TW201741451A - Compositions and methods for bioengineered tissues - Google Patents

Abstract

The present disclosure provides methods for producing bioengineered tissue along with an apparatus and other relevant compositions employed in generation thereof.

Description

Composition and method for bioengineered tissue Cross-reference to related applications

The present application claims priority to U.S. Patent Application Serial No. Serial No. No. No. No. No. No. No. No. No. No. No. No. No.

The present invention relates to a composition and method for bioengineered tissue

The following discussion of the background of the invention is only intended to aid the understanding of the invention and is not intended to

Spheroid and organism culture systems and other organ modeling methods contribute to the formation of cell conformations and polarity, which are closer to those found in native tissues. Although cultures derived entirely from clonal cell populations have certain advantages, more emphasis is placed on the importance of three-dimensional tissue, cell polarity, epithelial-mesenchymal interaction, and paracrine signals from epithelial-mesenchymal relationships in regenerative medicine. Sex, which is used to stabilize cells and their functions.

Conventional methods of generating organs and organism tissues are limited to micromodels because large numbers of cells cannot be maintained in the absence of vascular support and they are unable to mimic the blood vessel and tissue division of model organs. Therefore, still There is a need for a scalable, stable method of producing bioengineered tissue.

Aspects of the present disclosure relate to compositions, kits and methods for producing and using bioengineered tissues or micro-organs, as well as containers configured to produce bioengineered tissues or micro-organs.

Aspects of the present disclosure relate to containers for generating bioengineered tissue. In some embodiments, the generating comprises introducing epithelial cells and/or mesenchymal cells into or onto a biological matrix scaffold. In some embodiments, the generating comprises introducing a substantial and/or non-parenchymal cell. In some embodiments, the cells are partners in the lineage phase between each other. Aspects of the present disclosure relate to a three-dimensional scaffold comprising an extracellular matrix comprising (i) native collagen found in an organ and/or (ii) matrix residues of a vascular tree found in an organ. Things.

In some embodiments, the biomatrix scaffold comprises collagen. In some embodiments, the biomatrix scaffold comprises (1) (i) nascent collagen, (ii) collagen molecules that aggregate but not cross-link, (iii) cross-linked collagen, and (iv) different forms of these Collagen-bound factors (matrix components, signaling molecules, other factors) and/or (2) most of the cross-linked and uncrosslinked native collagen found in the tissue and matrix molecules that bind to these collagens And signal molecules. In some embodiments, the biomatrix scaffold is a three dimensional one. In some embodiments, the biomatrix scaffold comprises one or more collagen-related matrix components, such as laminin, nucleic acid, elastin, proteoglycan, hyaluronic acid, non-sulfated glycosaminoglycan, and sulfuric acid Glycosaminoglycans and growth factors associated with matrix components And cytokines. The biomatrix scaffold comprises greater than 50% of the matrix binding signal molecules found in vivo. In some embodiments, the matrix-bound signaling molecule can be epidermal growth factor (EGFs), fibroblast growth factor (FGFs), hepatocyte growth factor (HGFs), insulin-like growth factors (IGFs), transforming growth factor ( TGFs), nerve growth factors (NGFs), neurotrophic factors, interleukins, leukemia inhibitory factor (LIFs), vascular endothelial growth factor (VEGFs), platelet-derived growth factor (PDGFs), stem cell factors (SCFs), colony stimulation Factor, GM-CSF, erythropoietin, thrombopoietin, heparin-binding growth factor, IGF binding protein, placental growth factor and wnt signal. In some embodiments, the biomatrix scaffold comprises a matrix residue of the vascular tree of the tissue. In another embodiment, the matrix residue can provide vascular support of the cells in the bioengineered tissue.

In some embodiments, wherein the cell is in a seeding medium, the generating further comprises, after the initial incubation period, optionally replacing the seeding medium with a differentiation medium. In some embodiments, wherein the cells are in a seeding medium, they are introduced at a plurality of intervals, each interval followed by a rest period. In some embodiments, the interval is about 10 minutes and the rest time is about 10 minutes. In some embodiments, the seeding density is less than or about about 12 million cells per gram of wet weight of the biomatrix scaffold and is introduced at one or more intervals. In some embodiments, the cells in the inoculation medium are introduced at a rate of about 15 ml/min at one or more intervals. In some embodiments, the cells in the inoculation medium are introduced at intervals of 10 minutes, with each interval being accompanied by a resting period of 10 minutes. In some implementations In the example, cells in the inoculation medium were introduced at a rate of 1.3 ml/min after three intervals.

In some embodiments, the inoculating medium comprises a serum-free inoculation medium. In some embodiments, the inoculation medium is supplemented with serum, optionally from about 2% to 10% fetal serum such as fetal bovine serum (FBS). In some embodiments, serum supplementation media (eg, deactivation of the enzyme used to prepare the cell suspension) may be required. In some embodiments, the supplement occurs for a few hours.

In some embodiments, the seeding medium comprises a basal medium, a lipid, insulin, transferrin, and/or an antioxidant. In some embodiments, the inoculation medium may comprise one or more of the following: a basal medium, low calcium (0.3-0.5 mM); no copper, zinc, and selenium; insulin, transferrin/fe, and one or more purified free fatty acids, Wherein the one or more purified free fatty acids (eg, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, linolenic acid) are optionally complexed with purified albumin; and one or more lipid binding proteins such as high density Lipoprotein HDL). In some embodiments, the inoculation medium can be used to include or maintain a low oxygen concentration level (1-2%).

In some embodiments, the cells are cultured in the inoculation medium for 4 to 6 hours at 4 °C prior to the introduction step. In some embodiments, the cells can be isolated from fetal or neonatal organs. In some embodiments, the mesenchymal cells are stromal cells, endothelial cells, and hematopoietic cells. In some embodiments, the cells can be isolated from an adult or child donor. In some embodiments, the epithelial or parenchymal cells can be bile trunk cells, gallbladder-derived stem cells, hepatic stem cells, hepatoblasts, stereotyping Hepatocytes and biliary tract precursor cells, axin2+ precursor cells (eg, axin2+ liver precursor cells), mature parenchymal cells or epithelial cells, mature hepatocytes, mature cholangiocarcinoma cells, pancreatic stem cells, pancreatic committed progenitor cells, islet cells, and/or acinar cells Any one or more of; and/or stromal cells or non-parenchymal cells may be hemangioblasts, stellate precursors, stellate cells, mesenchymal stem cells, pericytes, smooth muscle cells, stromal cells, nerves Any of a quantum cell precursor, a neuronal cell, an endothelial cell precursor, an endothelial cell, a hematopoietic cell precursor, and/or a hematopoietic cell. In some embodiments, the epithelial or parenchymal cells may be stem cells from the biliary tree, liver, gallbladder, hepatopancreatic duct or progeny thereof; and/or the mesenchymal or non-parenchymal cells may be hemangioblasts, endothelium and / or stellate cell precursors, mesenchymal stem cells, stellate cells, stromal cells, smooth muscle cells, endothelial cells, bone marrow-derived stem cells, hematopoietic cell precursors and / or hematopoietic cells. In some embodiments, the epithelial or parenchymal cells can include differentiated parenchymal cells such as, but not limited to, axin2+ precursor cells (eg, axin2+ hepatocytes or liver precursor cells), mature cells (eg, mature hepatocytes, mature biliary cells), multiple Ipocytic cells (such as polyploid hepatocytes) and apoptotic cells. In some embodiments, mature cells may be associated with sinusoidal endothelium, some of which may be permeable to stromal cells (eg, endothelial cells). In some embodiments, axin2+ precursor cells (eg, axin2+ liver precursor cells) may be ligated In endothelial cells. In some embodiments, the epithelial or parenchymal cells are mature islets, optionally associated with mature endothelium and/or mature acinar cells; and/or optionally associated with a mature stroma. In some embodiments, the ratio of the cells is 80% and 20%: epithelial cells and mesenchymal cells or parenchymal cells and non-parenchymal cells. In some embodiments, the cell Is at least 50% of stem cells and / or precursor cells. In some embodiments, the cell does not comprise any terminally differentiated hepatocytes and/or pancreatic cells. In some embodiments, the epithelial or parenchymal cells can be one or more of a stem cell, a committed precursor cell, a diploid adult cell, a polyploid adult cell, and/or a terminally differentiated cell; and/or a stroma The non-parenchymal cells may be one or more of hemangioblasts, endothelial precursors, mature endothelium, matrix precursors, mature matrices, neuronal precursors and mature neuronal cells, hematopoietic precursors, and/or mature hematopoietic cells.

In some embodiments, the composition of the cells can be adjusted for the desired tissue, for example, for bioengineered liver tissue, hepatocytes can be used in a specific ratio, or a specific proportion of pancreas can be used for bioengineered pancreatic tissue. Dirty cells. For example, for the liver, epithelial cells can be stem cells (eg, bile trunk cells) and from biliary tree, liver, hepatopancreas, and/or gallbladder, biliary tree, gallbladder-derived stem cells, hepatic stem cells, hepatoblasts, committed hepatocytes And one or more of biliary progenitor cells, axin2+ precursor cells (eg, axin2+ liver precursor cells), mature hepatocytes, and/or progeny of mature cholangio cells; and/or interstitial or non-parenchymal cells may be hemangioblasts, stars One or more of a stellate precursor, a stellate cell, a mesenchymal stem cell, a smooth muscle cell, a stromal cell, an endothelial cell precursor, an endothelial cell, a hematopoietic cell precursor, and/or a hematopoietic cell. Similarly, these same mesenchymal or non-parenchymal cells can be used in the pancreas; and/or epithelial cells for the pancreas may include biliary tract cells (eg, from hepatopancreatic ducts), pancreatic stem cells, pancreatic progenitors Cells, islet cells, stem cells, and their progeny from biliary trees, hepatopancreatic ducts, or pancreas and/or acinar cells. In another embodiment, for In the liver, terminally differentiated hepatocytes can be excluded, and for pancreas, terminally differentiated pancreatic cells can be excluded.

