Human liver scaffold

Abstract

The present invention relates to a method for decellularizing human liver tissue to produce a human liver extracellular matrix (ECM) scaffold, for example, for use in therapy or disease modeling. The method involves, for example, freezing and thawing, followed by subjecting the liver tissue to multiple cycles of osmotic stress to mechanically disrupt cells in the tissue, detergent treatment, and protease and / or DNase treatment to produce a decellularized human ECM scaffold.

Description

[0001] This application is a divisional application of the following application: Application date: June 2, 2015; Application number: 201580035899.3; Invention name: “Human liver scaffold”.

[0002] The present invention relates to the preparation of human liver extracellular matrix (ECM) scaffolds, for example, for use in therapy, drug screening, biomarker identification or disease modeling.

[0003] Tissue engineering is an emerging field that aims to improve the quality of life for millions of people worldwide by restoring organ function. Tissue engineering combines cells and scaffolds to develop 3D structures that can regenerate organs and recapitulate disease in vitro. In addition to restoring organ function, tissue engineering offers important biomedical applications, including platforms for in vitro testing of therapeutic drugs and biologics, for testing drug toxicity, for evaluating new devices and interventional techniques, and for discovering new biomarkers.

[0004] The human liver is a single organ divided into multiple functional subunits, termed segments (eight segments with individual portal vein and arterial supply, hepatic venous and biliary drainage) and regions (further segments with shared vascular and biliary pedicles). These anatomical features allow the human liver to be divided into multiple functional subunits that can be used in the practice of liver resection and segmental liver transplantation. Traditionally, blood from the portal vein and hepatic arterial systems perfuses the human liver in an antegrade manner; blood is drained by the hepatic venous system, and bile is secreted by the liver through the biliary system.

[0005] The liver performs a wide variety of functions, including detoxification, protein synthesis, production of biochemicals required for digestion, and a key role in carbohydrate and lipid metabolism. The liver is essential for survival; currently, there are no long-term solutions to compensate for the absence of liver function. This unique function is achieved through the cooperative efforts of parenchymal and nonparenchymal cells, which are rigidly organized within a network of proteins called the extracellular matrix (ECM) that forms a 3D scaffold.

[0006] Liver disease, caused by viral infections, alcohol, and other conditions, is on the rise, currently resulting in 10,000 deaths annually in the UK and 32,000 in the US. Liver transplantation is the only effective treatment for advanced acute or chronic liver disease and is associated with excellent long-term survival and quality of life. However, the supply of livers available for transplant is limited, while demand is growing. Waiting times are increasing, and 15% to 25% of people eligible for a liver transplant die or become too ill while waiting. The development of an artificial liver could reduce deaths from liver disease and provide a solution for patients without an organ for transplant.

[0007] In addition, all models used to study the development of liver fibrosis (HF) and hepatocellular carcinoma (HCC) have inherent problems. Thus, studies on 2D monolayer single cell cultures or mixed cell cultures have formed the main axis of most in vitro studies, but are far from reality. In addition, most animal models, although sharing many features with human diseases, do not share the same gene expression patterns as humans, and their effectiveness as in vivo models is limited. Thus, there is an unmet need to develop models that reproduce the complexity of human diseases.

[0008] In recent years, the liver of large animals such as pigs and sheep has been assessed to obtain the possibility of a suitable scaffold for utilizing human cells to regenerate. However, except the inherent difference in the metabolic capacity of the liver, the extracellular ECM scaffold has different macroscopic features and microscopic features in different species. Specifically, the segmentation and lobulation of the human liver are not as obvious as other species. In addition, the microscopic lobule anatomy and structure of the human liver are unique, and are different from other mammals such as pigs, because the fibrotic boundary between the liver lobules does not exist in the human (and the human lobule structure does not have a formal structural boundary). The existence of the typical fibrotic boundary of the porcine liver represents a major problem in the transplantation of the scaffold obtained from the porcine liver after utilizing human cells to regenerate for the human. In fact, the different structures of the porcine may cause portal hypertension, because bypass can easily cause the flow obstruction in the normal human liver.

[0009] The in vitro development of 3D human liver tissues that recapitulate the composition and organization of 3D human ECM proteins is one of the major challenges in tissue engineering. One promising approach involves the creation of bioscaffolds through tissue decellularization. However, due to inherent differences in macro- and microstructural structure unique to humans and distinct from other mammals, previous reports by other authors have failed to produce 3D human liver scaffolds. Although decellularization of mouse, rat, ferret, sheep, and porcine livers has been reported, no publications have described the successful generation of human liver bioscaffolds.

[0010] The inventors have recognized that a method comprising one or more treatment cycles utilizing a panel of different cell-disrupting agents can be used to decellularize human liver tissue without disrupting the extracellular matrix. This may be useful for the reproducible production of acellular scaffolds that maintain the structure and morphology of the extracellular matrix of human liver.

[0011] One aspect of the present invention provides a method for producing a human liver scaffold, comprising

[0012] (i) providing human liver tissue,

[0013] (ii) mechanically disrupting cells in tissues,

[0014] (iii) exerting osmotic stress on cells in tissues,

[0015] (iv) optionally exposing the tissue to proteases and / or DNAses, and

[0016] (v) exposing the tissue to a detergent, and

[0017] (vi) repeating each of steps (iii), (iv) and (v), and optionally (ii) one or more times,

[0018] Thus, human liver scaffolds were produced.

[0019] In some embodiments, the method may include step (iv). In other embodiments, step (iv) may be omitted (ie, the method does not include step (iv)).

[0020] The human liver scaffolds produced by the claimed method comprise acellular human liver extracellular matrix (ECM) (ie, the scaffolds are decellularized) and retain the three-dimensional structure and bioactivity of the ECM of the source liver tissue.

[0021] After the cells are mechanically disrupted, the liver tissue is sequentially exposed to different decellularization reagents (i.e., osmotic stress reagent, protease / DNase, and detergent). Steps (iii), (iv), and (v), and optionally, step (ii), can be performed one or more times in any order to decellularize the scaffold. In some embodiments, multiple cycles of steps (iii), (iv), and (v), and optionally, any one, two, or all three of step (ii), can be performed on the liver tissue in any order.

[0022] In some embodiments, step (ii) can be repeated one or more times. For example, multiple cycles including step (ii) can be performed on liver tissue. This can be useful, for example, when non-freeze-thaw techniques such as ultrasound are used to mechanically disrupt cells.

[0023] Preferably, in steps (iii) to (vi), fluid shear stress is applied to the liver tissue, which promotes the penetration of the decellularization agent into the tissue, for example, into the liver sinusoids, and separates cells and cell debris from the extracellular matrix (ECM).

[0024] Any suitable technique may be used to generate fluid shear stress in liver tissue, including perfusion, agitation, or negative pressure. Preferably, fluid shear stress is generated in human liver tissue by perfusion or agitation.

[0025] The choice of technique for generating fluid shear stress can depend on the liver tissue or application. For example, a whole liver, a single lobe segment, a segment, or other liver structural unit can be perfused with a decellularization agent to generate fluid shear stress. A small liver sample, such as a liver tissue block (LTC), can be immersed in a decellularization agent and agitated to generate fluid shear stress.

