue.

The biological activity of the 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 compression, contractility and/or wall compression of the organ or tissue. In addition, the biological activity of cells attached to an organ or tissue, such as ion transport/exchange activity, cell division and/or cell viability, may also be monitored. See, e.g., Laboratytytytextbook of anatomi and Physiology handbook (2001, Wood, Presence Hall) and Current Protocols in Cell Biology (recent Protocols in Cell Biology) (2001, ed. by Bonifacino et al, John Wiley & Sons). As mentioned above, it is useful to simulate the active load on an organ during recellularisation. The computer readable storage medium of the present invention, in conjunction with a programmable processor, may be used to coordinate the components required to monitor and maintain the reactive load placed on an organ or tissue.

In one embodiment, the weight of an organ or tissue may be entered into a computer readable storage medium as described herein, which in combination with a programmable processor, may calculate the number of contacts and perfusion pressures for a particular organ or tissue. Such storage media may record pre-and post-load (pressure before and after perfusion, respectively) and flow rate. In this embodiment, the perfusion pressure, direction and/or type of perfusion fluid may be adjusted by one or more pumps and/or control valves, for example, in conjunction with a programmable processor.

According to the present invention, conventional molecular biology, microbiology, biochemistry and cell biology techniques within the skill of the art may be employed. These techniques are well described in the literature. The invention is further described in the following examples, which do not limit the scope of the invention as claimed.

Examples

Group A decellularization (part I)

Example 1 preparation of solid organs for decellularization

To avoid post-mortem thrombosis, donor rats were subjected to systemic heparinization with a dose of 400U heparin per kilogram of donor. After heparinization, the heart and adjacent large vessels were carefully enucleated.

The heart was placed in a physiological saline solution (0.9%) containing heparin (2000U/ml) and stored at 5 ℃ until further processing. Under sterile conditions, connective tissue is separated from the heart and large blood vessels. The inferior vena cava distal to the right and left atria and the left and right pulmonary veins are ligated with a non-absorbable monofilament ligature.

Example 2 intubation and perfusion of solid organs

The heart was fixed on a decellularization apparatus for perfusion (fig. 1). The descending thoracic artery was cannulated for retrograde coronary (arterial) perfusion (fig. 1, cannulated a). Branches of the thoracic artery (e.g., brachiocephalic trunk, left common carotid artery, left subclavian artery) are ligated. The pulmonary artery was cannulated before it was separated into left and right pulmonary arteries (fig. 1, cannulation B). The superior vena cava was cannulated (fig. 1, cannula C). This configuration enables retrograde and antegrade coronary (arterial) perfusion.

When positive pressure is applied to the aortic cannula (a), perfusion occurs from the coronary arteries through the capillary bed to the coronary venous system to the right atrium and superior vena cava (C). When positive pressure is applied to the superior vena cava cannula (C), perfusion from the right atrium, coronary sinus, and coronary veins through the capillary bed to the coronary arteries and aortic cannula (a) occurs.

Example 3 decellularization

After the heart is fixed to the decellularization equipment, antegrade perfusion is started with cold heparinized decalcified phosphate buffer containing 1-5 mmoles adenosine per liter of infusion solution to reestablish a continuous coronary flow. Coronary flow was assessed by measuring coronary infusion pressure and flow and calculating coronary resistance. After 15 minutes of stable coronary flow, detergent-based decellularization treatment was started.

The specific treatment method is as follows. Briefly, however, the heart is perfused anterograde with detergent. After perfusion, the heart is washed retrograde with buffer (e.g., PBS). The heart was then perfused with PBS containing antibiotics, and then perfused with PBS containing dnase I. The heart was then perfused with 1% benzalkonium chloride to reduce microbial contamination and prevent future microbial contamination, and the organs were then perfused washed with PBS to remove any residual cellular components, enzymes, or detergents.

Example 4 cardiac decellularization of dead rats

Hearts of 8 male nude rats (250-300g) were removed. Immediately after removal, the aortic arch was cannulated and retrograde perfused with the indicated detergent. The feasibility of 4 different detergent-based decellularization methods (below) were compared to the efficacy of (a) removing cellular components and (b) preserving vascular structure.

Decellularization typically comprises the following steps: stabilizing the solid organ, decellularizing, renaturing and/or neutralizing the solid organ, washing the solid organ, degrading any DNA left on the organ, disinfecting the organ and establishing organ homeostasis.

A) Decellularization method #1(PEG)

The hearts were washed without reflux with 200ml PBS containing 100U/ml penicillin, 0.1mg/ml streptomycin and 0.25. mu.g/ml amphotericin B. Manual reflux decellularization was then performed with 35ml of polyethylene glycol (PEG; 1g/ml) for up to 30 minutes. The organs were then washed with 500ml PBS for up to 24 hours under reflux with a pump. 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 under manual reflux. The organ was washed again with 500ml PBS for at least 24 hours.

B) Decellularization method #2(Triton X and Trypsin)

The heart was washed without reflux for at least about 20 minutes with 200ml PBS containing 100U/ml penicillin, 0.1mg/ml streptomycin, and 0.25. mu.g/ml amphotericin B. 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 for 24 hours each time under reflux with a pump. The heart was perfused with 35ml of DNase I (70U/ml) for 1 hour under manual reflux, and washed 2 times with 500ml of PBS for at least about 24 hours each under reflux with a pump.

C) Decellularization method #3 (1% SDS)

The heart was washed without reflux for at least about 20 minutes with 200ml PBS containing 100U/ml penicillin, 0.1mg/ml streptomycin, and 0.25. mu.g/ml amphotericin B. The heart was decellularized with 500ml of water containing 1% SDS for at least about 6 hours under reflux with a pump. The heart was then washed with deionized water for about 1 hour and PBS for about 12 hours. The hearts were washed 3 times with 500ml PBS for at least about 24 hours each time under reflux with a pump. The heart was then perfused with 35ml of DNase I (70U/ml) for about 1 hour under manual reflux, and washed 3 times with 500ml of PBS for at least about 24 hours each under reflux with a pump.

D) Decellularization method #4(Triton X)

The heart was washed without reflux for at least about 20 minutes with 200ml PBS containing 100U/ml penicillin, 0.1mg/ml streptomycin, and 0.25. mu.g/ml amphotericin B. Then, the heart was decellularized with 500ml of water containing 5% Triton X and 0.1% ammonia water for at least 6 hours under reflux with a pump. 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 3 times with 500ml PBS for at least about 24 hours each time under reflux with a pump. Then, the heart was perfused with 35ml of DNase I (70U/ml) for about 1 hour under manual reflux, and washed 3 times with 500ml of PBS for at least about 24 hours each.

For the first experiment, the decellularization equipment was set up in a laminar flow hood. At 60cm H₂The coronary perfusion pressure of O perfuses the heart. Although not required, the hearts described in the above experiments were fixed in a decellularization chamber, completely immersed in PBS containing antibiotics, and perfused with it in reflux mode at a continuous flow rate of 5 ml/min for 72 hours, washing away as much of the cellular components and detergent as possible.

The absence of myofilaments and nuclei in histological sections was defined as successful decellularization. Successful preservation of vascular structure was assessed by perfusion with 2% evans blue prior to embedding the histological sections.

Decellularization was extremely efficient when the heart was first perfused anterogradely with an ionic detergent (1% Sodium Dodecyl Sulfate (SDS), about 0.03M) in deionized water at a continuous coronary perfusion pressure, then perfused anterogradely with a non-ionic detergent (1% Triton X-100) to remove SDS, and presumably to renature extracellular matrix (ECM) proteins. Intermittently, the heart was perfused retrograde with phosphate buffer to unblock the obstructed capillaries and small blood vessels.

Example 5 evaluation of decellularized organs

To demonstrate the integrity of the vascular structure after decellularization, the decellularized heart was stained using evans blue using the landov perfusion method to stain the vascular basement membrane and quantify the macrovascular and microvascular density. In addition, polystyrene particles can be perfused into and through the heart to quantify coronary artery volume, vessel leakage water, and perfusion profiles assessed by analyzing coronary artery outflow and histological sections. The 3 criteria were evaluated in combination, compared to isolated non-decellularized hearts: 1) average distribution of polystyrene particles, 2) significant changes in leakage at some levels, 3) microvascular density.