In some embodiments, where a differentiation medium is used, depending on the cells used, the differentiation medium includes basal medium, lipids, insulin, transferrin, antioxidants, copper, calcium, and/or for epithelial cells, One or more beacons that multiply and/or maintain one or more of mesenchymal cells, parenchymal cells, and/or non-parenchymal cells. The aspect disclosed herein relates to the differentiation medium itself. In some embodiments, the differentiation medium can include Kubota medium; one or more lipid binding proteins (eg, HDL), one or more purified fatty acids (eg, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic oil) Acid, linolenic acid), one or more sugars (galactose, glucose, fructose), one or more glucocorticoids (such as, for example, dexamethasone or hydrocortisone), copper (for example, a concentration of about or about 10 ^{- 10} to about or about 10 ^-12 M); calcium (eg, 0.6 mM); selected from prolactin growth hormone, glucocorticoids, glucagon, thyroid hormones (eg, triiodothyronine or T3) , epidermal growth factor (EGFs), hepatocyte growth factor (HGFs), fibroblast growth factor (FGFs), insulin-like growth factor (IGFs), leukemia inhibitory factor (LIF), interleukin (IL) such as IL6 and IL11, wnt One or more hormones and/or growth factors for proliferation and/or maintenance of epithelial cells or parenchymal cells in ligands, bone morphogenetic proteins (BMPs) and/or cyclic adenosine monophosphates; and/or selected from angiopoietin Vascular endothelial growth factor (VEGFs), white Interleukins (ILs), stem cell factors (SCFs), leukemia inhibitory factor (LIF), colony stimulating factors (CSFs), thrombopoietin, platelet-derived growth factor (PDGFs), erythropoietin, insulin-like growth factors (IGFs) One or more hormones and/or growth factors for the propagation and/or maintenance of mesenchymal or non-parenchymal cells in fibroblast growth factor (FGFs), epidermal growth factor (EGFs). In some embodiments, a differentiation medium can be used that contains or maintains an oxygen content of about 5%.

In some embodiments, the container is a flow path designed to design a fluid for simulating cellular vascular support.

Aspects of the present invention relate to bioengineered tissues, including region-dependent phenotypic traits of native liver characteristics, including (a) having stem cells/precursor cells, diploid adult cells, and/or associated stroma Or a region surrounding the portal vein of a non-parenchymal trait; (b) a sinus plate with mature bile epithelial cells (eg, biliary cells) and/or associated mature stellate cells and stromal cells, mature parenchymal cells (eg, hepatocytes) And/or related mesenchymal cells, such as, but not limited to, intermediate acinar regions of sinusoidal endothelial and/or pericytes (ie, smooth muscle cell) traits; (c) centrally surrounding regions with terminally differentiated parenchymal cell traits, such as but not Limited to hepatocytes, which include polyploid hepatocytes and apoptotic hepatocytes and/or associated mesenchymal cells such as, but not limited to, transdermal endothelium and/or diploid axin2+ liver precursor cells bound to the endothelium. In some embodiments, the phenotypic trait of the tissue comprises a trait associated with diploid parenchyma and/or stromal cells of the area surrounding the portal vein. In some embodiments, the phenotypic trait of the tissue comprises traits of mature parenchymal cells (eg, mature hepatocytes) and/or stromal cells (eg, sinusoidal endothelium) found in the central acinar region of the native liver. In some embodiments, the phenotypic characteristics of the tissue include those found in the intermediate acinar region of the native liver. Traits of mature parenchyms (eg, mature hepatocytes) and/or stromal cells (eg, sinusoidal endothelium). In some embodiments, the tissue comprises (i) polyploid hepatocytes associated with permeabilizing endothelial cells; and/or (ii) diploid hepatic progenitor cells and/or axin2+ liver precursors linked to central venous endothelium One or more of the cells. In some embodiments, the periportal region of the tissue is rich in traits comprising stem/progressor cell niches of hepatic stem cells, hepatocytes, and/or committed precursor cells and/or diploid adult hepatocytes. In some embodiments, the parenchymal cells of the tissue further comprise precursors and/or mature forms of hepatocytes and/or cholangiocarcinoma cells. In some embodiments, the mesenchymal cells of the tissue further comprise precursors and/or mature forms of stellate cells, pericytes, smooth muscle cells, and/or endothelium. Similarly, aspects of the present disclosure are directed to a bioengineered tissue comprising a partition-dependent phenotypic trait of native pancreatic features and/or a trait comprising a region associated with pancreatic cells in the pancreatic head and a pancreas Traits of pancreatic cell-associated regions in the dirty tail. In some embodiments, the mesenchymal cells include a stroma, smooth muscle cells, endothelial cells, and hematopoietic cells; in other embodiments, these mesenchymal cells can be indicative of a region-dependent morphology.

Additional aspects involve three-dimensional micro-organs. Non-limiting examples include three-dimensional micro-organs that are generated in or included in the disclosed containers, as disclosed by the disclosed bio-engineered tissue. Kits for the generation and culture of these micro-organs are also considered herein.

Also provided herein is a method of assessing treatment of an organ comprising applying the treatment to a bioengineered tissue or a three-dimensional micro-organ.

Figure 1 shows the characteristics of a biomatrix scaffold after decellularization. A) Percent retention of different growth factors in the biomatrix scaffold compared to fresh tissue. B-E) Ultrastructure of a biomatrix scaffold imaged by scanning electron microscopy (SEM). B) Trisomy including the portal vein (PV), hepatic artery (HA), and bile duct (arrow). C) The sinusoidal region of the acinar in the biomatrix scaffold, which indicates that it has no cell D-E) collagen bundle (*) and adhesion molecules that bind to collagen (arrow). F-O) Identification of matrix molecules in appropriate regions of the liver acinus by immunohistochemical staining P) Quantitative analysis of collagen content in the scaffold compared to fresh tissue.

Figure 2 depicts RNA sequencing data obtained from three fetal liver tissues and used for relative gene expression between cells in a bioreactor

Figure 3 shows histology of human fetal liver stem/progenitor cells after 14 days of culture. A-D) Cell markers located in the area surrounding the portal vein. G) PAS staining (periodic acid; PAS) staining of hepatocytes showing hepatic sugar storage. H) Positive hepatocytes for Cyp3A4, P450 metabolic enzymes. I) SEM images of endothelial cells lined with blood vessels. The inserted image is a positive endothelial cell for CD31, also known as platelet endothelial cell adhesion molecule (PECAM).

Figure 4 depicts RNA-sequencing related performance of fetal liver, bioreactor tissue (Bio_T14) and adult liver samples. A) Matrix metal peptidases (MMPs) such as MMP-2 and -9 are enzymes involved in matrix remodeling. B-E) Expression of extracellular matrix molecules. The cells grown in the bioreactor performed significantly higher levels than the ECM molecules (p < 0.05) compared to the other samples. [Bio_T14=Bioreactor No. T14)

Figure 5 depicts markers of cells found in the area surrounding the portal vein The depicted RNA-sequencing relative gene expression. The cells cultured in the bioreactor showed a significant decrease in gene expression in stem cells and hepatoblast markers, and an increase in biliary cell markers (p < 0.05). This represents a shift towards a more mature phenotype (p < 0.05).

Figure 6 depicts RNA-sequencing relative gene expression of markers that find contour cells in the cardia region. At the same time as the reduction of stem cells and precursor cell markers, cells cultured in the bioreactor continue to differentiate into a mature liver phenotype, which is evident by an increase in expression of genes associated with mature metabolic traits. p<0.05

Figure 7 shows the results of the expression assay: A) RNA sequencing of genes associated with feedback loops and signal transduction pathways is referred to as the Salvador/Warts/Hippo (SWH) pathway, which is used to regulate organ size and involves Hippo ( "Phase-like" kinases and YAP (Yes-associated protein) cells cultured in bioreactors showed a decrease in Hippo kinase and an increase in YAP and related targeted genes compared to fetal and adult livers, indicating that regeneration is underway. the process of. B) Gene expression of the angiogenic marker after 14 days of culture and SEM images of fetal liver endothelial cells lined with the biomatrix scaffold. C) Hematopoietic and endothelial stem cell markers such as endothelial transcription factor, GATA-2, stem cell factor receptor (SCR) and interleukin 7R (IL7R) and mature hematopoietic genes such as recombinant activating gene 1 (Rag1), CD3 (T cell common Relative gene expression of receptor) and colony stimulating factor (CSF). The bioreactor sample has a CD3 gene expression level similar to that of the adult liver, and both are associated with T cells as Rag1 expression increases. CSF, a kind of bone The genes expressed by myeloid cells are significantly more expressed than those in the fetus and adult liver. p<0.05

Figure 8 shows the results of various assays: A) lactate dehydrogenase (LDH), full-length keratin 18 (FL-K18), cell necrosis index and cleaved cytokeratin 18 (ccK18), and apoptosis indicators are cells Viability; and B) cell production of alpha-fetoprotein (AFP) and albumin and urea secretion in culture for 14 days. The rise and fall in albumin levels appears to complement apoptosis data, suggesting cell cycle phenomena and regenerative responses.