[0026] Human liver tissue for decellularization as described herein can be obtained from human livers that are not suitable for clinical transplantation purposes. Suitable livers can be obtained in accordance with applicable national laws and ethical principles.

[0027] In some embodiments, the liver tissue may be normal tissue that exhibits no pathology associated with damage or disease.

[0028] In other embodiments, liver tissue can be a pathological tissue showing a pathology associated with injury or disease. For example, liver tissue can be fatty, fibrotic, inflamed or show one or more other features associated with disease or injury. In certain embodiments, pathological liver tissue can show the pathology associated with acute or chronic liver disease, including viral infection, such as hepatitis A, B, C, D or E, alcohol or toxin damage, liver fibrosis (HF), non-alcoholic fatty liver disease (NAFLD), primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), alcoholic liver disease, ischemic hepatitis, giant cell hepatitis, and liver cancer such as hepatocellular carcinoma (HCC). Alternatively, pathological liver tissue can show the pathology associated with other diseases affecting the liver, such as amyloidosis.

[0029] Liver scaffolds generated from pathological liver tissue may have different structures and compositions than those generated from healthy liver tissue. For example, the morphology of the pathological scaffold or the amount or relative amounts of ECM components such as collagen, tenascin, and laminin may be altered in scaffolds generated from pathological liver tissue compared to healthy liver tissue. This may be useful for obtaining disease-specific liver scaffolds for disease modeling.

[0030] Pathological liver tissue can be obtained from an individual suffering from liver disease or a disease affecting the liver (eg, a disease characterized by liver damage or altered liver function).

[0031] Methods for harvesting and storing liver tissue for decellularization as described herein are currently known. For example, the liver can be heparinized to prevent aggregation and / or perfused with cryoprotectants to reduce or prevent tissue destruction after thawing.

[0032] Human liver tissue suitable for decellularization as described herein can include the entire liver or portions of the liver, including functional units of the liver, such as lobes, sectors, segments or subsegments of the liver, or small samples or sections.

[0033] A portion of the liver may include one or more functional units of the liver, such as lobes, sectors, segments or subsegments. Suitable functional units may be vascularized and / or include a vascular-biliary pedicle. For example, the liver tissue may include a subsegment of S8 of the human liver, one or more segments of segments S1 to S8 of the human liver, or a multi-segment portion, such as the left lateral (S2+S3±S1), the left liver (S2+S3+S4+S1), the right liver (S5+S6+S7+S8), the right lateral liver (S6+S7), the right extended liver (S4+S5+S6+S7+S8+S1). In certain preferred embodiments, the liver tissue may be a left lateral (S2+S3±S1) or left liver (S2+S3+S4+S1) portion of the human liver.

[0034] The amount and quality of the decellularized liver tissue as described herein can depend on the intended use of the scaffold. For example, the quality of the decellularized liver tissue used for transplantation can depend on the weight of the individual and the amount of functional liver tissue in the individual.

[0035] In other embodiments, the portion may be a small non-vascularized liver slice or wedge (e.g., with a 0.008 cm width, length, and / or diameter) of 0.2-2 cm, preferably 0.2-1.25 cm, more preferably 0.2-1.0 cm. 3 Up to 2cm 3 , more preferably 0.008 cm 3 Up to 1cm 3 , for example, about 0.125 cm 3 Preferably, the slices are of a suitable size for manipulation in standard laboratory containers, such as multiwell plates, and may be, for example, roughly cubic with sides of approximately 0.5 cm.

[0036] Small, non-vascularized liver slices or wedges can be used to recapitulate the complexity of the 3D human microenvironment on a small scale for liver disease modeling, drug screening, biomarker identification and validation, and development of disease diagnostics. Suitable slices can be obtained using a tissue dicer, punch biopsy, needle biopsy, or by sectioning the parenchyma, such as a simple scalpel cut of a block.

[0037] Multiple liver sections for decellularization as described herein can be harvested from a single liver, such that a single donated human liver can be used for more than one patient or can be used for both clinical and disease modeling applications.

[0038] The method may comprise providing a discarded whole human liver or a portion thereof.Prior to decellularization as described herein, the whole human liver may be divided into one or more portions.

[0039] In some embodiments, the human liver tissue can be treated with a cryoprotectant prior to step (i). For example, the tissue can be treated with an intracellular and / or extracellular cryoprotectant prior to freezing. Suitable cryoprotectants include DMSO, ethylene glycol, propylene glycol, glycerol, 2-methyl-2,4-pentanediol (MPD), and sucrose.

[0040] In the first decellularization step, cells in human liver tissue are mechanically disrupted to facilitate their destruction and removal. The cells can be mechanically disrupted by any suitable technique that disrupts the cells of the liver tissue without affecting the extracellular matrix, including freeze / thaw, sonication, or high-intensity focused ultrasound (HIFU).

[0041] In some embodiments, mechanical disruption techniques may not be repeated after initial treatment. For example, freeze / thaw treatments, such as after exposure to other decellularization agents, may result in ECM disruption.

[0042] In other embodiments, after the initial treatment, the cells in the liver tissue may be mechanically disrupted one or more times (e.g., in step (vi) above). For example, the tissue may be subjected to HIFU or ultrasound treatment one or more times.

[0043] Preferably, the cells are mechanically disrupted by subjecting the human liver tissue to one or more freeze / thaw cycles. For example, the tissue can be frozen at -20°C or lower, preferably -50°C or lower, -60°C or lower, or -70°C or lower, and then thawed one or more times. The frozen tissue can be conveniently thawed at 4°C to 37°C. In certain preferred embodiments, the tissue can be thawed at approximately 4°C to minimize temperature gradients within the tissue that could damage the ECM. For example, the tissue can be frozen at approximately -80°C for 24 hours or longer and then thawed at approximately 4°C.

[0044] Preferably, the frozen / thawed human liver tissue is dried to prevent ECM damage. In some embodiments, the human liver tissue can be dried prior to the freeze / thaw step, for example, at room temperature for 5 to 30 minutes.

[0045] Liver tissue can be subjected to freeze / thaw treatment in an isotonic buffer, for example saline, such as 0.90% (w / v) NaCl or PBS.

[0046] After mechanical disruption, human liver tissue is decellularized by a series of sequential exposures to permeabilization agents, enzymes, and detergents (i.e., decellularization agents). These sequential exposures to different decellularization agents separate cells and cell debris from the extracellular matrix (ECM) and remove them from the liver tissue.

[0047] Osmotic stress can cause the cracking of the cell in liver tissue, and amplifies the effect of mechanical destruction.Osmotic stress can be produced by exposing tissue to one or more osmotic agents having different osmotic pressures (that is, non-isotonic agents) to the cells in tissue.Tissue can be exposed to one or more hypotonic agents with an osmotic pressure lower than that of cell, and cell is placed in a hypotonic environment and / or one or more hypertonic agents with an osmotic pressure higher than that of cell, and cell is placed in a hypertonic environment.In certain embodiments, hypotonic agents can be preferred.

[0048] Hypertonic agents can be useful, for example, in separating DNA from proteins. Suitable hypertonic agents are known in the art and include saline (e.g., >0.9% (w / v) NaCl, e.g., 3% to 7% (w / v) NaCl), which can optionally be buffered, for example, with phosphate, borate, or tris, and polyethylene glycol solutions.