Fiber orientation was evaluated using a polarized microscopy technique available in real time from Tower et al (2002, fiber imaging mechanical testing of soft tissues during mechanical testing of soft tissues), annual review of biomedical engineering (Ann biomedical Eng.), 30 (10): 1221-33) for samples under uniaxial or biaxial stress. During Landoff perfusion, the basic mechanical properties of the decellularized ECM (flexibility, elasticity, burst pressure) were recorded and compared to freshly isolated hearts.

Group B, acellular (part II)

Example 1 decellularization of rat Heart

100mg/kg ketamine (Phoenix Pharmaceutical, Inc.), 10mg/kg xylazine (Phoenix Pharmaceutical, Inc., St. Mo.) was intraperitoneally injected into male F344 Fisher rats (46229, Ind. Napolis, 29176 mailbox, Harron laboratories, Harlan Labs) 12 weeks old were anesthetized. Following systemic administration of heparin (American Pharmaceutical Partners, Inc., il.) via the left femoral vein, a median sternotomy was performed to open the pericardial membrane. The fat body behind the sternum was removed, the ascending thoracic aorta was dissected and its branches were ligated. The heart was removed from the chest cavity transecting the vena cava and pulmonary veins, pulmonary arteries and thoracic aorta. A pre-filled 1.8mm aortic cannula (lonoti Glass, montovaya, california) was inserted into the ascending aorta for retrograde coronary perfusion (landov). Heparinized PBS containing 10. mu.M adenosine (Hyclone, Rogen, Utah) at 75cm H₂Coronary perfusion pressure of O perfuses the heart for 15 minutes followed by 2-15 hours with deionized water containing 1% Sodium Dodecyl Sulfate (SDS) or 1% polyethylene glycol 1000(PEG 1000) (EMD Biosciences, rahoya, germany) or 1% Triton-X100 (Sigma, st. Followed by 15 minutes of deionized water and 30 minutes of deionized water containing 1% Triton-X (sigma, st louis, missouri). Then, the treatment is continued with PBS containing antibiotics (100U/ml penicillin-G (Cor. TM. (Gibco), Calsbarda, Calif.), 100U-ml streptomycin (Cor., Calsbauda) and 0.25. mu.g/ml amphotericin B (Sigma, St. Louis, Mo.) were infused for 124 hours.

The appearance appeared edematous and opaque after retrograde perfusion for 420 minutes with 1% PEG, 1% Triton-X100 or 1% SDS, PEG and Triton-X100 perfusate, whereas SDS perfusion achieved a more drastic change as opaque material was slowly washed away, resulting in a translucent graft. The hearts subjected to all 3 treatments remained sufficiently intact without rupture of the coronary arteries or insufficiency of the aortic valve during perfusion (at a sustained coronary perfusion pressure of 77.4 mmHg). Coronary flow decreases occurred during the first 60 minute perfusion period in all 3 methods, then returned to normal during the SDS perfusion period, while remaining increased during Triton-X100 and PEG perfusion. SDS perfusion first increased the calculated coronary artery resistance the most (up to 250 mmhg.s.ml)^-1) Followed by Triton-X (up to 200mmHg. s.ml.)^-1) And PEG (up to 150 mmhg.s.ml)^-1)。

With histological sections of detergent-perfused heart tissue, it was determined that neither PEG nor Triton-X100 treated hearts were completely decellularized for the observed time intervals; hematoxylin-eosin (H & E) staining showed nuclei and cross-striated filaments. In contrast, no nuclei or contractile filaments were observed in sections of SDS-perfused hearts. However, vascular structure and ECM fiber orientation were retained in SDS-treated hearts.

To remove ionic SDS from the ECM after the first decellularization, the organ was perfused with Triton-X100 for 30 minutes. In addition, to ensure complete washing out of all detergents and reconstruction of physiological pH, decellularized organs were perfused extensively with deionized water and PBS for 124 hours.

Example 2 decellularization of rat Kidney

To isolate the kidney, the entire abdominal contents were wrapped in wet gauze and carefully moved to the side, leaving the retroperitoneal space exposed. The mesenteric vessels were ligated and intercepted. The abdominal aorta was ligated and severed below the renal artery origin. The thoracic aorta was cut directly above the septum and cannulated with a 1.8mm aortic cannula (lens glass, incorporated, monto vica, california). The kidneys were carefully removed from the retroperitoneal cavity and immersed in sterile PBS (halon, luo gen, utah) to minimize traction on the renal arteries. The perfusion was carried out for 15 minutes with heparinized PBS, followed by 2-16 hours with deionized water containing 1% SDS (Invitrogen, Calsward, Calif.) and 30 minutes with deionized water containing 1% Triton-X (Sigma, St. Louis, Mo.). The perfusion of the liver with antibiotic-containing PBS (100U/ml penicillin-G (Cor., Gibco, Calsbauda, Calif.), 100U/ml streptomycin (Cor., Calsbauda, Calif.) and 0.25. mu.g/ml amphotericin B (Sigma, St. Louis, Mo.) was then continued for 124 hours.

SDS perfusion for 420 min followed by Triton-X100 perfusion yielded a fully decellularized renal ECM scaffold with intact vessels and organ structure. Evans blue perfusion confirmed that its intact vasculature was similar to the decellularized cardiac ECM. Movat's five-color staining of decellularized renal cortex revealed intact glomeruli without any intact cells or nuclei and proximal and distal renal convoluted tubule basement membranes. Staining of decellularized renal medulla revealed intact tubule and collecting duct basal membranes. SEM of decellularized renal cortex confirmed intact glomerular and tubular basement membranes. Features such as the bowman's capsule of the glomerulus and the glomerular capillary basement membrane within the glomerulus are delineated from the surrounding proximal and distal tubules and glomerulus. SEM images of decellularized renal medulla show a complete medullary cone with a complete collecting vessel basement membrane leading to the papilla, up to the renal pelvis. Thus, all major ultrastructures of the kidney are intact after decellularization.

Example 3 decellularization of rat Lung

The lungs (with trachea) were carefully removed from the chest cavity and immersed in sterile PBS (halon, rocko, utah) to minimize traction on the pulmonary arteries. The perfusion was carried out for 15 minutes with heparinized PBS, followed by 2-12 hours with deionized water containing 1% SDS (Invitrogen, Calsward, Calif.) and 15 minutes with deionized water containing 1% Triton-X (Sigma, St. Louis, Mo.). The lungs were then perfused with antibiotic-containing PBS (100U/ml penicillin-G (Cor., Gibco, Calsbad, California), 100U/ml streptomycin (Cor., Calsbad, California) and 0.25 μ G/ml amphotericin B (Sigma, St. Louis, Mo.) for 124 hours.

SDS perfusion for 180 minutes followed by Triton-X100 perfusion yielded a fully decellularized lung ECM scaffold with intact airways and vessels. Mollurgical five-color staining of tissue sections revealed the presence of ECM components in the lung, including major structural proteins such as collagen and elastin, as well as soluble components such as proteoglycans. However, no nuclei or intact cells remain. Airways remain from the main bronchi to the terminal bronchioles to the respiratory bronchioles, alveolar tubules and alveoli. The vascular bed from the pulmonary artery down to the capillary level and pulmonary vein remains intact. SEM micrographs of decellularized lungs showed retention of bronchial, alveolar and vascular basement membranes, with no evidence of cell retention. The network of elastic and reticular fibers that provide the major structural support for the alveolar septum, as well as the septal basement membrane (including the dense network of capillaries within the pulmonary interstitium) remain intact.

SEM micrographs of decellularized trachea show intact ECM structure with decellularized hyaline cartilage ring and rough luminal basement membrane without respiratory endothelial cells.