Figure 9 shows cells cultured in a bioreactor and undergoing saccharification or glycolytic effects. The shift in glucose production and consumption can also correspond to a shift in tissue engineered liver development. Glycogenogenesis occurs in cells surrounding the precursor and portal vein, and glycolytic effects are associated with cells in the area surrounding the heart. B) Multivariate analysis showed that although the metabolic behavior of the bioreactor was similar, it was still at different stages of metabolic function. C) The Variable Importance Projection (VIP) plot shows the metabolites that contribute. VIP>1.0 is considered important.

A to F of Fig. 10 are transmission electron microscope (TEM) images of the tissue engineered liver after 14 days of culture. A-C) Several hepatocytes that form bile duct (BC) cells and their sinus spaces between them (arrows). B) Possible secretory vesicles visible around the gallbladder (arrow). D) The cells adhere to the biomatrix scaffold. E, F) Connection complexes between cells, including: cell clusters, adhesions, and gap junctions (arrows).

Figure 11 is an image of the decellularization process in rat liver and the biomatrix scaffold used in the bioreactor experiment.

Figure 12 depicts albumin and urea secreted by hepatocytes when cultured in a serum-free, hormone-determining medium (BIO-LIV-HDM) designed for bioreactors or commercially available hepatocyte maintenance medium (HMM).

Embodiments in accordance with the present invention will be described more fully below. However, the disclosed embodiments may be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and the scope of the invention will be fully conveyed by those skilled in the art. The terminology used in the description herein is for the purpose of description

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning meaning meaning It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the context of the present application and the related art, and should not be idealized or overly formal unless explicitly defined herein. Ways to explain. For example, descriptive symbols can be used to refer to biological materials (eg, tissue, organoidoid samples) having specific organ characteristics, such as "liver" to describe liver-derived tissue or liver-like organs. Although not explicitly defined below, these terms should be interpreted according to their usual meanings.

The terminology used in the description herein is for the purpose of description All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety.

Unless otherwise indicated, the practice of the present technology will employ conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA within the skill of the art. See, for example, Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., NY MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; US Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Tra Nsfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology.

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be employed in any combination. Furthermore, the present invention also contemplates that in some embodiments, the features or combinations of features set forth herein may be eliminated or omitted. For purposes of illustration, if the specification indicates that the composite comprises components A, B, and C, it is specifically intended that one or a combination of A, B, or C may be omitted and not claimed, either singly or in any combination.

All numerical names such as pH, temperature, time, concentration, and molecular weight (including range) are suitably changed by an approximation of 1.0 or 0.1 in increments of (+) or (-), or by +/- 15%, or 10%, or 5%, or 2% change. It should be understood that although not always explicitly stated, all numerical names are preceded by the term "about." Although not always explicitly stated, it should be understood that the reagents described herein are merely exemplary and equivalents are known in the art.

I. Definition

The singular forms "a", "an", and "the"

When referring to measurable values such as quantity or concentration (eg, percentage of collagen in total protein in a biomatrix scaffold), etc., the term "about" means 20%, 10%, 5%, 1% of the specified amount. , 0.5% or even 0.1% change.

The terms "acceptable," "effective," or "sufficient" when used to describe a selection of any of the compositions, ranges, dosage forms, and the like disclosed herein are intended to be suitable for the purpose of disclosure.

Also, as used herein, &quot;and/or&quot; refers to and includes any and all possible combinations of one or more of the associated listed items, such as a combination that is absent when interpreted as an alternative ("or").

The term "bioengineering" is used to describe an artificial organ or tissue engineered to have similar or identical biological properties as the original organ or tissue in which it was found. In some aspects, this may require engineering using a particular device; in other respects, this may require the use of various biological factors.

The term "biological matrix scaffold" refers to an isolated tissue extract rich in extracellular matrices and, as described herein, optionally a plurality or a majority, retaining collagen and/or collagen binding factors that are native to the living tissue. Some of them. In some embodiments, the biomatrix scaffold comprises: collagen, fibronectin, laminin, nucleic acid/myelin, integrin, elastin, proteoglycan, glycosaminoglycan (sulfated and non-sulfated) - comprising a hyaluronic acid) composition, or consisting of the predominantly preceding materials, or a combination of the foregoing, all being part of a biomatrix scaffold (eg, included in the term biomatrix scaffold).

In some embodiments, the biomatrix scaffold lacks a detectable amount of specific collagen, fibronectin, laminin, nucleic acid/intron, elastin, proteoglycan, glycosaminoglycan, and/or the foregoing The combination in the middle. In some embodiments, substantially all of the collagen and the collagen binding factor are retained, and in other embodiments, the biomatrix scaffold includes all collagen known in the tissue.

The biomatrix scaffold can comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99. 5% or 100% of collagen, collagen-related matrix composition and/or matrix-bound growth factor, hormone and/or cytokine found in native tissue. In some embodiments, the biomatrix scaffold comprises at least 95% collagen and a majority of collagen-associated matrix and matrix-bound growth factors, biological tissue hormones and/or cytokines. The collagen described herein can be a neonatal (newly formed), non-crosslinked collagen. As disclosed herein, collagen is a triple helix composed of three amino acid chains such as hair (a region dominated by three amino acids: [glycine-valine-X] (where X can be various) Any of the different amino acids) forms a fibrillar domain of collagen and has a molecular end which has a unique amino acid chemistry of a different collagen type, thus resulting in a globular domain. Collagen molecules may be secreted Self-assembly to form collagen fibers (aggregated collagen molecules); self-assembly with non-collagen matrix composition and signaling molecules (cytokines, growth factors); then cross-linking to form the extracellular matrix. Exemplary collagen is briefly described below Protein and its extraction method.

Certain collagen molecules have amino acid chemistry that is unique to each of the 29 known collagen types. Collagen is secreted from cells and then one or both ends of the molecule are removed by specific peptidases, followed by polymerization of multiple collagen molecules to form collagen fibers or fibrils. The exception is "net collagen" which retains the spherical domain and then polymerizes the ends to the end to form a network of collagen molecules (ie, in a chicken-like structure). After polymerization into a fiber or mesh, the collagen is cross-linked by the action of lysyl oxidase, an extracellular copper-dependent enzyme, which produces collagen. A covalent bond between protein molecules (and between elastin molecules) to produce a crosslinked form of a composition that constitutes a very stable collagen aggregate and binds to collagen. The amount of collagen molecules per fibril in the fibrillar collagen and the pattern of attachment in the mesh collagen are determined by the exact amino acid chemistry of that particular collagen type.

Tissue extraction can be accomplished using a buffer at a neutral pH and a salt concentration of 1 M or greater, thereby isolating non-crosslinked and crosslinked collagen; maintaining the salt required for non-crosslinked collagen to dissolve The exact concentration depends on the type of collagen. For example, the rich type I and type III collagen in the skin requires about 1M salt; in contrast, the collagen in the amnion (such as type V collagen) requires 3.5 to 4.5M salt); non-crosslinking and crossing in the liver The combined collagen requires at least 3.4M salt. Therefore, most methods of preparing extracts rich in extracellular matrices do not retain all collagen, especially uncrosslinked collagen. In addition, some methods utilize the following methods: a) an enzyme that degrades the matrix composition; and/or b) a low salt or salt-free buffer that causes any factor of uncrosslinked collagen and any factor to which it binds (eg, , distilled water). Thus, there are various forms of extracts for matrix scaffolds that contain cross-linked collagen and any factors that bind to those cross-linked collagen but have no or minimal amounts of uncrosslinked collagen and its associated factors. Although extracts of cross-linked collagen, either primarily or separately, also have adhesion molecules and signaling molecules, these extracts do not readily interact with cells due to their orientation and position in the cross-linked matrix. In contrast, uncrosslinked collagen assembles itself with other matrix components and signaling molecules, which can be used to interact with cells. In some embodiments, The preparation of the biomatrix scaffolds disclosed herein avoids low ionic strength buffers, thereby maintaining both cross-linked and uncrosslinked collagen.

In some embodiments, the biomatrix scaffolds disclosed herein comprise substantially all collagen comprising neonatal (newly formed) collagen, aggregated collagen molecules prior to cross-linking, and cross-linked collagen. In addition, the biomatrix scaffold can optionally comprise other matrix components plus signal molecules composed of a matrix that binds to or binds to these collagens. In some embodiments, the ratio of collagen in the biomatrix scaffold is similar or identical to the ratio of collagen derived from the biomatrix scaffold in tissue. Non-limiting examples of suitable percentages of nascent collagen that mimic initial tissue include, but are not limited to, at least about or about 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25% , 30%, 35%, 40%, 45% or 50%.

As described herein, "most collagen-associated matrix composition and matrix-bound growth factors, biological tissue cytokines and/or hormones" means about 50%, 60%, 70%, 75%, 80%, 85% retention. , 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% collagen-related matrix composition and matrix-bound growth factors, hormones found in native (eg, untreated) biological tissues and/or Or cytokines. The terms "powder" or "powder" are used interchangeably herein to describe a biomatrix scaffold that has been ground into a powder. The term "three-dimensional biomatrix scaffold" refers to a decellularized scaffold that retains its native three-dimensional structure. Such a three-dimensional scaffold can be either the entire scaffold or its frozen section.

The term "buffer" and / or "rinsing medium" as used herein. Refers to the reagent used to prepare the biomatrix scaffold.

The term "cell" as used herein refers to a eukaryotic cell. In some embodiments, the cell is of animal origin and can be a stem cell or a somatic cell. The term "cell population" refers to a group of one or more cells having the same or different starting identical or different cell types. In some embodiments, such a population of cells can be derived from a cell line; in some embodiments, such a population of cells can be derived from a sample of an organ or tissue.