[0049] Hypotonic agents can be useful, for example, in causing cell lysis by simple osmotic effects, with minimal molecular and structural changes to the ECM. Suitable hypotonic agents are known in the art and include water, deionized water, and saline with <0.9% (w / v) NaCl.

[0050] In some embodiments, enzymes are used to disrupt cellular and subcellular structures in liver tissue and to disconnect cells from the ECM. For example, proteases can be used to degrade proteins in tissue. Suitable proteases are known in the art and include trypsin. Liver tissue can be exposed to 0.0025-0.25% (w / v) trypsin, for example, 0.025% trypsin. In some embodiments, liver tissue can be exposed to a protease in a solution containing a chelating agent, such as EDTA, that chelates, for example, Ca 2+ It can also destroy divalent metal ions such as β-catenin and disrupt cell adhesion to the ECM.

[0051] In a series of exposures to decellularization reagents, the liver tissue can be exposed to a protease prior to exposure to a detergent. This causes cells or cellular material that has been separated from the ECM by the protease to be washed away from the tissue by the detergent.

[0052] In other embodiments, no proteases may be used to decellularize the liver tissue.

[0053] In some embodiments, deoxyribonuclease (DNase) can be used to degrade the genomic structure and deoxyribonucleic acid in the tissue. Suitable DNases are known in the art and include DNase I. Liver tissue can be exposed to 1000-10000 KU DNase I, for example, approximately 2000 KU DNase I. DNase treatment can be particularly suitable for methods using mild detergents such as sodium deoxycholate. In some embodiments, the DNase can be in a salt-balanced solution, for example, 1M NaCl. In other embodiments, DNase may not be used to decellularize the liver tissue.

[0054] In some embodiments, liver tissue may be decellularized without the use of DNases or proteases.

[0055] Detergents solubilize lipids and fats in tissues and facilitate the removal of cellular debris from the ECM.

[0056] Detergents may include anionic detergents such as sodium dodecyl phosphate (SDS), sodium deoxycholate (SdC), and Triton TM For example, liver tissue can be exposed to 0.01 to 5% SDS, e.g., 0.01-1% SDS; 0.01 to 5% sodium deoxycholate (SdC), e.g., approximately 4% sodium deoxycholate; and / or 0.01 to 5% Triton X-200. TM X-200, e.g., 3% Triton TM X-200.

[0057] Detergents may include non-ionic detergents such as polyethylene glycol and Triton TM X100, for example, polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-diphenyl ether. For example, liver tissue can be exposed to 0.01 to 5% Triton TM X-100, or 1 to 3% Triton TM X-100, e.g., 3% Triton TM X-100.

[0058] Detergents may include zwitterionic detergents such as CHAPS (3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonic acid inner salt), sulfobetaine-10 (3-(decyldimethylammonium)propanesulfonic acid inner salt), sulfobetaine-16 (n-hexadecyl-N,N-dimethyl-3-amino-1-propanesulfonic acid inner salt), and tri(n-butyl)phosphate. For example, liver tissue may be exposed to 0.01 to 5% zwitterionic detergent, for example, 0.01-1% zwitterionic detergent.

[0059] Many other suitable detergents are known in the art and are available from commercial sources (eg, Sigma Aldrich, MO, USA).

[0060] In certain preferred embodiments, the liver tissue can be exposed to a first detergent and a second detergent in separate steps. For example, the method may include (a) exposing the tissue to an anionic detergent, such as SDS, and (b) exposing the tissue to a non-ionic detergent, such as Triton TM X-100.

[0061] In some embodiments, the concentration of the decellularization reagent used in each treatment step can be gradually increased to a maximum value. For example, the concentration of the detergent used can be gradually increased to a maximum value. The initial detergent concentration can be sufficiently low to avoid the formation of aggregates or clumps of cellular material that can clog blood vessels in the liver tissue. After the initial detergent treatment, the detergent concentration is increased to effectively remove all cellular material.

[0062] Thus, step (iii) can comprise exposing the liver tissue to gradually increasing concentrations of detergent. Preferably, when step (iii) is repeated, the highest concentration is used. For example, the liver tissue can be exposed to 0.005%-0.02% SDS, e.g., approximately 0.01% SDS, followed by exposure to 0.05% SDS to 0.2% SDS, e.g., approximately 0.1% SDS, followed by exposure to 0.5% SDS to 2% SDS, e.g., approximately 1% SDS.

[0063] The subsequent detergent treatment step (ie, a repetition of step iii) may use 0.5% SDS to 2% SDS, for example, about 1% SDS.

[0064] In certain preferred embodiments, the liver tissue can be exposed to the decellularization agent by perfusing the tissue with the agent. Preferably, the liver tissue is perfused in a retrograde direction, rather than in an antegrade direction.

[0065] Suitable techniques for performing tissue perfusion in vitro are known in the art. For example, liver tissue can be prepared for perfusion by inserting a cannula into a blood vessel, conduit, or cavity, such as the vena cava or hepatic vein. The cannulated tissue can then be perfused using a decellularizing agent via cannulation.

[0066] In a preferred embodiment, the rate of perfusion of the liver tissue with the decellularization agent is not constant. For example, the initial rate of perfusion of the tissue can be low (e.g., <0.99 ml / min / g tissue) to avoid structural damage to the liver tissue. During the decellularization process, as steps (iii) to (v) are repeated, the liver tissue becomes less resistant to perfusion, and the perfusion rate is increased (e.g., >1 ml / min / g tissue). This increases fluid shear stress in the tissue.

[0067] Preferably, the perfusion rate is gradually increased to a maximum value (compensation phase) and then maintained at or near this maximum value (stable phase) during two or more cycles of exposure to the decellularization agent. In some embodiments, the tissue can be initially perfused with the decellularization agent at a flow rate of 0.1-1.99 ml / min / g of tissue, e.g., 0.5 to 1.5 ml / min / g, and gradually increased to 2-20 ml / min / g of tissue, e.g., 2-8 ml / min / g. In other embodiments, the tissue can be initially perfused with the decellularization agent at a flow rate of 0.01-0.99 ml / min / g of tissue, e.g., 0.5 to 1.5 ml / min / g, and gradually increased to 1-10 ml / min / g of tissue, e.g., 2-8 ml / min / g, over 5 to 10 days, preferably about 9 days.

[0068] Liver tissue can be perfused with each decellularization agent for about 0.004 to about 5 hours per gram of solid tissue (for tissue samples >40 grams), or about 2 to about 12 hours per gram of solid tissue (for tissue samples <40 grams).

[0069] For example, decellularization as described herein can take 1-60 days, e.g., 7-60 days, depending on the initial quality of the liver tissue.

[0070] The order in which the tissue is perfused with the different decellularization agents can vary, depending on whether the liver tissue is perfused with each agent at least once, at least twice, or at least three times during the decellularization process.