Example 4 decellularization of rat liver

To isolate the liver, median dissection was performed to expose the vena cava, dissected and cannulated with a mouse aortic cannula (lens manufactured by lisuriti, montoriya, california). The hepatic artery and vein and bile duct were dissected, the liver carefully removed from the abdomen and immersed in sterile PBS (halon, luogen, utah) to minimize traction on the portal vein. The perfusion was carried out for 15 minutes with heparinized PBS, followed by 2-12 hours with deionized water containing 1% SDS (Invitrogen, Calsward, Calif.) and 15 minutes with deionized water containing 1% Triton-X (Sigma, St. Louis, Mo.). The perfusion of the liver with antibiotic-containing PBS (100U/ml penicillin-G (Gibco, Calsbauda, Calif.), 100U/ml streptomycin (Cico, Calsbauda), and 0.25 μ G/ml amphotericin B (Sigma, St. Louis, Mo.) was then continued for 124 hours.

SDS perfusion for 120 minutes followed by perfusion with Triton-X100 was sufficient to obtain a fully decellularized liver. Mollurgical five-color staining of decellularized liver confirmed retention of characteristic liver tissue with central vein and portal cavity containing hepatic artery, bile duct and portal vein.

Example 5 methods and materials for evaluating decellularized organs

Histological and immunofluorescence methods. The decellularized tissue embedded in paraffin was stained in mottle according to the manufacturer's instructions (American Mastertech Scientific, usa, loco, california). Briefly, a paraffin-removed slide was stained with Van' Hoff (Verhoeff) elastic dye, washed, resolved with 2% ferric chloride, washed, placed in 5% sodium thiosulfate, washed, blocked with 3% glacial acetic acid, stained with 1% Octanum blue solution, washed, stained with crocus sativa-acid fuchsin, washed, immersed in 1% glacial acetic acid, decolorized with 5% phosphotungstic acid, immersed in 1% glacial acetic acid, dehydrated, placed in alcohol saffron solution, dehydrated, fixed and coverslipped.

Immunofluorescent staining was performed on the decellularized tissue. Paraffin-embedded tissues (recellularized tissues) but not frozen sections (decellularized tissues) were subjected to the following antigen recovery: paraffin sections were dewaxed and rehydrated by immersion in xylene twice for 5 minutes each, followed by immersion in a continuous alcohol gradient, rinsing with cold running tap water. Then, the slide glass was placed in an antigen recovery solution (2.94g of trisodium citrate, 22ml of a 0.2M hydrochloric acid solution, 978ml of ultrapure water, and pH-adjusted to 6.0) to boil for 30 minutes. After rinsing with running cold tap water for 10 minutes, immunostaining was started. Prior to staining, cryosections were fixed with 4% paraformaldehyde (Electron microscopy Sciences, Hatfield, Pa.) in 1XPBS (Medium technology, Mediatech, Hellanden, Virginia) for 15 minutes at room temperature. Slides were blocked with 4% fetal bovine serum (FBS; Halon, Rogen, Utah) in 1XPBS for 30 minutes at room temperature. Samples were incubated with diluted primary and secondary antibodies (abs) for 1 hour each at room temperature. Between each step, the slides were washed 3 times with 1XPBS (5-10 minutes each time). Primary antibodies against collagen I (goat polyclonal IgG (catalog No.: sc-8788), san Cruz Biotechnology Inc. (Santa Cruz Biotechnology Inc.), san Cruz, Calif.), collagen III (goat polyclonal IgG (catalog No.: sc-2405), san Cruz Biotechnology Inc., san Cruz, Calif.), fibronectin (goat polyclonal IgG (catalog No.: sc-6953), san Cruz Biotechnology Inc., san Cruz, Calif.) and laminin (rabbit polyclonal IgG (catalog No.: sc-20142), san Cruz Biotechnology Inc., san Cruz, Calif.) diluted 1: 40 with blocking buffer were used. Bovine anti-goat IgG phycoerythrin (catalog No.: sc-3747, St. Cruis Biotechnology, Inc., St. Cruis, Calif.) and bovine anti-rabbit IgG phycoerythrin (catalog No.: sc-3750, St. Cruis Biotechnology, Inc., St. Cruis, Calif.) secondary antibodies diluted 1: 80 in blocking buffer were used. The slides were covered with coverslips (fisherbrandy (Fisherbrand)22x 60, pittsburgh, pa) and hardened mounting media containing 4', 6-diamidino-2-phenylindole (DAPI) (carrier Laboratories, ltd (Vectashield, Vector Laboratories, Inc.), burlingham, ca) was added dropwise. Images were recorded on a nikon Eclipse TE200 inverted microscope (freyer co. inc., illinois), Huntley, illinois, with ImagePro +4.5.1 software (media control, silvestris, maryland).

Scanning electron microscopy normal and decellularized tissues were fixed by perfusion with 0.1M cacodylate buffer (electron microscopy, hunter, pa) containing 2.5% glutaraldehyde (electron microscopy, hunter, pa) for 15 minutes. The tissue was then washed twice with 0.1M cacodylate buffer for 15 minutes each. Post-fixation was performed with 1% osmium tetroxide (electron microscopy, intel. pa) for 60 minutes. Tissue samples were then dehydrated with increasing concentrations of EtOH (50% 10 min, 70% 10 min twice, 80% 10 min, 95% 10 min twice, 100% 10 min twice). The tissue samples were then critical point dried in tossmis (tousmis) Samdri-780A (tossmis corporation, rockville, maryland). A30 second gold/palladium sputter coating was performed on a Denton DV-502A Vacuum evaporator (Denton Vacuum, Moore, N.J.). Scanning electron microscope images were taken using a Hitachi S4700 field emission electron microscope (Hitachi technologies, USA, Calif.) and the scanning electron microscope was used.

And (5) mechanical testing. A cross of myocardial tissue was cut from the left ventricle of the rat, the central region being approximately 5mm x 5mm, the axis of the cross being aligned both circumferentially and longitudinally with the heart. The original thickness of the center of the cruciform tissue was 3.59. + -. 0.14mm as measured by a micrometer. Cruciform myocardium with the same size central region was also excised in the same direction from the left ventricular tissue of decellularized rats. The original thickness of the decellularized sample was 238.5 + -38.9 μm. In addition, another tissue engineering scaffold for constructing blood vessels and heart tissue, the mechanical properties of fibrin gel, was examined. The fibrin gel was made into a cross-type with a final concentration of 6.6 mg fibrin/ml. The average thickness of the fibrin gel is 165.2 ± 67.3 μm. All samples were attached with tweezers to a biaxial mechanical testing machine (Instron Corporation, norwood, massachusetts) immersed in PBS with equal strain to 40% in the biaxial direction. To accurately explore the hydrostatic mechanical properties, the samples were stretched to increase their strain by 4%, with a rest of at least 60 seconds at each strain value. The force is converted to engineering stress by scaling the force value to a specific axial cross-sectional area (5mm x original thickness). The engineering stress is calculated as a displacement normalized to the original length. To compare the data between the two axes and between the sample sets, the tangent modulus was calculated according to the following formula:

[ T (40% Strain) -T (36% Strain) ]/4% Strain

Where T is the engineering stress and e is the engineering strain. The values of the tangent modulus were averaged and the differences between the two axes (circumferential and longitudinal) and the group were compared.

Example 6 evaluation of the biocompatibility of decellularized organs

To assess biocompatibility, 100,000 mouse embryonic stem cells (mESC) were suspended in 1cc of standard expansion Medium (IMDM cell culture Medium) (gumbo (Gibco), carlsbad, california), 10% fetal bovine serum (halon, luo kojic), 100U/ml penicillin-G (mebo, carlsbad, california), 100U/ml streptomycin (mebo, carlsbad, california), 2mmol/L L-glutamine (yiweichu, carlsbad, california), 0.1 mmol/L2-mercaptoethanol (mebo, carlsbad, california)) and seeded onto ECM sections and control plates without specific growth factor stimulators or feeder cell supports. 4', 6-diamidino-2-phenylindole (DAPI) was added to the cell culture at a concentration of 10. mu.g/ml to label the nuclei for quantification of the attached and expanded cells. Images under UV light and phase differences after baseline, 24, 48, and 72 hours were recorded on a nikon Eclipse TE200 inverted microscope (frel ltd, hunter, illinois) using ImagePro +4.5.1 (media control, inc., west furiprin, ma).