The term "precursor cell" or "precursor" as used herein is broadly defined to include stem cells and their progeny; in certain aspects of the invention, the term "stem cell/precursor cell" will be used herein in connection with "predecessor", Precursor cells or "precursors" are used interchangeably. "Progeny" can include pluripotent stem cells or unipotent cells that can differentiate into a particular lineage that results in one or more mature cell types. Non-limiting examples of precursor cells include, but are not limited to, embryonic stem (ES) cells, induced multifunctional (iPS) cells, germ layer stem cells, definitive stem cells, perinatal stem cells, amniotic fluid stem cells, mesenchymal stem cells, transient fast growing cells Or stereotyped precursor cells of any tissue type. When used in conjunction with a descriptor such as "single energy," "pluripotency," and/or "stationation," is the ability of a cell to differentiate into one or more adult fates - for example, embryonic stem cells are pluripotent and can cause All adult fate of the germ layer (ectoderm, mesoderm, endoderm); determines that stem cells are pluripotent and can produce two or more adult fates; whereas stellate precursors or endothelial precursor cells are single energy Examples of precursor cells are therefore stereotyped in a particular cell lineage.

As used herein, a "parenchymal cell" is an epithelial cell that is usually an organ. In the liver, they can contain hepatocytes and biliary cells; In the pancreas, they may include acinar cells and islets; in the liver and pancreas, as well as other endoderm organs (eg, thyroid, intestine, lung), they may be derived from endoderm stem cells. Their phenotypic traits are lineage traits associated with the earliest collection of traits found in cells of zone 1 of the liver acinus, transferred to the central acinar zone (liver zone 2) and terminated in the central zone (liver zone 3 Terminally differentiated cells. Furthermore, it has recently been discovered that a population of diploid parenchymal cells linked to the endothelium forming the central vein has unipotent precursor cell properties. Non-limiting exemplary parenchymal cells are bile trunk cells, hepatic stem cells, hepatocytes, committed hepatocytes and biliary precursor cells, axin2+ precursor cells (eg, axin2+ liver precursor cells), mature parenchymal cells (hepatocytes, biliary cells, and A unipotent derivative of pluripotency or a subset of its stem cells). Other non-limiting examples include, in particular, hepatic pancreatic ducts, pancreatic stem cells, pancreatic progenitor precursor cells from the hepatic pancreatic duct and pancreatic duct gland, islets, and acinar cells. These exemplary embodiments are applicable to, for example, the liver and the pancreas, respectively.

As used herein, "non-parenchymal cells" are derived from mesodermal and ectodermal stem cells and their lineage progeny, including mature mesodermal and ectodermal cell types. Progeny derived from mesoderm stem cells include: precursor cells of hemangioblasts, endothelial cells and stellate cells, mature endothelial cells, mature stellate cells, stromal cells, smooth muscle cells, pericytes, hematopoietic stem cells, and precursor cells and their progeny, It includes Kufu's cells, natural killer cells (Pit cells), bone marrow cells, lymphocytes and various other hematopoietic cells. Ectodermal stem cell progeny include neuronal precursors and mature neuronal cells.

"Epithelial cells" are known in the art to be from epithelial cells. As used herein, the term "interstitial cells" refers to those that are derived from mesoderm. Quality cells. There is an epithelial stroma partnership that forms the center of the organization's relationship, and it may be lineage-related; this is that epithelial stem cells work with mesenchymal stem cells, and their offspring mature in a coordinated manner. This relationship is sustained by a "cross-talk" of signals (paracrine signals) consisting of a soluble signal and an extracellular matrix that dynamically and synergistically functions to regulate epithelial cells and interstitial cells. The biological response of the cells. For example, hemangioblasts, a type of mesenchymal stem cell population, cooperate with hepatic stem cells. They produce endothelial cell precursors and their progeny that are combined with the hepatocyte lineage and, at the same time, produce astrocyte precursors and their progeny that are combined with the biliary cell lineage. The stellate cells and endothelial cell population undergo a maturation process that is corresponding and coordinated with the epithelial cells to which they bind. Thus, the phenotypic properties of these cells are lineage dependent and vary depending on whether the cells are in the early, middle or late stages of the lineage. This is roughly translated as whether the cell is from the liver acinar region 1 (early), region 2 (middle) or region 3 (late). Non-limiting exemplary non-parenchymal cells are hemangioblasts, mesenchymal stem cells, stellate cell precursors, stellate cells, pericytes, stromal cells, smooth muscle cells, neuronal cell precursors, neuronal cells, endothelial cell precursors. , endothelial cells, hematopoietic precursors and hematopoietic cells.

The term "biliary stem cells" (BTSC) refers to stem cells found throughout the biliary tract that include the gallbladder and have the ability to transform into liver and/or pancreatic stem cells and their progeny precursor cells. They are found in the extra-biliary bile ducts (PBGs) - licking the surface of the bile duct; and between the walls of the PBGs - within the bile duct wall. The offspring of PBG-associated BTSCs are located in the gallbladder and are located in the gallbladder The vesicle hair or its bottom is in the tickle corresponding to the intestinal crypt. There are multiple BTSC subpopulations that form a lineage of hepatic stem cells (HpSCs) found in PBGs that are transferred to the giant intrahepatic bile duct and that are connected to a catheter plate (fetal and neonatal tissue) and Converted to Herring's tube (pediatric and adult organizations). HpSCs produce hepatoblasts located adjacent to or near the Herring tube and transform the committed hepatocytes and biliary cell progenitors, which are matured into hepatocytes and biliary cells. In addition, the progeny of BTSCs produce pancreatic stem cells found throughout the biliary tract, but mainly in the PBGs of the hepatopancreatic duct, and these are in turn transformed into committed pancreatic precursor cells found in the pancreatic duct glands in the pancreas. . Biomarkers for all BTSC subpopulations include: endoderm transcription factors (SOX9, SOX17, FOXL1, HNF4-α, ONECUT2, PDX1), pluripotency genes (eg, OCT4, SOX2, NANOG, SALL4, KLF4, KLF5, BMI) -1); one or more CD44 isoforms (both CD44 and CD44v), hyaluronic acid receptor isoforms; CXCR4; ITGB1 (CD29), ITGA6 (CD49f), ITGB4, and cytokeratin 8 and 18 . The isoforms of CD44, such as CD44S, were found to be more expressed by both stem cells and mature cells, while multiple CD44 variant isoforms (CD44v) were predominantly present in the stem cell subpopulation. Currently, three stages of the BTSC subgroup have been identified: Phase 1 BTSCs express sodium iodide symporting transport protein (NIS), some CD44v isoforms found in stem cells, and CXCR4; they do not express LGR5 or EpCAM; Stage 2 BTSCs express specific isoforms of CD44 variants found in stem cells, less NIS but increase LGR5 expression but not EpCAM; Stage 3 BTSCs (only found in the gallbladder and throughout the biliary tract) Discovered BTSCs) express LGR5 and EpCAM, and are more mature A mixture of CD44v and CD44s found in the cells. Stage 3 BTSCs are precursors to hepatic stem cell precursor cells and pancreatic stem cells.

The term "hepatic stem cells" (HpSCs) refers to stem cells found in a Herring tube that connects the ends of the PBGs of the large intrahepatic bile duct of the biliary tract to the liver plate. HpSCs retain the ability to self-replicate and are versatile. Biomarkers for these cells include epithelial cell adhesion molecules (EpCAM; found in the cytoplasm and cell membrane), neural cell adhesion molecules (NCAM), and very low albumin levels (if any) that express SOX9, SOX17, CD29 (ITBG1), HNF4-alpha, ONECUT2, low to medium levels of one or more pluripotency genes (OCT4, SOX2, NANOG, KLF5, SALL4) and expression of cytokeratin 8, 18 and 19. They do not express PDX1 or alpha-fetoprotein (AFP) or P450-A7 or the secretin receptor (SR).

The term "hepatogenic cells" refers to dual-energy liver stem cells that produce hepatocytes and biliary cells. They have minimal self-replication ability under conditions that allow BTSCs and HpSCs to self-replicate. However, they will be broadly separated from the treatment of additional cytokines and growth factors, but division may include a degree of differentiation characterized by overlapping biologys profiles with HpSCs but different from BTSCs. It includes HNF4-α, CPS1, APOB, EpCAM (mainly in cell membrane), P450-A7, cytokeratin 7, 19, 8 and 18, secretin receptor, albumin, high level of AFP, intercellular adhesion molecule (ICAM-1), but not NCAM, DLK1 and the lowest (if any) pluripotency gene.

As used herein, the term "typed precursor cell" refers to a single-energy precursor cell that produces a single cell type, eg, a certain type of hepatocyte precursor cell. (Normally recognized by albumin, AFP, glycogen, ICAM-1 expression, various enzymes involved in glycogen synthesis) and hepatocytes are produced. Stereotypes of biliary (or biliary) cells (generally recognized by EpCAM, cytokeratin 7 and 19, aquaporin, CFTR, production of membrane pumps associated with the production of bile (the synthesis of bile salts by hepatocytes)) Generate bile duct cells.