[0071] The order of exposure to different detergents is based on their different mechanisms of action. Mechanical disruption (first step) promotes strong cell disruption. Exposure to a hypotonic solution (second step) enhances cell lysis when flushing cellular material. Then, the liver tissue can be optionally exposed to proteolytic enzymes (third step) to remove cellular material attached to the ECM. Finally, the liver is exposed to different detergents (fourth step) to effectively flush out cellular material. These four steps can be repeated as a function of flow rate.

[0072] The mechanical destruction of step (i) promotes the strong cell fragmentation in human tissue. Typically, step (i) is performed once at the beginning of decellularization, but, in some embodiments, HIFU or ultrasound-mediated mechanical destruction can be introduced more than once during decellularization. The cell lysis caused by mechanical destruction is enhanced by the osmotic stress in step (ii). Exposure to osmotic solution also washes cell debris from tissue. Then, optionally, in step (iii), liver tissue is exposed to DNA enzyme, or preferably, proteolytic enzyme, to remove cellular material attached to ECM. Finally, in step (iv), liver is exposed to one or more detergents, to wash out cellular material from tissue. Increased fluid shear stress can be utilized to repeat steps (iii) to (iv) once or many times.

[0073] In some embodiments, the method may comprise repeating steps (iii) to (v) by perfusing the liver tissue in the following order: (iii), (iv), (iii), (iv)(v), [(iii), (v)] n ,(iii),(iv),[(iii),(v)] n , wherein n is 1 to 25. Suitable perfusion decellularization protocols are shown in Table 1. Optionally, in this method, step (ii) may be repeated one or more times.

[0074] The liver as a whole or in its functional units can be perfused with decellularizing agents to generate an ECM scaffold that matches the size and structure of the source tissue. In some embodiments, this scaffold can then be repopulated with cells to obtain, for example, functioning liver tissue suitable for direct transplantation into humans.

[0075] In some embodiments, the liver tissue can be pathological. Perfusion of pathological liver tissue with a decellularizing agent can be used, for example, to generate an ECM scaffold that can be repopulated with cells for 3D disease modeling.

[0076] In other preferred embodiments, human liver tissue can be exposed to a decellularization reagent by immersing the tissue in the reagent and agitating it, for example, on a rotary mixer.

[0077] Slices or samples of liver tissue can be stirred in a decellularization reagent to generate a scaffold that reproduces the complexity of the 3D human microenvironment on a small scale for modeling liver disease. The sample can be normal or pathological liver tissue. Suitable sample width / length or diameter ranges can be between 0.2-2 cm or 0.2-1.0 cm. For example, the sample can be a slice or block of approximately 5 mm wide, referred to herein as a "liver tissue block" (LTC).

[0078] Liver tissue samples for decellularization by agitation protocols can be obtained according to standard techniques using a tissue dicer, needle biopsy, punch biopsy, or by simple scalpel cuts of slices or cubes of human liver parenchyma.

[0079] Agitation of the liver tissue immersed in the decellularization agent can apply fluid shear stress to it. Suitable agitation can include rotary shaking, for example, in a rotary mixer or magnetic stirrer at a speed of 100-1000 rpm, preferably about 900 rpm.

[0080] In some embodiments, the agitated tissue may be treated with one or more cycles of:

[0081] (a) exposing the tissue to an osmotic stress agent, such as a hypotonic or hypertonic agent,

[0082] (b) exposing the tissue to proteases and / or DNA enzymes, and

[0083] (c) exposing the tissue to a detergent,

[0084] In each cycle, (a), (b), and (c) can occur in any order.

[0085] For example, tissue may be treated with one or more cycles of:

[0086] (a) exposing the tissue to a hypotonic agent,

[0087] (b) exposing the tissue to a detergent, and

[0088] (c) exposing the tissue to DNase,

[0089] For example, 1 to 25 cycles may be performed.

[0090] The tissue may be exposed to a hypotonic agent, such as water, for 12 to 36 hours, preferably about 24 hours. The tissue may be exposed to a hypotonic agent at 4°C.

[0091] The tissue may be exposed to a detergent, such as SdC, such as 4% SdC, for 4 to 12 hours, preferably about 6 hours. The tissue may be exposed to the detergent at ambient temperature.

[0092] The tissue can be exposed to the DNA enzyme for about 3 hours. The tissue can be exposed to the DNA enzyme at ambient temperature. For example, the tissue can be exposed to 2000 KU of DNA enzyme 1 in 1M NaCl.

[0093] The method may further include exposing the tissue to a cleaning solution after exposure to any of the decellularization reagents proposed above. The tissue may be washed between one or more of the steps above and / or between cycles, for example, between steps (a) and (b), between steps (b) and (c) and / or after step (c) and before any cycle is repeated. For example, the tissue may be rinsed with phosphate buffered saline (PBS) or PBS supplemented with an antibiotic-antifungal agent for 5 to 30 minutes, preferably for about 5 minutes, at ambient temperature. Suitable antibiotic-antifungal agents are known in the art.

[0094] In other embodiments, tissue may be treated with one or more cycles of:

[0095] (a) exposing the tissue to a hypotonic agent such as water,

[0096] (b) Exposing the tissue to a protease such as trypsin, preferably in dH2O (deionized water).

[0097] (c) exposing the tissue to a first detergent, for example, an anionic detergent such as SDS,

[0098] (d) exposing the tissue to a second detergent, such as Triton TM Ionic detergents such as X-100.

[0099] The tissue can be exposed to a hypotonic agent, such as water, at ambient temperature for 15 to 30 minutes.

[0100] The tissue can be exposed to a protease, such as 0.025% trypsin in dH2O (deionized water) or EDTA, for 12 to 36 hours at ambient temperature.

[0101] The tissue can be exposed to the first detergent, eg, 0.01-1% SDS, at ambient temperature for 12 to 96 hours.

[0102] The tissue can be exposed to a second detergent, e.g., 0.01-3% Triton, at ambient temperature. TM X100, for 12 to 72 hours.

[0103] Between one or more steps in the above steps and / or between multiple cycles, tissue can be washed. Can be between one or more steps in the above steps and / or between multiple cycles, for example, between step (a) and (b), between step (b) and (c), between step (c) and (d), and / or after step (d) and before any cycle repeats, tissue can be washed. For example, tissue can be rinsed at ambient temperature with phosphate buffered saline (PBS) or PBS supplemented with antibiotic-antimycotic agent for 5 minutes to 3 hours, for example, 30 minutes to 1 hour.

[0104] For example, the method may include subjecting the agitated tissue to one or more cycles of:

[0105] (a) Expose the tissue to deionized water,

[0106] (b) Wash the tissue in PBS,

[0107] (c) exposing the tissue to SdC,

[0108] (d) Wash the tissue in PBS,

[0109] (e) exposing the tissue to DNase, and

[0110] (f) Wash tissue in PBS.

[0111] Steps (a) to (f) may be repeated 4-8 times, preferably about 4 times.

[0112] Suitable agitation may be provided by an orbital shaker at 600-1200 rpm, eg, about 900 rpm; or by a magnetic stirrer at 150-600 rpm, eg, 300-400 rpm.

[0113] In some embodiments, the tissue may be exposed to water and glucose solution alternately for 4-12 hours, eg, 8 hours, prior to step (a).

[0114] In other embodiments, the method described above may further include:

[0115] (f) Exposing the tissue to a hypertonic agent such as 9% saline.