The decellularized ECM is compatible with cell survival, attachment, and proliferation. Seeded mescs were transplanted onto ECM scaffolds and invasion of the matrix started within 72 hours of cell seeding.

Example 7 evaluation of decellularized organs

The reactivity and integrity of the aortic valve of the arterial vascular bed of SDS decellularized rat hearts was assessed by performing a lanchov perfusion with 2% evans blue dye. No dye was observed to enter the left ventricle indicating that the aortic valve was intact. Visual inspection confirmed that the dye filled the coronary arteries up to the fourth branch point without dye leakage. Perfusion to large (150 μm) and small (20 μm) arteries and veins was then determined in tissue sections from the red fluorescence of vascular basement membrane stained by evans blue.

To confirm that the major cardiac ECM component was retained, the SDS decellularized ECM scaffold was immunofluorescent stained. It was determined that major cardiac ECM components such as collagen I and III, fibronectin and laminin are present, but there is no evidence of alpha actin retained with intact nuclear or contractile components including the cardiac myosin heavy chain or myofibrils.

Scanning Electron Micrographs (SEM) of SDS decellularized cardiac ECM demonstrated that fiber orientation and composition were retained in the aortic wall and aortic valve leaflets, while no cells were found throughout the tissue thickness. The decellularized left and right ventricular walls retain ECM fiber architecture (interlacing, struts, helices) and orientation, while completely eliminating myofibrils. Within the ECM retained by both ventricles, intact vascular basement membranes of varying diameters were observed with no endothelial or smooth muscle cells observed. Furthermore, a thin layer of dense epicardial fibers remains beneath the intact epicardial basal lamina.

To assess the mechanical properties of decellularized heart tissue, a biaxial test was performed and compared to fibrin gel, which is commonly used as an artificial ECM scaffold in heart tissue engineering. The stress-strain behavior of normal rat ventricles and decellularized samples showed a high degree of anisotropy. In contrast, the stress-strain characteristics in the two main directions are very similar in the fibrin gel sample. Orientation-dependent stress-strain behavior was observed in all samples in the normal rat ventricle and decellularized groups, and a typical characteristic of all samples in the fibrin gel group was stress-strain isotropy.

To compare the stress-strain characteristics between these two groups and between the cardiac spindles, the tangential moduli at 40% strain in the circumferential and longitudinal directions were calculated (see equation of example 5). It was noted that the modulus of the decellularized sample group was significantly higher than that of the normal rat ventricle and fibrin gel sample group in both directions. However, the difference between the modulus in both directions of normal rat ventricles and acellular matrix was significant, whereas fibrin gel did not.

For intact left ventricular tissue, the stress at 40% strain was 5-14kPa longitudinally and 15-24kPa circumferentially, consistent with the previously disclosed data. In both rat ventricular tissue and decellularized rat ventricular tissue, the circumferential direction is stiffer than the longitudinal direction, which is most likely determined by the muscle fiber orientation of the heart. Although the orientation of the fibers varies with the thickness of the heart tissue, most of the fibers are circumferential and, therefore, are expected to be stiffer in this direction. Acellular tissue is significantly stiffer than intact tissue. This is also thought to be because the extracellular matrix is stiffer than the cells themselves, and the combination of ECM and cells may not be as stiff as ECM alone. Although it appears that the tangential modulus values of decellularized tissues are quite large, they are only slightly greater than the Young's modulus of purified elastin (about 600kPa) and less than the Young's modulus of simple collagen fibers (5MPa), so that the values identified herein are within reasonable ranges.

Example 8 decellularization of other organs or tissues

Similar results were obtained with the perfusion decellularization methods described herein applied to skeletal muscle, pancreas, small and large intestine, esophagus, stomach, spleen, brain, spinal cord and bone, except for rat heart, lung, kidney and liver.

Example 9-decellularization of pig kidney

Porcine kidney was isolated from heparin-administered male animals. To perfuse the isolated organ, the renal artery was cannulated and perfused with PBS for more than 15 minutes to wash out the blood. The perfusion was carried out with 27L of 1% SDS in deionized water under a pressure of 50-100mmHg for 35.5 hours. SDS on the ECM scaffold was removed by priming with 1% Triton-X in deionized water. The decellularized kidney was then perfused with PBS containing antibiotics for 120 hours for washing and buffering to remove the detergent, making the pH biocompatible.

Organ clarity was observed within 2 hours of the initial perfusion. Became noticeably clear white within 12 hours of perfusion. Decellularization was terminated when the organ became white translucent.

Example 10 transplantation of decellularized Heart

Preparation was performed by cannulating the aorta distal to the aortic valve of the heart of the F344 rat and ligating all the large and pulmonary vessels except the left branch of the pulmonary trunk (distal to its branches) and the Inferior Vena Cava (IVC). The acellular process is completed by adopting a Landaf retrograde arterial perfusion method and perfusing for more than 12-16 hours by 2 liters of 1% SDS. The heart was then renatured by perfusion with 35mL of 1% Triton-X-100 for 30-40 minutes, followed by washing with PBS containing antibiotics and antifungal agents for 72 hours. IVC was ligated prior to transplantation.

A large (380-400 g) RNU rat was prepared for transplantation into the decellularized heart. The anastomotic region was isolated by clamping the IVC and abdominal aorta of the host animal with an obtuse angle mosquito clamp. The aorta of the decellularized heart was anastomosed to the abdominal aorta proximal and below the host renal branches using 8-0 suture. The left branch of the pulmonary stem of the decellularized heart was anastomosed to the proximal portion of the host IVC, minimizing the physical stress on the pulmonary stem.

When both vessels are sutured into the host animal, the clamp is released and the blood of the host animal fills the decellularized heart. The abdominal aortic pressure of the recipient animal was visually observed in the decellularized heart and aorta. The decellularized heart becomes swollen and bright red due to the filling with blood. Bleeding from the anastomotic site is minimal. Heparin was administered 3 minutes after releasing the clamps (start perfusion), the heart was photographed and placed on the abdomen to reduce the pressure at the anastomotic site. The abdomen was closed in a sterile manner and the animal was monitored until it recovered. Animals were euthanized 55 hours after transplantation and decellularized hearts were explanted for observation. Animals that did not receive heparin in LV were found to develop massive thrombi after dissection and evaluation. Blood clots are also observed in the coronary arteries on the right and left sides of the heart.

In other transplantation experiments, the clamp was released after suturing both vessels into the host animal and the blood of the host animal was engorged into the decellularized heart. The abdominal aortic pressure of the recipient animal was visually observed in the decellularized heart and aorta. The decellularized heart becomes distended and bright red with minimal bleeding at the site of anastomosis. Heparin (3000U) was administered by intraperitoneal injection 3 minutes after releasing the clamp (start perfusion). The heart was photographed and placed in the abdomen to reduce pressure at the anastomotic site. The abdomen was closed in a sterile manner and the animal was monitored until it recovered. Animals were found to die from bleeding approximately 48 hours after transplantation. The current time for transplantation is 55-70 minutes.

Recellularization of group C

Example 1 recellularization of cardiac ECM slices

To assess the biocompatibility of decellularized ECM, 1mm thick decellularized heart sections were cultured with myogenic and endothelial cell lines. 2x 10⁵Skeletal myoblasts from individual rats, myoblasts from C2C12 mice, human umbilical endothelial cells (HUVEC) and Bovine Pulmonary Endothelial Cells (BPEC) were seeded onto tissue sections and co-cultured for 7 days under standard conditions. Myogenic cells migrate and expand within the ECM, aligning with the original fiber orientation. These myogenic cells show increased proliferation and complete regeneration of most of the ECM sections. Endothelial cell lines exhibit a less invasive growth pattern, forming a monolayer on the surface of the graft. No antiproliferative effect was observed under these conditions.