The descriptor "mature" used to describe a cell refers to a differentiated cell. For example, "mature hepatocytes" refers to the main parenchymal cells in the liver, which are diploid in the portal vein region, diploid and polyploid in the middle gland region, and mostly in the central surrounding region. Polyploid in the middle. Gene expression profiles may be regionally lineage dependent, including: region 1 genes (representatives are transferrin mRNA (without the ability to translate proteins), connexin 28 and enzymes involved in glycogen synthesis), region 2 genes ( Representative are tyrosine aminotransferases, which are capable of translation into protein transferrin mRNA and albumin with the highest expression levels; and region 3 genes (representative of late P450s such as P450-3A4 and a gene associated with apoptosis). Please refer to, for example, Turner et al; Human Liver Stem Cells and Liver Lineage Biology. Hepatology, 2011; 53: 1035-1045 (a more detailed list of genes expressed in a pattern associated with the hepatic acinar region), which is incorporated herein by reference. The final parenchymal cell layer in region 3 is diploid, axin2+ A monoenergetic liver precursor cell that is connected to the endothelium of the central vein.

The term "angioblasts" is used to describe the formation of pluripotent precursors of the endothelium, stellate cells, and pericytes associated with mesenchymal stem cells. These cells can express one or more biomarkers, such as CD117, VEGF receptor, Von Willebrand factor, CD133. Please refer to, for example: Geevarghese A. and Herman I., Transl Res. 2014; 163(4): 296-306 (discussing the overlap of biomarkers between meta-mass systems), which is incorporated herein by reference. Angioblasts can also produce mesenchymal stem cells (MSCs), which cause pericytes to be in the form of smooth muscle cells wrapped around the endothelium, and with their contractile forces help to move blood from region 1 to region 3, and then Enter the central vein. They produce many factors involved in angiogenesis, including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), endothelin, IGF II, epidermal growth factor (EGF), and acidic fibroblast growth factor (a-FGF). ) and neurotrophic proteins. See Geevarghese (2014), Figure 13.

The term "stellate cell precursor" refers to a unipotent precursor of a stellate cell; one of an interstitial partner for hepatocytes and an interstitial ligand for a precursor cell of a committed cholangiocarcinoma. Biomarkers for these cells include CD146 (also known as Mel-CAM), alpha-smooth muscle actin and myomeric protein. It is known that stellate cell precursors are abundant paracrine signals required for the production of hepatic osteoblasts and committed precursor cells, including growth factors such as hepatocyte growth factor (HGF) and matrix-derived growth factor (SDGF) and early lineages. Stage matrix composition, such as laminin and type IV collagen.

The term "endothelial cell precursor" refers to a unipotent precursor of endothelial cells; other interstitial partners used to form hepatocytes and is also an interstitial partner for the characterization of hepatocyte precursor cells. Biomarkers for these cells include the VEGF receptor, Von Willebrand factor, CD133, and CD31 (also known as PECAM). These cells are known to produce paracrine signals, which also include growth factors (eg, VEGFs, angiopoietin) and matrix components (eg, type IV collagen). Protein, laminin and heparan sulfate proteoglycans).

The term "mature stellate cells" means a stromal cell partner for cholangiocarcinoma cells. The biomarkers of these cells include: as shown in the above figure, α-smooth muscle actin and myotropin, mature stellate cells, but not precursors, expressing significant levels of retinoids (vitamin A derivatives), colloids Fibrillary acidic protein (GFAP), type I and III collagen and other mature matrices and other markers of mature stellate cells.

The term "endothelial cells" is used to describe interstitial cell partners between hepatocytes. Their phenotypic traits are transformed from the formation of a trait of a complete basement membrane with hepatocytes in the vicinity of the portal vein triad to a permeability ("window") endothelium that forms a gap between the cell and the matrix with the central vein. Biomarkers include high levels of CD31 and VEGF-receptors.

The term "hematopoietic cells" (this is a British term; the US term for hematopoietic cells "hemopoietic") is a cell that is produced in the liver in the fetal and maternal stages, and then produced in the bone marrow. Terms include, but are not limited to, hematopoietic stem cells, lymphocytes, granulocytes, monocytes, macrophages, platelets, native killer cells (called Pit cells in the liver), and red blood cells.

The term "comprising", as used herein, is intended to mean that the compositions and methods include the listed elements, but do not exclude other elements. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is to be construed as including the listed materials or steps, and does not substantially affect the basic and novel embodiments. Feature. Reference In Re Herz, 537 F.2d 549,551-52,190 U.S.P.Q.461,463 (CCPA 1976) (emphasis in the Original); also refer to MPEP § 2111.03. Therefore, the term "consisting essentially of" as used herein shall not be construed as equivalent to "including". The term "consisting of" means excluding minor constituents other than the other ingredients and the substantial method steps of applying the compositions disclosed herein. Aspects defined by each of these transition terms are within the scope of the invention.

As used herein, the term "container" refers to a device that is specifically configured to contain cells and/or tissue. In some embodiments, such a container may be a bioreactor designed to hold a biomatrix scaffold. In further embodiments, the container can be configured to treat decellularization and/or recellularize the scaffold.

The term "culture" or "cell culture" refers to the maintenance of cells in an artificial, in vitro environment, in some embodiments, as adherent cells (eg, monolayer cultures) or as spheroids or organoids. Aggregate cultures maintain cells. The term "spheroid" refers to a floating aggregate of cells of all cell types (eg, aggregates from a cell line); "organids" are floating aggregates of cells including a plurality of cell types. In some embodiments, this will be epithelial cells and their mesenchymal companion cells, typically endothelial cells and/or stromal cells. The cell can be a dry/progenitor cell of these classes of cells, or can be a mature cell. The term "cell culture system" as used herein refers to a culture condition in which a cell population can be cultured.

As used herein, "medium" refers to a nutrient solution used for the cultivation, growth or proliferation of cells. The medium may be characterized by functional properties such as, but not limited to, the ability to maintain cells in a particular state (eg, pluripotent state, resting state, etc.) to mature cells - in some instances, specifically, promoting The precursor cells differentiate into cells of a particular lineage. Non-limiting examples of media are Kubota media and liver hormone defining media, which are further defined below. In some embodiments, the medium can be an "inoculation medium" for presenting or introducing cells into a given environment. In other embodiments, the medium may be a "differentiation medium" for promoting cell differentiation. Such a medium may be composed of a "basal medium" or a mixture of nutrients, minerals, amino acids, sugars, and trace elements, and may be used to maintain cells in vitro.

More specifically, the "basal medium" is a buffer composed of an amino acid, a sugar, a lipid, a vitamin, a mineral, a salt, and various nutrients, which simulates a room around the cell. The chemical composition of the liquid. Such media may optionally be supplemented with serum to provide the necessary signaling molecules (hormones, growth factors) required to drive biological processes (eg, proliferation, differentiation). Although serum can be compared to autologously derived from the cell types used in culture, the most common serum is serum from routine slaughter of animals used for agriculture or food purposes, such as from cows, sheep, goats, horses, etc. serum. The serum-supplemented medium may optionally be referred to as serum supplementation medium (SSM).

Many commercially available basal media forms are available for epithelial stem/progenitor cells, but modifications must be made to maintain stem cell traits in the cells. Studies (Kubota et al, PNAS, 2000; 97(22): 12132-12137) have shown that in order to keep endoderm epithelial cells in an undifferentiated state, ie, stem cells, serum-free medium can be used; low oxygen content (1-2 %); no copper, no cytokines and growth factors; calcium content less than 0.5 mM; supplemental insulin and transferrin/iron, and purified free fatty acid mixture, pure The mixture of free fatty acids is complexed with related carrier molecules, such as albumin, and is optimized (but not strictly required) for lipoproteins, such as high density lipoproteins. Such an optimized medium for stem cells has been developed for endoderm stem cells, and is hereinafter defined as "Jumbotian medium". It enables endoderm stem cells to be expanded in a self-replicating manner for several months. (Kubota and Reid PNAS 2000; 97(22): 12132-12137) If the cells are cultured in a Kubota medium and a hyaluronic acid matrix or a hyaluronic acid hydrogel or a medium supplemented with hyaluronic acid, as a stem cell The stability of epithelial cells is optionally enhanced. Y. Wang, H. L. Yao, C. B. Cui et al. Hepatology. 2010; 52(4): 1443-54, US 8,802, 081, incorporated herein by reference.

The post-mature lineage stage of the precursor, such as hepatocytes and committed precursor cells, has limited ability to self-replicate, but has considerable expansion capacity; this condition is amplified by Kubota medium and cells supplemented with various growth factors. Factors such as HGF, EGF, and forms of FGF, IL-6, IL-11, and the like, and the use of a matrix underlayer comprising type III and/or type IV collagen and laminin. (See, for example, Kubota and Reid PNAS 2000; 97(22): 12132-12137; Turner et al; Journal of Biomedical Biomaterials. 2000; 82(1): pp. 156-168; Y. Wang, HLYao, CBCui et Al. Hepatology. 2010 Oct 52(4): 1443-54 is incorporated herein by reference.

As used herein, "differentiation" refers to the specific conditions that allow cells to mature into an adult cell type that produces an adult-specific gene product.

When referring to a specific molecular, biological or cellular material, the terms "equivalent" or "bioequivalent" are used interchangeably and mean those have A substance that has minimal homology but still retains the desired structure or function.

As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or transcribed mRNA which is subsequently translated into a peptide, polypeptide or protein. If the polynucleotide is derived from a genetic DNA, expression may include splicing of the mRNA in eukaryotic cells. The expression level of the gene can be determined by measuring the amount of mRNA or protein in the cell or tissue sample; in addition, the expression levels of the plurality of genes can be determined to establish an expression profile of the particular sample.

The term "extracellular matrix" or "ECM" as used herein, refers to a composite scaffold secreted by a cell, comprising various biologically active molecules, adjacent to one or more cell surfaces, and involving cells and tissues or organs including the same. Structural and / or functional support. The specific matrix composition and its concentration are related to specific tissue types, histological structures, organs, and other supercellular structures. Compositions of extracellular matrices associated with the present invention include, but are not limited to, collagen, collagen-related matrix composition, and growth factors.