[0116] The duration of each cycle of exposure to the decellularization agent can be 2-3 days.The total time to decellularize the scaffold using the agitation protocol can be 2.5-25 days, for example, about 8 days.

[0117] After decellularization, the scaffold can be sterilized, for example, by exposure to a sterilizing agent. Suitable sterilizing agents include gamma irradiation, electrolyzed water, and chemical agents such as peracetic acid. The scaffold can be exposed to 0.01% peracetic acid and 4% ethanol, for example, for 30 minutes to 2 hours.

[0118] As described herein, the stent may be perfused with a sterilizing agent or immersed in a sterilizing agent and agitated.

[0119] Following decellularization and sterilization, the liver scaffold can be tested, for example, for the absence of cells and / or the presence of ECM components, such as collagen, laminin, elastin, proteoglycans, hyaluronic acid, fibronectin, growth factors, and extracellular proteases.

[0120] Suitable techniques, including macroscopic visualization, microscopy, and immunohistochemistry, are known in the art.

[0121] Decellularized human liver scaffolds may lack myofilaments, endothelial cells, smooth muscle cells, as well as cell debris and nuclei detectable in tissue sections using standard tissue staining methods.

[0122] The decellularized human liver scaffolds produced as described here retain the 3D organoid morphology and architecture as well as ECM bioactivity of the source liver tissue.

[0123] In some embodiments, the structure and morphology of the decellularized liver scaffolds produced by the methods described above can be confirmed by electron microscopy.

[0124] Depending on the source tissue, the liver scaffold may include normal ECM or may be disease-altered ECM. For example, the liver scaffold may include one or more structural changes characteristic of one or more liver diseases or pathologies.

[0125] Human liver scaffolds allow for efficient attachment, migration, proliferation, and three-dimensional organization of cells cultured within the scaffold. Decellularized human liver scaffolds can also provide bioactive molecules and biosensing properties that maintain cell phenotype and functional properties and promote the production of tissue-specific matrices.

[0126] Following the generation of a decellularized human liver scaffold as described herein, methods can include repopulating the scaffold with cells to generate artificial liver tissue.

[0127] Suitable cells include human primary and cell line hepatocytes (e.g., hepatic stellate cells, Kupffer cells, sinusoidal cells), primary hepatocytes, endothelial cells, iPSCs (induced pluripotent stem cells) or cells derived from patient-specific iPSCs, embryonic stem cells (hESCs), mesenchymal stem cells (hMSCs), hepatocyte-derived mesenchymal stem cells (HDMSCs), fetal stem cells (e.g., amniotic fluid stem cells and fetal hepatocytes), cancer cells, and endothelial progenitor cells (EPCs).

[0128] In some embodiments, a decellularized human liver scaffold can be repopulated with autologous human cells obtained from a patient, for example, to produce artificial liver tissue for implantation into a patient. In other embodiments, a decellularized human liver scaffold can be repopulated with heterologous human cells, i.e., cells derived from a different human individual, for example, to produce artificial liver tissue for implantation into a patient. In some embodiments, the heterologous human cells can be screened prior to implantation to check for immunocompatibility with the patient.

[0129] Decellularized scaffolds can be repopulated by seeding cells within the scaffold and culturing them under appropriate conditions. For example, cells can be injected directly into the parenchyma of the decellularized scaffold; perfused through major vascular access; and / or dripped onto the surface of the decellularized scaffold.

[0130] Other aspects of the present invention utilize the methods described above, mutatis mutandis, to provide for the decellularization of liver tissue from non-human animals, such as non-human primates, pigs, sheep, horses, or cows, to produce non-human liver scaffolds. As described above, the non-human scaffolds can be repopulated with human cells to produce artificial liver tissue comprising human cells within a non-human ECM scaffold. This can be used to produce artificial liver tissue for implantation into a patient.

[0131] In some embodiments, the decellularized human liver scaffold or artificial liver tissue produced as described herein can be segmented to remove one or more liver tissue structures such as ducts and blood vessels. The structures removed from the decellularized human liver scaffold can optionally be repopulated with cells as described above. The structures removed from the liver scaffold or tissue can be used for transplantation, for example, to treat diseases as described below, or for disease modeling.

[0132] Other aspects of the present invention provide human liver scaffolds or artificial liver tissues produced by the methods described above.

[0133] The human liver scaffolds produced as described herein are acellular and display the extracellular matrix pore structure and morphology of the source liver tissue. Human liver scaffolds produced from fatty source liver tissue exhibit increased lipid content characteristic of the source tissue. Human liver scaffolds produced from fibrotic source liver tissue exhibit increased ECM components characteristic of the source tissue.

[0134] Scaffolds or tissues can be used for disease modeling. As described above, suitable scaffolds can be derived from normal liver tissue or pathological liver tissue.

[0135] A disease modeling method may include:

[0136] providing a human liver scaffold or artificial human liver tissue produced as described above,

[0137] Determine the effects of a compound, drug, biologic, device, or therapeutic intervention on a scaffold or tissue, or the cells therein.

[0138] The methods described here can be used to model liver diseases or conditions affecting the liver, such as liver fibrosis, liver cancer and metastasis, liver drug toxicity, post-transplant immune responses, and autoimmune hepatitis.

[0139] Biologics may include viruses such as HBV and HCV.

[0140] Decellularized liver scaffolds can be used for the diagnosis of liver disease. Suitable scaffolds can be derived from liver tissue from individuals suspected of having liver disease.

[0141] A method of diagnosing liver disease in a human subject may comprise:

[0142] A sample of a liver scaffold produced as described above is provided from an individual

[0143] The presence and amount of one or more liver scaffold proteins in the sample is determined.

[0144] The presence and amount of liver scaffold proteins in a sample can indicate the presence of liver disease in an individual.

[0145] The scaffold or tissue may be used therapeutically, for example, to replace or supplement liver tissue in an individual.

[0146] Treatment for liver disease may include:

[0147] The human liver scaffold or artificial human liver tissue produced as described above is implanted into an individual in need thereof.

[0148] The implanted scaffold or tissue can replace or supplement an individual's existing liver.

[0149] Another aspect of the present invention provides a human liver scaffold or artificial human liver tissue produced as described above for use in the treatment of a liver disease or dysfunction in a subject.

[0150] For example, a human liver scaffold or artificial human liver tissue can be implanted into an individual's body to regenerate a complete new liver or improve repair of a damaged liver, or can support an individual's liver function from outside the body.

[0151] Other aspects of the present invention provide for decellularization of human pancreatic tissue using the methods described above, mutatis mutandis, to produce a human pancreatic scaffold. As described above, the human pancreatic scaffold can be repopulated with human cells to produce artificial pancreatic tissue comprising human cells within a human ECM scaffold. This can be used to produce artificial pancreatic tissue for implantation into a patient.

[0152] For example, another aspect of the present invention provides a method for producing a human pancreatic scaffold, comprising

[0153] (i) providing human pancreatic tissue,

[0154] (ii) mechanically disrupting cells in tissues,

[0155] (iii) applying osmotic stress to said cells in the tissue,

[0156] (iv) exposing the tissue to proteases and / or DNAses, and

[0157] (v) exposing the tissue to a detergent, and

[0158] (vi) repeating each of steps (iii), (iv) and (v), and optionally (ii) one or more times,

[0159] Thus, human pancreatic scaffolds were produced.