Practice ofExample 2 recellularization of cardiac ECM by coronary perfusion

To determine the efficacy of seeding regenerative cells onto or into the ECM surface of decellularized heart by coronary perfusion, decellularized heart was transferred into organ chambers under cell culture conditions (5% CO)₂60% humidity, 37 ℃), and is continuously perfused with oxygenated cell culture fluid. At 40cm H₂Injecting into 120x 10 under O coronary artery perfusion pressure⁶Individual PKH-labeled HUVECs (suspended in 50ml endothelial cell growth medium). Coronary effluent was collected and cells were counted. The effluent is then refluxed and re-perfused to maximize cell number. Reflux was repeated twice. After the third passage, approximately 90X 10 remained in the heart⁶And (4) cells. Perfusion was continued with 500ml of refluxing oxygenated endothelial cell culture medium for 120 hours. The heart was then removed and placed in a cryostatic chamber. HUVEC are present only in the arteries and veins that remain within the cardiac range and do not completely diffuse into the extravascular ECM.

Example 3 recellularization of decellularized rat Heart with neonatal rat Heart cells

Isolation and preparation of rat neonatal cardiomyocytes. On the first day, 8-10 SPF Fisher-344 newborn puppies (Harron Laboratories, Indianapolis, Ind.) that were 1-3 days old were anesthetized by inhalation of 5% isoflurane (Abbott Laboratories, North Chicago, Illinois) and sprayed aseptically with 70% EtOH for a rapid sternotomy. The heart was removed and immediately placed in a 50ml conical tube containing HBSS (reagent #1 in neonatal cardiomyocyte separation system (worthington biochemical Corporation)) on ice. The supernatant was removed and the whole heart was washed once with cold HSS by vigorous stirring. The hearts were transferred to 100mm plates containing 5ml of cold HBSS, connective tissue was removed, and the remaining tissue was cut to less than 1mm²Of the chip (a). Additional HBSS was added to bring the total plate volume to 9ml, and 1ml trypsin (reagent #2, Huawa)Kit of the company, Shengton) to a final concentration of 50. mu.g/ml. The plates were incubated overnight in a 5 ℃ cooler.

The next day, the plates were removed from the cooler and placed on a sterile lid on ice. The buffer containing tissue and trypsin was transferred with a wide mouth pipette into a 50ml conical tube on ice. Trypsin inhibitor (reagent #3) and 1ml of HBSS (reagent #1) were recombined, added to a 50ml conical tube and gently mixed. The tissue was oxygenated by passing air over the surface of the liquid for 60-90 seconds. The tissue was then warmed to 37 ℃ and collagenase (300 units/ml) reconstituted with 5ml of Leibovitz (Leibovitz) L-15 was slowly added. The tissue was placed in a hot (37 ℃) shaking bath for 45 minutes. The tissue was then titrated 10 times with a 10ml pipette to release the cells (3 ml/sec) and then filtered through a 0.22 μm filter. The tissue was washed with an additional 5ml L-15 medium, titrated a second time, and collected in the same 50ml conical tube. The cell solution was then incubated at room temperature for 20 minutes and 50Xg centrifuged for 5 minutes to pellet the cells. The supernatant was gently removed and the cells resuspended in the desired volume with neonatal cardiomyocyte medium.

Culture solution and solution. All the culture fluids were sterile filtered and stored in a 5 ℃ cooler protected from light. The separation Kit (Worthington Isolation Kit) of washington corporation contains the suggested culture solution for culture: lebovistz L-15. This culture broth was used only for the next day of tissue treatment. Alternative calcium-containing media as described herein were used for plating. Lebovisz L-15 culture from Washington, Inc. (Worthington); and 1L of cell-raising grade water is used for reconstructing powder of the Labowitz culture solution. The Lebovistz L-15 broth contained 140mg/ml CaCl, 93.68mg/ml MgCl, and 97.67mg/ml MgS. Culture solution of neonatal cardiomyocytes: IMDM cell culture medium (Megaku, catalog No.: 12440-. amphotericin-B (final concentration 0.25. mu.g/ml) was added as needed. The culture medium may be supplemented with 1.2mM CaCl (Fisher-Elekeri)School, catalog No.: c614-500) and 0.8mM MgCl (sigma, catalog No.: m-0250).

In vitro analysis of recellularized cultures. As an advance towards the creation of bioartificial hearts, isolated ECM was recellularized with neonatal cardiac cells. 50x 10⁶A combination of freshly isolated rat neonatal cardiomyocytes, fibroblasts, endothelium and smooth muscle cells were injected into a fully decellularized heart (constructed as described herein). Cardiac tissue is then sectioned and the sections cultured in vitro to test the biocompatibility of the decellularized ECM and the ability of the resulting construct to develop into a myocardial membrane ring.

After 24 hours, minimal shrinkage of the resulting loop was observed under the microscope, demonstrating that the transplanted cells were able to attach and engraft onto the decellularized ECM. Under the microscope, cells grew in the direction of ECM fibers. Immunofluorescent staining confirmed that cardiomyocytes expressing the heavy chain of cardiac myosin had survived and successfully engrafted. Within 4 days, a population of contractile cell patches was observed on the acellular matrix, which by day 8 developed into synchronously contractile tissue loops.

On day 10, the rings were held between two measuring bars and their contractile force was measured under different preload conditions. The ring can be electrically paced to a frequency of 4Hz producing a contractile force of up to 3mN at a preload of up to 0.65 g. Thus, recellularization using the in vitro tissue culture methods described can result in contractile tissue that can produce forces equivalent to those produced by optimally engineered cardiac tissue loops made with artificial ECM constructs.

Recellularization of decellularized hearts by perfusion. Sterile cardiac tissue culture conditions (5% CO)₂，60％H₂O, 37 ℃ C.), will recellularize (50X 10)⁶Freshly isolated rat neonatal cardiomyocytes, fibroblasts, endothelial and smooth muscle cells) scaffolds were fixed on a perfusable bioreactor (n-10) simulating rat cardiac physiology, including pre-and post-load with gradient increase (day 1:pre-load 4-12mmHg, post-load 3-7mmHg), pulsatile left ventricular dilation, pulsatile coronary flow (day 1: 7 ml/min) and electrical stimulation (day 2: 1 Hz). The perfused organ culture lasts for 1-4 weeks. Throughout the incubation period, 30 seconds of pressure, flow and EKG data were recorded every 15 minutes. Videos of neonatal artificial hearts were recorded at days 4, 6 and 10 after cell inoculation.

On day 10 post-cell inoculation, more in-depth functional assessments were performed including Left Ventricular Pressure (LVP) recording by inserting a pressure probe into the left ventricle, video recording of wall motion as the frequency of stimulation was increased from a 0.1-10Hz gradient, and drug stimulation with Phenylephrine (PE). The recellularized heart exhibits a contractile response to a single pace, which is a spontaneous contraction following a pacing contraction and a corresponding increase in LVP. After one pacing, the heart exhibits 3 spontaneous contractions, which then switch to fibrillar state. Similar to stimulated contraction, spontaneous depolarization also causes a corresponding increase in LVP and a recordable QRS complex, which may indicate the formation of a stable pattern of conductance during development.

Once the stimulation frequency was increased to 0.4Hz, on average two spontaneous contractions occurred after each induced contraction; when the pacing rate is up to 1Hz, only 1 spontaneous contraction occurs; spontaneous contractions did not occur at a pacing rate of 5 Hz. The maximum capture frequency was 5Hz, consistent with the 250ms refractory period of mature myocardium. After perfusion with 100 μ MPE, regular spontaneous depolarization occurs when the frequency is 1.7Hz, accompanied by a corresponding increase in LVP.