Examples of collagen include any and all types of collagen such as, but not limited to, type I to XXIX collagen. The biomatrix scaffold can comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more in the native tissue. Collagen found in. In some embodiments, the collagen is crosslinked and/or uncrosslinked. The amount of collagen in the biomatrix scaffold can be determined by various methods known in the art and described herein, such as, but not limited to, determining the hydroxyproline content. An exemplary method of determining the presence or absence of cross-linking or non-cross-linking properties of collagen, for example, based on the observation of its solubility properties. Reference is made, for example, in DREyre, ^* M. Weis, and J. Wu. Advances in collagen cross-link analysis Methods, 2009; 45(1): 65-74 (analysis of cross-linking is described by standard methods in the field of collagen chemistry). For example, collagen can be determined to be cross-linked based on whether it is dissolved in a 1 M salt concentration or a buffer below 1 M salt concentration.

Exemplary collagen-related matrix components include, but are not limited to, adhesion molecules; adhesion proteins; L- and P-selectin; heparin-binding growth-related molecules (HB-GAM); platelet activin type I repeats (TSR); amyloid P(AP); laminin; nestin/entactins; fibronectin; elastin; vimentin; proteoglycans (PGs); chondroitin sulfate PGs (CS-PGs); dermatan sulfate- PG (DS-PGs); members of the small leucine-enriched proteoglycan (SLRP) family, such as biglycan and mancotin; heparin PGs (HP-PGs); heparan sulfate-GG ( HS-PG), such as proteoglycans, proteoglycans and phosphatidylinositol proteoglycans; and glycosaminoglycans (GAG) such as hyaluronic acid, heparan sulfate, chondroitin sulfate, keratin sulfate and heparin .

In some embodiments, the biomatrix scaffold is primarily or consists of collagen, fibronectin, laminin, nestin/nestin, elastin, proteoglycan, glycosaminoglycan (combined with various substrates) GAGs), growth factors, hormones, and cytokines (in any combination). The biomatrix scaffold can comprise at least about 50%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more of one or more in protists Collagen-associated matrix composition, hormones and/or cytokines found in tissues and/or may be found in native tissue One or more of these compositions are at least about 50%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more.

In some embodiments, the biomatrix scaffold comprises all or part of a collagen-related matrix composition, hormone and/or cytokine known in the tissue. In other embodiments, the biomatrix scaffold comprises, consists essentially of, or consists of one or more collagen-associated matrices found in native tissue, hormones and/or cytokines in native tissue to approximate The concentration consists of (for example, a concentration of about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 100%).

Exemplary matrix-bound signaling molecules include, but are not limited to, epidermal growth factor (EGFs), fibroblast growth factor (FGFs), hepatocyte growth factor (HGFs), insulin-like growth factors (IGFs), transforming growth factors (TGFs). ), nerve growth factors (NGFs), neurotrophic factors, interleukins, leukemia inhibitory factor (LIFs), vascular endothelial growth factor (VEGFs), platelet-derived growth factor (PDGFs), bone morphogenetic factors, stem cell factors (SCFs), Colony stimulating factors (CSFs), GM-CSFs, erythropoietin, thrombopoietin, heparin-binding growth factor, IGF binding protein, placental growth factor, and Wnt signaling.

Exemplary cytokines include, but are not limited to, interleukins, lymphokines, monokines, colony stimulating factors, chemokines, interferons, and tumor necrosis factor (TNF). The biomatrix scaffold can comprise at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 100% or more (any combination) One or more of the matrixes found in the native tissue are bound to growth factors and/or cytokines and/or may have at least about 30% concentration of the native tissue in the growth factors and/or cytokines (any combination) Concentrations of 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 100% or more are present.

The biomatrix scaffold comprises a plurality of or a portion of a matrix-bound growth factor at a physiological level or near a physiological level, a hormone and/or a cytokine known to be detected in the native tissue and/or in the tissue, in other embodiments, the organism The matrix scaffold comprises one or more matrix binding growth factors, hormones and/or cytokines at concentrations similar to or close to those found in the native tissue (eg, by no more than about 50%, 40% difference) 30%, 25%, 20%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%). The amount or concentration of growth factors or cytokines present in the biostromal scaffold can be determined by various methods known in the art and as described herein, such as, but not limited to, various antibody assays and growth factor assays.

As used herein, the term "functional" can be used to modify any molecular, biological or cellular material in order to achieve a particular, specific effect.

The term "gene" as used herein is intended to include broadly any nucleic acid sequence that is transcribed into an RNA molecule, whether or not the RNA encodes (eg, mRNA) or non-encodes (eg, ncRNA).

As used herein, the term "generating" and its equivalent (eg, production, production, etc.) may be combined with "produced" when referring to method steps for rendering the micro-organ or bio-engineered tissue of the present invention. And its equivalent Used interchangeably.

As used herein, "hormone-defining medium for the liver" or "HDM-L" includes typical factors for the differentiation of stem cells into mature cells; this medium usually includes a basal medium supplemented with a mixture of hormones, growth factors and various nutrients. And serum-free is used to amplify or differentiate specific cell types - for example, parenchymal cells. In some embodiments, it can be prepared by supplementing Kubota medium, which is formulated for stem cells, and with additional hormones and factors required for cell differentiation. Exemplary growth factors for such differentiation media are disclosed in Y. Wang, H. L. Yao, C. B. Cui, and the like. The entire disclosure of U.S. Patent Application Serial No. 4,404, filed on Jan. The disclosed aspect relates to a specific HDM-L designated as "BIO-LIV-HDM" in the experiment, which refers to stem cells and precursors designed to distinguish between parenchymal and non-real mass spectrometry and/or epithelial and inter-mass mass spectrometry. Cells to produce mature liver tissue. In addition, the BIO-LIV-HDM-L further complements the growth factors and hormones required for various non-parenchymal cell types, including mesenchymal cells (stellate cells, pericytes, endothelial cells), their precursors, and mature forms, Both neuronal cells, their precursors and mature forms, as well as hematopoietic cells, their precursors and mature forms.

The term "hyaluronan" or "hyaluronic acid" as used herein refers to a polymer of uronic acid and amino sugar [1-3] which is passed by β 1-4, β 1- The 3-bond and its salt-linked glucosamine and hyaluronic acid disaccharide units are composed. Thus, the term hyaluronic acid refers to hyaluronic acid both native and synthetic.

As used herein, "hydrogel" is intended to mean a polymer chain. In a three-dimensional network, the polymer chain retains a majority of the aqueous medium in the three-dimensional network without being dissolved in the aqueous medium.

The term "isolated" as used herein, refers to a molecule or organism or cellular material that is substantially free of other materials.

As used herein, "Jumbotian's medium" refers to a serum-free, hormone-defining medium designed for endoderm stem cells and enables it to replicate in a self-replicating manner (for example, on a hyaluronic acid substrate or in a hyaluronic acid-containing medium). Buffered in the buffer). Kubota can refer to any basal medium comprising copper free, low calcium (<0.5 mM), insulin, transferrin/iron, purified free fatty acid mixture in combination with purified albumin, and optionally high density lipoprotein . Kubota medium or its equivalent is serum free and contains only a mixture of purified and defined hormones, growth factors and nutrients. In certain embodiments, the medium comprises copper free, low calcium (<0.5 mM) supplemented with insulin (5 μg/mL), transferrin/iron (5 μg/mL), high density lipoprotein (10 μg) /mL), serum-free basal medium (eg, selenium (10 ^-10 M), zinc (10 ¹² M), nicotinamide (5 μg/mL), and a mixture of purified free fatty acids bound to the purified albumin form (eg , RPMI1640 or DME/F12). Non-limiting exemplary methods for preparing such media have been disclosed elsewhere, for example, Kubota H, Reid LM, Proceedings of the National Academy of Sciences (USA) 2000; 97: 12132-12137, Y. Wang, HLYao, CBCui Et al. Hepatology. 2010; 52(4): 1443-54, Turner et al; Journal of Biomedical Biomaterials. 2000; 82(1): pp. 156-168; Y. Wang, HLYao, CBCui et al. Hepatology. 2010 Oct 52 (4): 1443-54, the disclosure of which is incorporated herein by reference. Kubota media can be designed for specific cell types by providing specific factors and supplements to allow for specific amplification under serum-free conditions. For example, Kubota's medium for hepatoblasts is designed for hepatocytes and their progeny, committed precursor cells, and promotes their expansion under serum-free conditions. Extensions may occur with self-replication, but usually replicate to a minimum extent (if it occurs). This medium is particularly effective if the cells are on a matrix of type IV collagen and laminin.

The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, ie, deoxyribonucleotides or ribonucleotides or the like. . A polynucleotide can have any three-dimensional structure and can perform any function known or unknown. The following are non-limiting examples of polynucleotides: genes or gene fragments (eg, probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.

Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure modification can be imparted before or after assembly of the polynucleotide. The nucleotide sequence can be interrupted by non-nucleotide components. After polymerization, the polynucleotide can be further modified, for example, by binding to a labeling component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any aspect of this technique that becomes a polynucleotide is to include both double-stranded forms and two known or predicted double-stranded forms. Each of the complementary single-chain forms.

As used herein, the term "organ" refers to the structure of a particular portion of a single organism in which certain functions or functions of a single organism are performed locally and are morphologically separate. Non-limiting examples of organs include skin, blood vessels, cornea, thymus, kidney, heart, liver, umbilical cord, intestine, nerves, lungs, placenta, pancreas, thyroid, and brain. Organs can be used as a source of tissue, for example, fetal, neonatal, young, child or adult organs can be used to derive a population of cells of interest in the disclosed applications.