[0160] Human pancreatic scaffolds can be generated according to any of the methods described above for the liver scaffolds, with the terms "liver" and "hepatic" being replaced with "pancreas" and "pancreatic."

[0161] The decellularized human pancreatic scaffolds generated by the methods described above can be repopulated with cells to generate artificial pancreatic tissue.

[0162] Aspects of the present invention provide decellularized human pancreatic scaffolds and artificial pancreatic tissue produced by the methods described herein.

[0163] Human pancreatic scaffolds and artificial pancreatic tissue can be used for disease modeling, diagnostic and therapeutic methods as described above for liver scaffolds and tissue, mutatis mutandis.

[0164] Another aspect of the present invention provides a bioreactor comprising:

[0165] Human liver scaffolds were generated as described above;

[0166] an input conduit for introducing culture medium into the scaffold; and

[0167] Output tubing for channeling culture medium out of the scaffold.

[0168] Preferably, the liver scaffold is seeded with human cells for repopulation of the scaffold. This can be used to create artificial liver tissue.

[0169] Suitable cells for seeding scaffolds are described above.

[0170] The bioreactor may further comprise a culture medium reservoir. The input conduit may be connectable or adapted to be connected to the reservoir so as to perfuse the scaffold in the bioreactor with culture medium from the reservoir so as to allow recellularization of the scaffold by the cells seeded into the scaffold.

[0171] The bioreactor may further include a tissue chamber for accommodating the liver scaffold.

[0172] The scaffold can be contained in a suitable culture medium in a bioreactor.

[0173] The liver scaffold in the tissue chamber and the culture medium in the reservoir can be maintained at 37°C under normoxic conditions to allow repopulation.

[0174] The bioreactor may further include one or more sensors for determining or monitoring the presence or amount of liver function in the scaffold as it is repopulated with the seeded cells.

[0175] Another aspect of the present invention provides a bioreactor comprising:

[0176] Artificial human liver tissue produced as described above;

[0177] an input tube for introducing blood into the artificial human liver tissue; and

[0178] Output tubing used to channel blood away from the artificial human liver tissue.

[0179] Preferably, the bioreactor is outside the body.

[0180] The input and output tubing may be connectable or adapted to connect to the individual's vascular system so that the artificial human liver tissue in the bioreactor is perfused with the individual's blood to provide liver function to the individual, e.g., to reduce the accumulation of toxins caused by loss of endogenous liver function.

[0181] The bioreactor may further include a tissue chamber for accommodating the artificial human liver tissue.

[0182] The artificial human liver tissue can be contained in a suitable culture medium in a bioreactor.

[0183] Another aspect of the present invention provides a decellularization system and apparatus suitable for use in producing human liver scaffolds using the methods described above.

[0184] Decellularization systems can include:

[0185] Decellularization equipment, including

[0186] a tissue chamber for receiving human liver tissue for decellularization, and

[0187] Three or more reagent reservoirs for containing decellularization reagents for introduction into the tissue cavity.

[0188] The reagent reservoir may be operably connected to the tissue cavity so that the reagent from the reservoir may be introduced into the cavity.

[0189] The reagent reservoir may contain a hypotonic reagent, a protease and / or DNase, and a detergent.

[0190] The tissue cavity may contain liver tissue.

[0191] In some embodiments, the system may further include a sampler adapted to generate liver tissue slices from a human liver sample and load them into the tissue chamber of the decellularizer.

[0192] The decellularization apparatus may further comprise one or more pumps for driving the decellularization reagent from the reagent reservoir into the tissue chamber, and optionally removing the decellularization reagent from the tissue chamber.

[0193] The chamber may include an inlet for introducing a decellularization reagent and an outlet for removing the reagent.

[0194] In some embodiments, the human liver tissue in the tissue cavity can be immersed in a decellularization reagent or physiological solution introduced into the cavity from a reservoir through an inlet. After exposure to the decellularization reagent, the reagent can be removed through an outlet according to the decellularization protocol described herein, and the decellularization reagent can be further introduced into the cavity. The system can further include a stirrer for agitating the human liver tissue immersed in the decellularization reagent in the tissue cavity.

[0195] In other embodiments, human liver tissue in a tissue cavity can be perfused with a decellularization agent from a reagent reservoir in a retrograde direction. Multiple decellularization agents can be perfused sequentially into the tissue according to the decellularization protocol described herein. During the perfusion process, the liver tissue can be immersed in a physiological solution or culture medium.

[0196] The decellularization device may include at least one cannula device for cannulating the liver tissue in the tissue cavity to allow perfusion in a retrograde direction. Suitable cannula devices may include hollow tubes of appropriate size for introduction into blood vessels, ducts, and / or cavities of human liver tissue. Typically, one or more blood vessels, ducts, and / or cavities are inserted into tissue in the tissue cavity. In some embodiments, the device may include an inlet cannula device for introducing a decellularization agent into the liver tissue, and an outlet cannula device for leading the decellularization agent from the liver tissue.

[0197] The decellularization apparatus may further include a perfusion device for perfusing the liver tissue through the catheter. The perfusion device may include a mechanism (e.g., a pump, air pressure, gravity) for moving a decellularization reagent from a reservoir through the human liver tissue via one or more catheters and tubing, an adapter, and / or a connector for perfusing the cannulated liver tissue in the tissue cavity with the decellularization reagent from the reagent reservoir.

[0198] Cannulation and perfusion are techniques known in the art.

[0199] The decellularization device can be adapted to maintain a sterile environment for the liver tissue in the tissue chamber. During the decellularization process, sterility can be maintained using various techniques known in the art, such as controlling and filtering airflow and / or perfusing with, for example, antibiotics, antifungals, or other antimicrobial agents to prevent the growth of undesirable microorganisms. Suitable antimicrobial compounds are known in the art.

[0200] The decellularization device can be adapted to monitor certain perfusion characteristics (e.g., pressure, volume, flow pattern, temperature, gas, pH), mechanical forces (e.g., ventricular wall motion and stress). For example, the system can include sensors that monitor the system (e.g., bioreactor) and / or liver tissue. Sensors can be used to monitor the pressure of the fluid flowing in the cannulated liver tissue; the ambient temperature in the system and / or the temperature of the liver tissue; the pH and / or flow rate of the fluid flowing in the cannulated liver tissue; and / or the biological activity of the recellularized liver tissue. In addition to having sensors for monitoring such characteristics, the system for decellularizing and / or recellularizing liver tissue can include controls for maintaining or adjusting such characteristics.

[0201] Controls may include components such as thermometers, thermostats, electrodes, pressure sensors, relief valves, valves for opening and closing fluid connections to decellularization reagents and changing the rate and direction of fluid flow.

[0202] To help ensure stable conditions (eg, temperature), the chamber, reservoir, and tubing may be water jacketed.

[0203] The system for generating human liver scaffolds can be controlled by a programmable processor. For example, the processor can receive and process information from one or more sensors. The processor can transmit this information and instructions back to the bioreactor and / or liver tissue.