Histological analysis on day 10 revealed that cells were dispersed and implanted throughout the thickness of the left ventricular wall (0.5-1.2 mm). Cardiomyocytes align along the ventricular fiber orientation, forming dense, organized graft regions resembling mature myocardium and less dense, immature graft regions resembling developing myocardium. The cardiomyocyte phenotype was determined by immunofluorescence staining for the cardiac myosin heavy chain. A high density of capillaries was maintained throughout the newly developed myocardium, with an average distance between capillaries of about 20 μm, similar to that reported for mature rat myocardium. Endothelial cell phenotype was determined by immunofluorescence staining for von willebrand factor (vWF). Cell survival was maintained throughout the thickness of the graft, indicating that sufficient oxygen and nutrients could be provided by coronary perfusion.

Group D-other Decellularization and recellularization

Example 1 rat liver isolation procedure

Each rat was anesthetized with 75mg/kg body weight ketamine and 10mg/kg body weight xylazine. The abdomen of the rats was shaved and disinfected with povidone iodine. Large doses of heparin sodium (100. mu.L heparin (1,000UI/mL stock solution)/100 g body weight) were infused intravenously into the rat stomach.

When heparin was effective, bioreactor flasks were assembled. Briefly, a 250ml flask was fitted with a tygon (tygon) tube (fitted on the bottom side) and a reducer union (used for drainage in the washing step described below) was fitted to the tube. When heparin is effective, the catheter is fitted with a rubber stopper; the PBS was placed in a 12cc syringe and a 3-way plunger was attached to the syringe. The syringe was fitted with a 18 gauge needle and pushed through a 8 gauge rubber stopper. To ensure that the liver lays flat in the container, the needle is preferably kept even (even) at the bottom of the rubber stopper. A short length of polyethylene tubing (e.g., PE160) with the skirt melted is sterilized with alcohol and slid into the free end of the tubing. A small amount of PBS was pushed into the cannula to expel the alcohol and a sufficient amount of PBS was poured into a 10cm dish to cover the detached liver.

After heparin circulation, the abdominal skin is incised to expose the underlying abdominal muscles. A mid-section laparotomy is performed, followed by a lateral incision or a midline incision along the abdominal wall, followed by retraction to expose the liver. The ligaments connecting the liver to the duodenum, stomach, diaphragm and anterior abdominal wall were excised gently (the capsule of gleason is very fragile). The common bile duct, hepatic artery and portal vein are excised, leaving sufficient length of cannula, and finally the hepatic superior and inferior vena cava are excised. The liver was removed from the remaining joined superior and inferior vena cava and placed in a dish with PBS. Any remaining ligaments are excised.

Example 2 liver Decellularization

The prepared cannula was inserted into the portal vein and tied with a pronil suture. Confirm integrity of the wire with PBS in the syringe (Mg-free)²⁺And Ca²⁺) Perfusing the liver to remove occult blood. The liver-rubber plug was placed in the bioreactor. The flask was placed on a collection reservoir and connected to a 1% SDS container (1.6L) via a sufficiently long line to produce a column with a maximum pressure of about 20 mmHg. After 2-4 hours of perfusion, the 1% SDS container was emptied and refilled with 1.6L of 1% SDS. The liver is usually perfused with a total of 4 batches of 1.6L of 1% SDS. After decellularization, the liver appeared transparent white in appearance, and blood vessels were visible.

On day 2, the SDS reservoir was disconnected and replaced with dH₂O60 ml syringe. Rinsing with water, then 60ml of 1% Triton X-100, then 60ml of dH₂And (4) rinsing by using O. The rinsed liver is washed again and perfusion with a sterilant (e.g., penicillin-streptomycin (e.g., Pen-Strep)) via a small pump (Masterflex, 50% maximum capacity, about 1.5 ml/min) is initiated) PBS). A length of tympan tube was removed from the bioreactor/flask and drained into the PBS reservoir. A length of tubing was connected by pump to a 0.8 micron filter connected to a 3-way piston on the flask. An 18 gauge needle was attached to the line in the PBS reservoir, making it lower than the sample line. After 6 hours, the organs were washed overnight by changing to 1 Xconcentration of fresh PBSw/Pen-Strep, changing the 0.8 micron filter.

On day 3, two more washes were performed with 1 Xconcentration of 500ml PBSw/Pen-Strep. The 0.8 micron filter was also replaced each time the PBS was replaced. The 3 rd wash was started in the morning, the solution was changed after 6 hours, and finally the wash was again overnight. On day 4, the liver was ready for recellularization.

On average, 14.27% of DNA remained in livers washed twice with 1.6l of 1% SDS, while on average 5.36% remained in livers washed four times with 1.6l of 1% SDS. That is, two washes with 1% SDS removed about 86% DNA (compared to cadavers), while 4 washes with 1% SDS removed about 95% DNA (compared to cadavers).

FIG. 3A shows decellularization of rat liver and rat kidney, and FIG. 3B shows decellularization of rat heart and rat lung. The middle part includes the photographs of the decellularized in progress, and the right and left photographs are SEM images of the decellularized organ.

FIG. 4 shows decellularized pig kidney and rat kidney perfused with dye, and also shows EM photographs of glomeruli and tubules of the decellularized kidney. Figure 5 shows whole rat cadavers decellularized as described herein.

Example 3 liver recellularization

Recellularization was performed as follows: cells (40X 10)⁶Primary liver-derived cells or HepG2 human cells) were suspended in warmed medium (37 ℃) at a concentration of about 8x10⁶One cell/ml (typically 5ml) was loaded into the syringe. Cells are injected via the portal vein into the liver, which is placed in a bioreactor or petri dish. Note that cells may also be injected through other vascular portals, or directly into the mesenchyme.

Adult rat liver was enzymatically digested with a womington enzymatic kit to obtain primary liver-derived cells. Briefly, rat liver was perfused through the portal vein with 1 Xcalcium-free magnesium-free Hanks' balanced saline (kit No. 1 tube) at 20 ml/min for 10 minutes, after which the liver was removed from the rat. Then, the liver was recirculated for 10-15 minutes at 20 ml/min with 100ml of L-15 containing MOPS buffer containing enzymes (collagenase (22,500 units) elastase (30 units) and nuclease I (1,000 units)) from kit Nos. 2 and 3. The organ is then mechanically disrupted, releasing the cells. Cells were centrifuged at 100g, resuspended in culture, and repeated 2 times before being used for recellularization.

Perfusion rates are controlled by the signs observed during the procedure (e.g., tension of perfused lobes, cell escape from the liver, and distribution of cells in the target lobe). After recellularization, the liver in the bioreactor was placed at 37 ℃ in 5% CO₂In the incubator. Inoculating into an oxygen-containing culture solution reservoir (containing 50ml of culture solution); in the reservoirThe medium was bubbled with humidified carbopol gold (95% oxygen, 5% carbon dioxide). The culture broth (37 ℃) was recirculated through the liver at a rate of 2-10 ml/min using a peristaltic pump. The re-cellularized rat liver medium was changed daily for 7 days (although the experiment could simply be terminated at a convenient time). Samples of the culture broth were collected at daily changes and stored at-20 ℃ for albumin and urea determination. On day 7, cytochrome P-450 experiments were performed.

FIG. 6 shows recellularisation of the liver of decellularised rats. Primary hepatocytes were injected into one lobe of liver by syringe through portal vein cannula. FIG. 7 shows targeted delivery of primary rat liver cells to the hepatic tail lobe (A) or the right lateral upper/lower lobe (B) of the liver in decellularized rats.

FIG. 8 shows a Scanning Electron Micrograph (SEM) of recellularized rat liver cultured for 1 week. These data show the similarity of cadaveric liver to re-cellularized liver at the micro-structural level. The cells were integrated into a matrix bed, with a shape similar to freshly isolated cells from cadaver tissue. FIG. 9 shows Mayer's trichrome (A) and H & E (B) staining, and FIG. 10A shows the Mayer's trichrome staining of recellularized rat liver 1 week after injection of rat hepatocytes into the tail knob. These results show that hepatocytes can be delivered into and retained within the matrix and can survive nutrient perfusion.