The terms "protein", "peptide" and "polypeptide" are used interchangeably and, in their broadest sense, refer to a compound of two or more subunits of an amino acid, an amino acid analog or a peptoid. . This subunit can be linked by a peptide bond. In another aspect, the subunit can be joined by other bonds (eg, esters, ethers, etc.). The protein or peptide must comprise at least two amino acids and is not limited to the maximum number of amino acids which may comprise the sequence of the protein or peptide. As used herein, the term "amino acid" refers to both native and/or non-native or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptoids.

As used herein, the term "subject" is intended to mean any animal. In some embodiments, the subject may be a mammal; in further embodiments, the subject may be a human, mouse or rat.

The term "tissue" is used herein to mean a tissue of an alive or dead organism or any tissue derived or designed to mimic a living or dead organism. The tissue may be healthy, diseased, and/or have a genetic mutation. The term "native tissue" or "biological tissue" as used herein and its variations Allogeneic means that the biological tissue exists in its native state; or exists in a state in which it is unmodified from the state derived from the organism. "Microorgans" are a part of the "bioengineering organization" that simulates "native tissue."

The biological tissue can include any single tissue (eg, a collection of cells that can be interconnected) or a group of tissues that make up an organ or portion or region of the organism. The tissue may comprise a homogeneous cellular material, or it may be a composite structure, for example, found in the area of the body including the chest, for example, may include lung tissue, bone tissue, and/or muscle tissue. Exemplary tissues include, but are not limited to, those derived from the liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary tract, duodenum, abdominal aorta, axillary vein, heart, and intestine, and include any of them combination.

As used herein, "treating" or "treatment" of a subject's condition means (1) preventing symptoms or diseases from occurring in a subject susceptible or not showing symptoms; (2) inhibition The disease either prevents its development; or (3) improves or causes the disease or symptoms of the disease to subside. As understood in the art, "treatment" is the route of obtaining beneficial or desired results, including clinical outcomes. For the purposes of the present technology, beneficial or desired results may include one or more, but not limited to, alleviating or ameliorating one or more symptoms, reducing the extent of a condition, including a disease, and stabilizing the condition (including disease) (ie, , non-deteriorating); delaying or slowing the state of the condition (including the disease), the course of the disease; improving or ameliorating the state of the condition (including the disease), status and remission (whether part or all), whether detectable or undetectable.

II. Abbreviation

Part of the disclosure of the present invention is to use abbreviations to indicate certain terms. Abbreviations for cell populations In this context, the species can be represented by lower case letters: r for rats; m for mice; H for humans. If the acronym of a molecule is printed in italics, it refers to the gene; if it is printed in a normal font, it refers to the protein encoded by the gene.

The following is a non-limiting list of abbreviations used herein: ACOX, acyl-CoA oxidase; APOL6, apolipoprotein L6; AFP, alpha-fetoprotein, a characteristic gene expressed by hepatoblasts; ASMA, alpha-smooth muscle Actin; ALB, albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ccK18, cleaved caspase K18, an indicator of cell necrosis when secreted; C/ EBP, CCAAT/enhancer binding protein alpha; CD, co-determinant; CD31, platelet endothelial cell antigen (or PECAM), surface markers of endothelial cells; CD34, hematopoietic stem/precursor cell antigen; CD45, in most hematopoietic cells Common leukocyte antigens found in subpopulations; CD133, prostaglandin, surface markers found on endothelial and parenchymal cell precursors; CSF, colony-stimulating factor; CYP, cytochrome P450 monooxygenase, which catalyzes many Related reactions to drug metabolism and/or cholesterol synthesis, steroids and lipids; in the early lineage stages of constitutive cells (eg CYP 1A1, CYP2C8, CYP3A4) (CYP3A7 and possibly CYP1B1) and other late lineage stages Form; CK, cytokeratin; CK7, cytokeratin associated with bile cells; CK8 and 18, cytokeratin associated with all epithelial cells; EGF, epidermal growth factor; EpCAM, epithelial cell adhesion molecule; FBS, fetal Bovine serum; FGF, fibroblast growth factor; bFGF, alkali Fibroblast growth factor; GAGs, glycosaminoglycans, dimers (uronic acid and amino sugar) polymers of carbohydrate chains, most of which have specific sulfation patterns and are involved in signal transduction Protein synergistically plays different roles; GATA, a zinc-binding DNA binding region) and a transcription factor acting on a DNA sequence, GATA; GATA-2, GATA binding protein 2, a regulator of hematopoietic gene expression; HBs, hepatoblasts ; HDL, high density lipoprotein; hGH, human growth hormone; HGF, hepatocyte growth factor; HpSCs, hepatic stem cells; HDM, hormone-defining medium; H&E, hematoxylin and eosin; HNF, hepatocyte nuclear factor; HNF1 α; Hepatocyte nuclear factor homologous 匣A; HNF1b, hepatocyte nuclear factor homolog 匣B, which was found to be involved in hepatic pancreas development; HPLC, high performance liquid chromatography; IGF, insulin-like growth factor Is homologous to insulin and acts as a mitogen or differentiation signaler based on its specific GAG; IGF-I, insulin-like growth factor I, known as mature hepatocytes Key regulators; IGF-II, insulin-like growth factor II, a major regulator of fetal liver cells; IL, interleukin; IL7-R, interleukin-7 receptor, essential for lymphocyte development; JAG1, Jagged1 Called CD339 (involving key genes in the gap signaling pathway in fate determination); K18, total cytokeratin 18, if it is released by cells, means cell death or necrosis; KM, Kubota medium; LGR5, leucine-rich Repeated G-protein coupled receptor 5, an important stem cell marker in the intestine, liver and pancreas; LDH, lactate dehydrogenase; LDLR, low density lipoprotein (LDL) receptor; LYVE-1, lymph Endothelial hyaluronic acid cell receptor; MST1, macrophage stimulation 1; MMP, matrix metal Protease (or peptidase); MMP2, matrix metalloproteinase-2, 72kDa type IV collagenase or gelatinase A (GELA); MMP9, matrix metalloproteinase 9, also known as 92kDa type IV collagenase or gelatinase B (GELB) Is a matrix metal, an enzyme belonging to the zinc-metalloproteinase family, which is involved in the degradation of extracellular matrix; MRP2, multidrug resistance-associated protein 2; NMR, nuclear magnetic resonance; PAR, protease activated receptor; PAS, iodine Acid Schiff stain; PDGF, platelet-derived growth factor; RAG1, recombinant activating gene 1; SEM, scanning electron microscopy; SCF, stem cell factor; SCTR, secretin receptor; SLC4A2, solute carrier family 4 (anion exchanger) , member 2; TGF, transforming growth factor; TEM, transmission electron microscopy; VEGF, vascular endothelial growth factor.

III. Mode for implementing the invention

Aspects of the present disclosure pertain to compositions and methods for producing bioengineered tissue and containers configured to generate the bioengineered tissue.

DETAILED DESCRIPTION OF THE INVENTION With regard to a method for producing bioengineered tissue, comprising: (a) introducing a cell suspension in a seeding medium into or onto a biomatrix scaffold, and (b) replacing the cell with a differentiation medium after the initial culture period The inoculation medium. In some embodiments, the method is performed in a vessel dedicated to performing such processing. Aspects of the present disclosure pertain to the container. In some embodiments, the container is configured with a flow path dedicated to simulating cellular vascular support. In other embodiments, this can be accomplished by using a three-dimensional biomatrix scaffold comprising matrix residues of the vascular tree.

In some embodiments, the vaccination occurs at multiple intervals and then With rest periods; these intervals and rest periods may vary from about 1 minute to about 15 minutes, such as about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes and/or 15 minutes. The number of cells introduced and their concentrations can also vary. For example, in some embodiments, a stent can be introduced at a given interval of between about 10 and 12 million cells per wet weight. In some embodiments, for a given number of intervals (one, two, three, four or more intervals), the rate of introduction may be 15 milliliters per minute, then after a given number of intervals, Reduce to a rate of, for example, 1.3 ml/min.

In some embodiments, the cells and seeding medium can be pre-incubated prior to introduction, for example, at 4 °C for 4 to 6 hours.

In some embodiments, the biomatrix scaffold can be derived from a particular organism that can be the same as or different from the organism from which the progenitor cells are derived.

In some embodiments, the biomatrix scaffold is a composition that decellularizes tissue by filling a biological tissue sample and rinsing medium with a plurality of buffers to merely or predominantly retain an extracellular matrix that produces a matrix scaffold from the tissue, and Prepared from biological tissue and is the underlying structure that maintains the histology of the tissue. In another embodiment, a complete biomatrix scaffold can be obtained from a commercial source.

The medium for acceptable, bioengineered tissue can be selected based on the desired trait of the tissue, for example, can be stimulated to differentiate and/or grow in a population of progenitor cells entering a particular organ or tissue type of cells. Cultures are selected in the presence of certain factors, as described, for example, in Y. Wang, H. L. Yao, C. B. Cui et al. Hepatology. 2010 Oct 52(4): 1443-54, the entire contents of which is incorporated herein by reference. In addition, different media may be relevant at different stages of the process of producing bioengineered tissue - such as inoculation media or differentiation media.

The invention is disclosed in U.S. Patent Application Serial No. U.S. Patent Application Serial No. Serial No. No. No. No. No. No. No. No. No.. In certain embodiments, the medium is a medium that promotes cell differentiation.