[0204] The processor can be modified or programmed to calculate the exposure time and perfusion pressure for each decellularization reagent for a particular liver tissue based on the weight and internal resistance of the liver tissue according to the decellularization method as described herein. The processor can change the decellularization reagent and change the perfusion pressure through one or more pumps and / or valve controls in the system. The processor can record the preload and afterload (pressure before and after perfusion, respectively) and flow rate for each decellularization step.

[0205] The system can be adapted to monitor the biological activity of liver tissue undergoing decellularization. For example, the mechanical activity, mechanical pressure, and / or wall stress of the liver tissue can be monitored.

[0206] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure.

[0207] Other aspects and embodiments of the present invention provide the aspects and embodiments described above with the term "comprising" replaced by the term "consisting of," as well as the aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of.

[0208] It should be understood that the present application discloses all combinations of any one of the above-described aspects and embodiments, unless the context requires otherwise. Similarly, the present application discloses all combinations of preferred and / or optional features, either individually or together with any one of the other aspects, unless the context requires otherwise.

[0209] Modifications to the above-described embodiments, further embodiments, and modifications thereto will be apparent to those skilled in the art upon reading this disclosure and, as such, are intended to be within the scope of the present invention.

[0210] All files and sequence database entries mentioned in this specification are incorporated herein by reference for all purposes.

[0211] As used herein, "and / or" is understood as specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" is understood as specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0212] Certain aspects and embodiments of the present invention will now be described by way of example and with reference to the figures described below.

[0213] Figure 1 Shown are flow rates (in ml / min) at different stages of perfusion over 14 days relative to the decellularization process.

[0214] Figure 2 Shown is a 125 mm 3 Liver tissue cube (LTC).

[0215] Figure 3 Shown is a histological comparison of native tissue and decellularized LTC (dLTC) after 4 decellularization cycles. H&E (hematoxylin and eosin) staining shows the removal of cells after LTC decellularization, and SR (Sirius Red) staining shows the retention of collagen (red) and the removal of cellular material (yellow).

[0216] Figure 4 Quantitative measurements of collagen (top) and DNA (bottom) after decellularization are shown. Quantification of collagen after different agitation speeds (900C1-900C4) demonstrates the preservation of collagen content when compared to fresh tissue. Decellularization is efficient, with DNA content significantly reduced (p<0.01) after 1 treatment cycle (bottom).

[0217] Figure 5Shown is the repopulation of a human liver scaffold with the human hepatic stellate cell line LX2. H&E staining shows progressive LX2 cell migration into the LTC scaffold when comparing day 1 to day 21 after recellularization (upper panel). The repopulation process is characterized by significant cell proliferation detected by immunostaining for the proliferation marker Ki67 (lower panel).

[0218] Figure 6 Shown is an increase in total cell counts after 14 days of repopulation with LX2. Importantly, total cell counts in human liver scaffolds increased significantly between 14 and 21 days (LH group). KI67 immunohistochemistry showed that more than 85% of cells proliferated at all different time points (RH group).

[0219] Figure 7 Shown is the repopulation of decellularized human liver scaffolds using the human hepatocellular carcinoma cell line SK-Hep. H&E and HVG (Hematoxylin Van Gieson) staining show cell attachment after 1 day of bioengineering and progressive cell migration into the human liver scaffold when comparing 1 day to 14 days after recellularization.

[0220] Figure 8 Shown are FACS staining of SK-Hep with anti-human Nanog (right) and isotype control (left).

[0221] Figure 9 The repopulation of decellularized human liver scaffolds using the human hepatocellular carcinoma cell line SK-Hep is shown after 1 and 7 days. Nanog expression was analyzed by immunohistochemistry. Clearly, cells attached to the ECM on day 1 and cells migrating within the scaffold on day 7 are Nanog-positive cells.

[0222] Figure 10 Shown is the repopulation of a decellularized human liver scaffold using the human hepatocellular carcinoma cell line SK-Hep after 14 days. Nanog expression (brown) was analyzed by immunohistochemistry, and cells were counterstained with hematoxylin (blue). The repopulation process after 14 days is characterized by Nanog-positive and Nanog-negative cells (bottom). Notably, at this stage, Nanog-positive cells surround the repopulated scaffold (top), a characteristic similar to the development of human hepatocellular carcinoma.

[0223] Figure 11 The macroscopic appearance of a decellularized human left liver lobe is shown using two different light backgrounds to highlight the preservation of the vascular tree.

[0224] Figure 12Shown are tissue sections of decellularized blood vessels (portal vein, hepatic artery, and hilar plate) and the biliary tree. H&E shows the absence of cells in the decellularized tissue. SR and EVG staining show the retention of collagen (red) and elastin (blue), respectively.

[0225] Figure 13 Shown is a histological comparison of fresh liver tissue and decellularized human liver left lobe segments (S1, S2, S3, and S4). H&E staining shows the removal of cells after decellularization, while SR and EVG (Elastica van Gieson) staining show the retention of collagen (red) and elastin (blue), respectively.

[0226] Figure 14 Shown is the repopulation of a human liver scaffold using the human hepatic stellate cell line LX2. H&E and HVG staining show cell attachment after 1 day of bioengineering as well as progressive cell migration into the human liver scaffold when comparing 1 day to 14 days after recellularization.

[0227] Figure 15 Shown is a low magnification (90x) SEM image comprising the portal tract surrounded by typical lobular structures.

[0228] Figure 16 A 300x SEM image is shown confirming the acellular nature of the scaffold and clearly defining the spaces once occupied by hepatocytes (ie, hepatocyte-free spaces).

[0229] Figure 17 Shown is a high magnification (2500x) SEM image showing a well-preserved three-dimensional network of connective tissue fibers that create spaces devoid of hepatocytes.

[0230] Abbreviations: SdC sodium deoxycholate; PBS / AA PBS + antibiotics and antimycotics; T / E 0.025% trypsin / 0.025% EDTA; SDS sodium dodecyl sulfate; TX100 Triton X 100; RT room temperature; PAA peracetic acid; EtOH ethanol.

[0231] 1. Methods

[0232] 1.1 Human liver collection and cannulation

[0233] Discarded human liver organoids (DHLOs) unsuitable for liver transplantation were heparinized according to standard transplantation protocols. DHLOs were fully prepared by subdividing into multiple blocks of small, non-vascularized liver units (liver tissue cubes, LTCs) of 0.2-1.0 cm and / or by segmental or subsegmental preparation of units with vascular biliary pedicles. Whole human livers or, alternatively, the left lobe (segments 2-3-4±1), right lobe (segments 5-8), or left lateral liver (segments 2-3±1) and liver tissue cubes (LTCs) were frozen at -80°C for at least 24 hours to facilitate cell lysis.

[0234] 1.2 Whole-mount perfusion decellularization of the left lobe of human liver

[0235] First, whole human liver lobes were thawed in PBS overnight at 4°C. Second, the vena cava or hepatic vein was cannulated to initiate a retrograde perfusion system.

[0236] Finally, 5CDF was applied to achieve complete organ decellularization, as shown in Tables 1, 2, and Figure 1 Two perfusion phases were used: a) a rapid increase in flow rate to compensate for resistance and b) a steady flow rate as decellularization progressed. Figure 1 Two stages of flow rate are shown in .