FIG. 11 shows the Mashimura trichrome staining of recellularized rat liver 1 week after injection of the human liver cell line (HepG2) in the caudate process (A) or the upper/lower right lateral lobe (B). FIG. 12 shows cell retention of primary rat hepatocytes (1-6) and human HepG2 cell lines (7 and 8). Counting the cells prior to injection to obtain the total number of cells perfused into the liver, counting unbound cells that flow through the matrix and eventually appear in the culture dish; the difference indicates the cells remaining in the matrix. FIG. 13 shows that human HepG2 remained active and proliferated after injection into the liver of decellularized rats.

Example 4 liver function

The function of decellularized and recellularized liver was evaluated as follows.

Urea production was assessed in livers recellularized with primary rat hepatocytes (fig. 14); aluminum production (fig. 15); and cytochrome P-450IAI (ethoxyisophenazolone-O-deethylase (EROD)) activity (FIG. 16). Urea production was determined using a fibrate/colorimetric test kit (Point Scientific Inc.) and albumin production and EROD activity were determined using a method modified according to tissue engineering cell culture (Vunjak-Novakovic & Freshney eds., 2006, Wiley-Liss). These experiments show that liver-derived cells maintain liver-specific function during culture.

Example 5 cell survival after recellularization

FIG. 17 shows embryonic and adult-derived stem/progenitor cells propagated on decellularized heart, lung, liver and kidney for at least 3 weeks. Cell proliferation was determined by counting DAPI-stained nuclei/high power field. FIG. 18 shows the proliferation of mouse embryonic stem cells (mESCs) and proliferative adult muscle progenitor cells (skeletal muscle myoblasts; SKMB) on decellularized heart, lung, liver and kidney. Cell viability was determined by measuring the extent of apoptosis after 3 weeks using the TUNEL assay versus the number of nuclei stained with total DAPI.

Human Embryonic Stem (ES) cells and human Induced Pluripotent Stem (iPS) cells were propagated on the acellular cardiac matrix for at least 1 week. Briefly, human ES cells (H9, from WiCeIl research institute; WA09, from the National Stem Cell Bank (NSCB)) were compared on an acellular matrix; IMR90 subclones of human iPS cells (generated as described by OCT4, SOX2, NANOG, and LIN28 lentivirus transgenes such as Zhang et al, 2009, Circ. Res., 104: e30-e41, obtained from Timothy Kamp, university of Wisconsin).

H9 cells and iPS cells (containing 20-50% cardiomyocytes, with the remainder being proliferating fibroblasts) were plated at a density of 200,000 cells and 90,000 cells, respectively, in wells containing chamber-specific (right or left atrium or ventricle) portions of rat acellular cardiac matrix that had separated to expose the interior of the matrix. The cells were simply placed on an acellular matrix. Cells were cultured in medium containing 20% serum for 3 days, then the serum was reduced to 2%, and cultured for an additional 4 days, consistent with the in vitro "switching" of proliferating muscle cells to a beating muscle cell phenotype. Control cells were placed in the same wells coated with gelatin (0.1%) and cultured under the same conditions. Cells were grown in EB20 medium. Daily microscopy assessed cultures and beating cells were recorded with a video camera. After 1 week of culture, live/dead experiments were performed to examine the cell viability. In addition, immunohistochemical staining was performed to show the presence of heart-related proteins. Cells grown on the acellular matrix were observed to beat by days 3-4, while cells on gelatin did not beat. On day 5, the cells on the matrix had spread and a larger area of beating cells was observed. Cells cultured under the same conditions on gelatin are either sparse or absent.

Example 6 Recellularization Process

Cell separation

LV and RV were isolated from rat pups by the Wominton method and dissected approximately in the center of the heart. The part branching from the bottom to the second LAD was discarded and the remaining part was placed in about 10ml HBSS. Optionally, LV and RV portions of the heart may be incubated overnight at 5 ℃ in trypsin for 18-22 hours. After aspiration of the cells into a syringe for injection into the acellular matrix, the remaining cells are used as controls (e.g. 10ml of culture medium is added, the cells are plated).

Extracellular matrix

A well-washed decellularized extracellular matrix (ECM) was obtained. For example, the cardiac extracellular matrix is washed with at least 2000ml of PBS solution for 3-4 days. The heart was cannulated with a 18 gauge cannula (in: left ventricle through heart valve, out: aorta) and sutured with 4-0 sutures. The LV cannula is pushed into proximity with the apex of the heart within the LV cavity (e.g., the apex of the LV cannula is about 0.7cm from the valve). The structure is checked for the absence of leaks. Optionally, a "high speed" test can be performed to ensure a sealed ECM attachment by turning the pump on to a [ precordial ] flow probe (mode) at a flow rate of 25-28 ml/min for at least 5-10 seconds prior to introduction of the cells.

Cell injection

100-120ml of culture medium was placed in a bioreactor, and a 60mm culture plate was placed under the apex of the heart to catch excess cells and avoid blockage of the coronary arteries and disruption of apoptotic signaling of the cells. Cells were injected with 27 gauge needle and 1ccTB syringe. Approximately 70 microliters of cells were injected into the ventricular wall at a needle entry angle of 15 degrees for each injection and under normal conditions. Cells were injected 10-12 times into the anterior wall of the left ventricle and 3-4 times into the apex. The total volume of cells injected should be about 1.3-1.5 ml. Some backflow and cell loss is expected. The heart was lowered into the bioreactor and the pump and tank were opened (95% O)₂And 5% CO₂) Monitoring seepage in the heart, flow problems and any other technical problems. The next day, the reactor is opened and the pacemaker electrode wire is connected. Pacing (continuous) begins as follows: frequency: 1 Hz; delaying: 170 MS; and (3) continuously: 6 MS; voltage range: 45-60V; flow (in): 18-22 mL/min; flow (out): 14-18 mL/min; diffusion was about 6-7 mL/min.

Culture solution

The following formulation is for 1 liter. Adding 100mL of FBS 10% into IMDM; 5mL Pen Strep; 10mL L-Glut; 168 μ L Amp-B; 1mL of B-Mercap; 20mL of horse serum; 180mg Ca²⁺；96mgMg²⁺(ii) a And 50mg vitamin C.

NNCM (NEO) cells

Neonatal cardiomyocytes (NNCM or NEO cells) were obtained from the womington kit preparation. NEO cells are sensitive to temperature; if the temperature drops below about 35 c, they do not beat. If there is no excessive confluency, the NEO cells start to beat within 24 hours on 2D plates. As the NEO cells grow and beat, they begin to grow on top of each other and begin to beat in synchrony; eventually the cell itself mechanically restricts and stops beating, usually during days 10-16.

Example 7 comparison of the Structure of decellularized and recellularized organs with cadaveric organs

Fig. 19 is SEM photographs of a decellularized heart (right side) and a cadaveric heart (left side). SEM photographs of the Left Ventricle (LV) and Right Ventricle (RV) were obtained. As seen in the photographs, the perfusion decellularized heart lacks cellular components, but retains the spatial and structural features of the intact myocardium, including the blood vessels. In addition, in perfusion of decellularized matrix, it can be seen that the structural features including corrugations (w), convolutions (c) and layers(s) are retained, although complete loss of cells within the matrix is observed.

Figure 20 shows histological (top) and SEM (bottom) comparisons between decellularized and recellularized rat liver (right) and cadaveric rat liver (left) as described herein. These results demonstrate the morphological similarity and structural organization between healthy stem cells from intact liver and hepatocytes cultured or seeded on decellularized liver. The H & E images show that cells in the re-cellularized liver begin to organize radially around blood vessels, similar to the architecture observed in freshly isolated healthy (cadaveric) liver. It also demonstrates that cells distribute and/or migrate throughout the mesenchyme, start the tissue and remain within the matrix as long as the experiment continues. SEM images demonstrate that cellular tissue is similar even at the suprastructure level in cadaveric and recellularized matrices.

Group E perfusion decellularization vs infiltration decellularization

Example 1 decellularization by infiltration

Organs (rat liver, kidney, heart, lung, muscle, skin, bone, brain and vessels; pig liver, gall bladder, kidney and heart) were decellularized using the perfusion methods described herein.

Organs (rat liver, heart and kidney) were decellularized using the infiltration method described in U.S. patent nos. 6,753,181 and 6,376,244. Briefly, the organ is placed in dH₂O, stirring was carried out with a magnetic stirrer rotating at 100rpm at 4 ℃ for 48 hours, then the organs were transferred to a solution of ammonium hydroxide (0.05%) and Triton X-100 (0.5%), and the solution was stirred with a magnetic stirrer (100rpm) for another 48 hours. The solution was replaced, and the organ was decellularized by repeated infiltrations with ammonium hydroxide and Triton X-100 for 48 hours if necessary (cell-free instruments were typically visually inspected)Officer). Approximately 5 replicates of ammonium hydroxide and Triton X-100 were used on the liver to create a visual cell-free organ. Following the decellularization process, organs were transferred to dH₂O, stirring for 48 hours (still stirring at 100 rpm); finally, a final wash with PBS was performed at 4 ℃ with stirring.

Example 2 comparison of perfusion and infiltration

Fig. 21A shows a photograph of perfusion of decellularized pig liver, and fig. 21B and 21C show SEM of vessels and mesenchymal matrix of perfusion decellularized pig liver, respectively. These photographs show the integrity of blood vessels and matrix in the perfused decellularized organ. On the other hand, fig. 22 shows a full view of an infiltrated decellularized rat liver, in which matrix exfoliation is visible at both low (left) and high (right) magnifications.

FIG. 23 shows SEM of infiltrated decellularized rat livers (A and B) and perfused decellularized rat livers (C and D). These results clearly indicate that infiltration decellularization significantly damaged the organ capsule (gleason's capsule), while perfusion decellularization retained the capsule. In addition, FIG. 24 shows the histology of infiltrated decellularized livers (A for H & E staining; B for trichrome staining) and perfused decellularized livers (C for H & E staining; D for trichrome staining). The liver of the rat infiltrated with decellularized cells failed to retain cells or dye after injection.

Figure 25 shows a comparison between rat cardiac infiltration decellularization (top panel) and perfusion decellularization (bottom panel). The left column of photographs shows the complete organ. As can be seen from these two photographs, the perfusion decellularized organ (bottom left) is much more transparent than the infiltration decellularized organ (top left), which retains the iron-rich "red-brown" color of the cadaver muscle tissue and still appears to contain cells. The photographs in the middle panel show the H & E staining pattern of the decellularized tissue. Staining showed that after infiltration decellularization (upper middle), many cells in the mesenchymal and vascular walls were retained, whereas after perfusion decellularization (lower middle) almost all cells and cell debris were removed, even though the unclosed vessels were evident. In addition, scanning electron micrographs in the right column show significant differences in the superstructure of the matrix after infiltration (top right) and perfusion (bottom right) decellularization. Moreover, complete retention of cellular components in the myocardial cross-section is observed in all walls of the infiltrating decellularized heart, whereas in the perfusion decellularized heart these cellular components are almost completely lost, and the spatial and structural features of the intact myocardium, including the blood vessels, are retained. For example, perfusion of decellularized matrix retains the structural features within the matrix, including waviness (w), curls (c), and layers(s), while the cells are completely lost.

Figure 26 shows a comparison between decellularization with rat kidney infiltration (top panel) and perfusion decellularization (bottom panel). Unlike the heart, the whole kidney infiltrated with decellularized cells (upper left) appeared substantially similar to the whole kidney perfused with decellularized cells (lower left), all quite transparent. However, in perfusion decellularized kidneys, the vascular network within the perfusion decellularized organ is more pronounced and more branched than is visible in the infiltrated decellularized construct. In addition, the perfusion decellularized kidney retains the intact organ capsule, surrounded by the mesentery, and is capable of decellularizing with the attached adrenal gland as shown. The photographs in the middle panel show the H & E staining pattern of both tissues. Staining showed that cellular components and/or debris and possibly even intact nuclei (stained purple) remained after infiltration decellularization (top middle), while almost every cell and/or all cellular debris was removed after perfusion decellularization (bottom middle). Also, SEM photographs showed that infiltrating decellularized kidney stroma (top right) was much more severely damaged than perfusion decellularized kidney stroma (bottom right). In the infiltrated decellularized kidney, the organ capsule is missing or damaged, and thus the superficial "pores" or matrix flaking is evident, whereas in the perfused decellularized organ the capsule is intact.

Figure 27 shows SEM photographs of decellularized kidneys. Fig. 27A shows perfusion of a decellularized kidney, while fig. 27B shows infiltration of a decellularized kidney. Fig. 28A shows SEM photographs of perfusion decellularized hearts, while fig. 28B shows SEM photographs of infiltration decellularized hearts. Fig. 29 shows SEM photographs of infiltrated decellularized liver. These images further demonstrate the damage inflicted by infiltration of decellularized super junction constructs to the organ and the survival of the matrix after perfusion decellularization.

Other embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims (16)

1. A method of manufacturing a liver, comprising:

providing a decellularized liver, wherein said decellularized liver comprises a decellularized extracellular matrix of said liver, wherein said extracellular matrix comprises an outer surface, wherein said extracellular matrix comprising a vascular tree substantially maintains a morphology of said extracellular matrix prior to decellularization, wherein said extracellular matrix is substantially intact; contacting said decellularized liver with about 40,000 or more of said regenerative cells under conditions wherein said cells engraft, proliferate and/or differentiate within and on said decellularized liver.

2. The method of claim 1, wherein the decellularized liver is contacted with 23x10⁶Or more regenerative cell contacts.

3. The method of claim 1, wherein the decellularized liver is contacted with 30x10⁶Or more regenerative cell contacts.

4. The method of claim 1, wherein the decellularized liver is contacted with 35x10⁶Or more regenerative cell contacts.

5. The method of claim 1, wherein said regenerative cells are hepatocytes.

6. The method of claim 1, wherein the regenerative cells are perfused into the decellularized liver through the portal vein.

7. The method of claim 1, wherein said regenerative cells are injected into said decellularized liver.

8. A method of making a liver lobe comprising:

providing a decellularized liver or a liver lobe containing portion, wherein said decellularized liver or liver lobe containing portion comprises a decellularized extracellular matrix of said liver or liver lobe containing portion, wherein said extracellular matrix comprises an outer surface, wherein said extracellular matrix comprising a vascular tree substantially maintains the morphology of said extracellular matrix prior to decellularization, wherein said extracellular matrix is substantially intact; contacting said decellularized liver lobe or lobe-containing fraction with said population of regenerative cells under conditions wherein said regenerative cells engraft, proliferate and/or differentiate within and on said decellularized liver lobe.

9. The method of claim 8, wherein the regenerative cells are primary hepatocytes.

10. The method of claim 8, wherein said regenerative cells are perfused into said lobe of the liver through the portal vein.

11. A method of decellularizing an organ, comprising:

providing the organ; cannulating one or more lumens, vessels, and/or ducts of the organ, thereby creating a cannulated organ; perfusing the cannulated organ through the one or more cannulas with a first cell disrupting medium; and determining the amount of nucleic acid remaining in the decellularized organ as compared to the corresponding cadaveric organ.

12. The method of claim 11, wherein the perfusion is about 2-12 hours per gram of organ tissue.

13. The method of claim 11, wherein the perfusion step is continued until 5% or less of the nucleic acid remains in the decellularized organ.

14. The method of claim 11, wherein said cell disruption medium comprises 1% SDS.

15. The method of claim 11, wherein the perfusion is multidirectional from the lumen, vessel and/or duct of each cannula.

16. A decellularized mammalian adrenal gland comprising:

a decellularized extracellular matrix of said adrenal gland, wherein said extracellular matrix has an outer surface, wherein said outer surface comprising a vascular tree substantially maintains the morphology of said extracellular matrix prior to decellularization, and wherein said outer surface is substantially intact.