[0237] 1.3 Agitated Decellularization of Human LTCs

[0238] Thaw LTC for 1-1.5 h at 37° C. The protocol for decellularization of LTC is shown in Tables 3 to 5.

[0239] 2. Results

[0240] After 4 cycles of decellularization, native liver tissue and decellularized LTC (dLTC) were compared histologically. It was found that the decellularization cycles removed cells and cellular material from LTC while preserving collagen ( Figure 3 ).

[0241] Collagen and DNA were quantified in dLTC after decellularization. Figure 4 Top), it was found that the amount of collagen in dLTC was retained at different agitation speeds. Decellularization was efficient, and after only 1 treatment cycle ( Figure 4 Bottom) DNA content was significantly decreased (p<0.01).

[0242] Decellularized human LTCs were repopulated using the human hepatic stellate cell line LX2. LX2 cells were found to migrate progressively into the LTC scaffolds within 21 days after recellularization. Figure 5The total cell count in the human liver scaffolds was found to increase after 14 days of repopulation with LX2 and to increase significantly between 14 and 21 days ( Figure 6 LH group). Immunostaining for the proliferation marker Ki67 showed that the repopulation process was characterized by significant cell proliferation ( Figure 5 At all different time points, more than 85% of cells proliferated ( Figure 6 RH group).

[0243] Decellularized human liver scaffolds were repopulated with the human hepatocellular carcinoma cell line SK-Hep. Cell attachment was observed 1 day after bioprocessing, and within 14 days after recellularization, SK-Hep cells gradually migrated into the human liver scaffolds ( Figure 7 Cells attached to the ECM on day 1 and cells migrating within the scaffold on day 7 are shown as Nanog-positive cells ( Figure 8 and 9 After 14 days, the repopulation process was characterized by Nanog-positive and Nanog-negative cells ( Figure 10 Notably, after 14 days, Nanog-positive cells were found surrounding the repopulated scaffolds, a characteristic similar to the development of human hepatocellular carcinoma ( Figure 10 ; upper group).

[0244] The vascular tree of the left lobe of the human liver was found to be preserved after decellularization ( Figure 11 ). It was found that the decellularized tissue had no cells, while collagen and elastin were retained ( Figure 12 ).

[0245] Comparison of fresh liver tissue and decellularized human left lobe liver segments (S1, S2, S3, and S4) confirmed the removal of cells and the preservation of collagen and elastin after decellularization ( Figure 13 ).

[0246] The human hepatic stellate cell line LX2 was used to repopulate the decellularized left lobe of the human liver. LX1 cells were found to attach to the decellularized scaffold after 1 day and to migrate into the scaffold gradually within 14 days. Figure 14 ).

[0247] The decellularized human liver scaffolds were analyzed by scanning electron microscopy. SEM images confirmed the acellularity of the scaffolds and showed the presence of clearly defined spaces once occupied by hepatocytes (i.e., hepatocyte-free spaces). The three-dimensional network of connective tissue fibers that constructed the hepatocyte-free spaces, as well as the portal and lobular structures, were found to be well preserved ( Figure 15-17 ).

[0248]

[0249]

[0250] Table 1

[0251]

[0252] Table 2

[0253] time Reagents temperature rpm 24h dH20 4℃ 900 4-6h SdC 4% RT 900 5min PBS RT 900 3h Dnase RT 900 5min PBS / AA RT 900

[0254] Table 3

[0255] time Reagents temperature rpm 12-36h dH2O 4℃ 100-1000 4-12h SdC4% RT 100-1000 5-30 minutes PBS RT 100-1000 3h Dnase RT 100-1000 5-30 minutes PBS / AA RT 100-1000

[0256] Table 4

[0257] time Reagents temperature rpm 15-30 minutes dH20 RT 100-1000 12-36h T / E 0.025% RT 100-1000 12-72h SDS 0.01-1% RT 100-1000 12-72h TX100 3% RT 100-1000 5-30 minutes PBS / AA RT 100-1000

[0258] Table 5

[0259]

[0260]

[0261] Table 6

Claims

1. A method for producing a human pancreatic scaffold, comprising: (i) mechanically destroying cells in healthy or pathological human pancreatic tissue, including the entire pancreas or its functional units, (ii) applying osmotic stress to said cells in said tissue by exposing said tissue to a hypotonic agent or a hypertonic agent, (iii) exposing the tissue to a protease and / or a DNase, (iv) exposing the tissue to a detergent, and (v) repeating each of step (ii), step (iii) and step (iv) one or more times in any order, This produced human pancreatic scaffolds, During steps (ii) to (v), the human pancreatic tissue is subjected to fluid shear stress, which is generated by perfusing the human pancreatic tissue in a retrograde direction at a perfusion rate, and in two or more repetitions of steps (ii) to (v), the perfusion rate is gradually increased from an initial value of 0.1-1.99 ml / min / g tissue to a target perfusion rate of 2-20 ml / min / g tissue and then maintained.

2. The method according to claim 1, wherein (i) mechanically disrupting the cells by subjecting the tissue to one or more cycles of freezing and thawing, or (ii) mechanically disrupting the tissue by subjecting the tissue to HIFU or sonication.

3. The method according to claim 1, wherein The hypotonic agent is deionized water and / or the hypertonic agent is water or saline.

4. The method according to claim 1, wherein In step (iii), the tissue is exposed to a protease.

5. The method according to claim 4, wherein The protease is trypsin or pronase.

6. The method according to claim 1, wherein The detergent is an anionic detergent.

7. The method according to claim 6, wherein: The detergent is sodium dodecyl phosphate (SDS) or sodium deoxycholate (SdC).

8. The method according to claim 1, wherein The detergent is a non-ionic detergent.

9. The method according to claim 8, wherein The detergent is polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-diphenyl ether (Triton X100 TM ).

10. The method according to claim 1, wherein Step (v) comprises: a) Repeating steps (ii) to (iv) by perfusing the pancreatic tissue in the following order: (ii), (iii), (ii), (iii)(iv), [(ii), (iv)] n ,(ii),(iii),[(ii),(iv)] n , wherein n is 1 to 25; or b) repeating steps (ii) and (iv) 1 to 25 times by perfusing the pancreatic tissue.

11. The method according to claim 1, wherein The pancreatic tissue was subjected to the perfusion protocol set forth in the table below.

12. The method of claim 1, comprising sterilizing the stent after step (v).

13. The method of claim 1, comprising repopulating the scaffold with cells to produce artificial pancreatic tissue.

14. The method according to claim 13, wherein The cells are human primary and cell line pancreatic cells, human primary hepatocytes, endothelial cells, human induced pluripotent stem cells (iPSCs), human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs), human fetal stem cells, human cancer cells, and human endothelial progenitor cells (EPCs).

15. The method according to claim 13, wherein The cells are cells derived from patient-specific iPSCs.

16. A human pancreatic scaffold produced by the method according to any one of claims 1 to 12 or an artificial pancreatic tissue produced by the method according to any one of claims 13 to 15.

17. Use of a human pancreatic scaffold produced by the method according to any one of claims 1 to 12 in the preparation of a medicament for treating pancreatic diseases or disorders.

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