In some embodiments, the medium further comprises one or more cell growth or differentiation factors, such as those previously described.

In some embodiments, the seeding medium comprises one or more of the following: calcium at a concentration of about 0.3 mM to 0.5 mM, trace elements (such as selenium and zinc, but not copper), purified free fatty acids (eg, palmitic acid, palmitoleic acid) a mixture of stearic acid, oleic acid, linoleic acid, linolenic acid, one or more lipid binding proteins (eg HDL), insulin and transferrin/iron. In some embodiments, the inoculating medium comprises serum, optionally for deactivating an enzyme for preparing a cell suspension. A non-limiting example of such a serum is fetal bovine serum (FBS). In some embodiments, serum is added thereto, replaced with serum-free medium (eg, serum-free HDM), typically within about 6 hours to 24 hours and/or replaced as soon as possible.

In some embodiments, the differentiation medium comprises one or more of the following: calcium, trace elements, ethanolamine, glutathione, at a concentration of at least about 0.5 mM, Ascorbic acid, minerals, amino acids and sodium pyruvate, a mixture of purified free fatty acids, one or more lipid binding proteins (eg HDL), one or more sugars, one or more glucocorticoids, insulin, transferrin/iron One or more hormones and/or growth factors - such as but not limited to those used to promote and/or maintain parenchymal cells, including: prolactin, growth hormone, glucagon, and thyroid hormones (eg, triiodothyronine or T3), epidermal growth factor (EGFs), hepatocyte growth factor (HGFs), fibroblast growth factor (FGFs), insulin-like growth factors (IGFs), bone morphogenetic proteins, Wnt ligands, and cyclic adenosine monophosphate And/or for promoting and/or maintaining non-parenchymal cells, including: angiopoietin, vascular endothelial growth factor (V EGFs), nerve growth factor, stem cell factor, leukemia inhibitory factor (LIF), colony stimulating factor ( CSFs), thrombopoietin, platelet-derived growth factor (PDGFs), erythropoietin, insulin-like growth factors (IGFs), fibroblast growth factor (FGFs), and epidermal growth Child (EGFs).

In some embodiments, the suspension of cells can be derived from a particular organism, which can be the same organism as the organism from which the biological matrix scaffold was derived. Stem or precursor cells are available from commercially available sources including, but not limited to, direct commercial retailers or repositories, such as the American Type Culture Collection (ATCC, http://www.atcc.org/). Alternatively, methods of producing and/or isolating stem or precursor cells from a sample disclosed in the art. Exemplary methods include those disclosed in U.S. Application Serial No. 12/926, the entire disclosure of which is incorporated herein by reference. Non-limiting example sources of cells include: liver, biliary tract, gallbladder, hepatopancreatic duct, pancreas, Duodenum, bone marrow, and endothelium (eg, liver or biliary stem cells from the biliary or gallbladder, bone marrow stem cells, and endothelial stem cells). Other examples include embryonic stem (ES) cells or induced pluripotent stem (iPS) cells from any source.

In some embodiments, the population of suspended cells can be a homogeneous population of cells - including only cells of the same type or heterogeneous populations of cells - including different types of cells. The number and concentration of cells in the population of suspension cells of the cultured cells can be determined based on the suspension cells, culture medium, culture size, desired organ/tissue characteristics, and other relevant factors. In some embodiments, the number of cells in the precursor cell population is determined by the growth rate and differentiation conditions of the dry/precursor cells. In some embodiments, the number of cells in the dry/precursor cell population is determined by the growth factors and other components present in the culture medium.

In some embodiments, the suspension of cells comprises parenchymal cells (eg, BTSCs, HpSCs, hepatoblasts, pancreatic stem cells, liver or pancreatic progenitor cells, hepatocytes, biliary cells, islets, acinar cells) and non-essential cell. Non-parenchymal cells include subpopulations of mesenchymal cells (eg, hemangioblasts or precursors of stellate or endothelial cells, mature stellate or mature endothelial cells), neuronal precursors and mature neuronal cells, and pre-hematopoietic cells. Body and mature hematopoietic cells (eg, precursors of lymphocytes, bone marrow cells, native killer cells, platelets, red blood cells, or mature counterparts thereof). In some embodiments, the ratio of these cells is about 10%/90%, 20%/80%, 30%/70%, 40%/60%, 50%/50%, 60%/40%, 70%. /30%, 80%/20%, 10%/90%. In some embodiments, the suspension of cells can include at least about 50% of precursors and/or stem cells. In some embodiments, the cell suspension does not include terminally differentiated hepatocytes.

In some embodiments, the gene or protein expression of the culture can be monitored for a time sufficient to produce bioengineered tissue. In certain embodiments, the gene or protein expression profile of a population of precursor cells cultured at a particular time point can be compared to a gene or protein expression profile of a population of cells selected from: (i) earlier or later time A population of precursor cells cultured, (ii) a population of control samples of precursor cells, (iii) a population of differentiated cells derived from organs or tissues. Similarly, the histology of the tissue can be compared to the early or late stages of development of the desired target tissue.

Without being bound by theory, it is envisioned that the gene or protein expression profile and/or histology of the cultured cells will be converted to resemble an organ or tissue or a less differentiated precursor within a time sufficient to produce bioengineered tissue. Gene or protein expression profiles and/or histology of differentiated cell populations.

The disclosed aspects relate to a three-dimensional biomatrix scaffold comprising a matrix residue of a vascular tree of an organ, wherein the scaffold is derived from the organ. In some embodiments, the scaffold further comprises native collagen found in an organ from which the scaffold is derived.

Another aspect of the present disclosure pertains to bioengineered tissues and/or micro-organs produced using the compositions and methods disclosed herein. In some embodiments, the resulting tissue demonstrates a mature lineage-dependent or mature region-dependent phenotypic characteristic of the native liver, such as, but not limited to, (a) a periportal region, (b) a parenchymal cell and a mesenchymal cell. The area of the sinus plate. Phenotypic traits may further include periportal traits associated with diploid cells, mature parenchyma and interstitial cell traits found in the middle acinar region of the native liver, parenchyma of central peripheral regions, and traits of mesenchymal cells. Bioengineering Tissues and/or micro-organs may also include (i) polyploid hepatocytes associated with endothelial cells and/or (ii) twice the endothelium attached to central veins and/or biliary cells associated with stellate cells Body liver cells. If the bioengineered tissue and/or micro-organ is designed for the pancreas, it can also include acinar and islet cells.

In some embodiments, the periportal region of the bioengineered tissue and/or micro-organ is rich in traits including stem/progressor cell niches of hepatic stem cells, hepatic cells, and committed precursor cells. In some embodiments, the parenchymal cells of the bioengineered tissue and/or micro-organ further comprise young (diploid) hepatocytes and biliary cells. In some embodiments, the mesenchymal cells of the micro-organs of the bioengineered tissue and/or the area surrounding the portal vein further comprise stellate cells, pericytes, smooth muscle cells, and precursors of endothelial cells. In some embodiments, the central acinar region of the bioengineered tissue and/or micro-organ is a trait that is enriched in mature parenchymal cells including mature hepatocytes and biliary cells. In some embodiments, the parenchymal cells of the bioengineered tissue and/or micro-organ also include hepatocytes and biliary cells. In some embodiments, the mesenchymal cells of the micro-organs of the bioengineered tissue and/or the area surrounding the portal vein further include stellate cells, pericytes, smooth muscle cells, neuronal cells, and endothelial cells. In some embodiments, the central surrounding area of the bioengineered tissue and/or micro-organ is enriched with mature parenchymal cells, traits of hepatocytes expressing late genes such as late P450 (eg, P450-3A), some of which are polyploid, Some are those who experience apoptosis. In some embodiments, the mesenchymal cells of the centrally surrounding region of the bioengineered tissue and/or micro-organ further comprise a transpermeable endothelium.

In some embodiments, the bioengineered tissue disclosed herein and / Or three-dimensional micro-organs can be used in vivo or in vitro. Non-limiting examples of potential uses include: research purposes for studying tissue morphogenesis, cell migration, clonal lineage, cell fate potential, cross-species development timing, and cell type-specific genomic expression; use of organ-like organs as specific organs, cell replacement therapy Or high-throughput drug screening models for other types of organ-specific treatments; and transplantation.

Aspects of the present disclosure also provide kits comprising suitable containers and/or media for the production of bioengineered tissue or micro-organs. In other embodiments, the kit may further include instructions on how to generate bioengineered tissue or micro-organs.

IV. Examples

The following examples are non-limiting and illustrative and may be used to effect the invention in various circumstances. In addition, all references disclosed below are hereby incorporated by reference in their entirety.

The reagents and supplies disclosed for research are available from: Abcam, Cambridge, MA; ACD Labs, Toronto, CA; Acris Antibodies, Inc., San Diego, CA; Advanced Bioscience Resources Inc. (ABR), Rockville , MD; Agilent Technologies, Santa Clara, CA; Alpco Diagnostics, Salem, NH; BD Pharmingen, San Jose, CA; Becton Dickenson, Franklin Lakes, NJ; Bethyl Laboratories, Montgomery, TX; BioAssay Systems, Hayward, CA; Isotope Laboratories, Tewksbury, MA; Carl Zeiss Microscopy, Thornwood, NY; Carolina Liquid Chemistries, Corp., Winston-Salem, NC; Charles River Laboratories International, Inc., Wilmington, MA; Chenomx, Alberta, Canada; Cole-Parmer, Court; Vernon Hills, IL; DiaPharma, West Chester Township, OH; Fisher Scientific, Pittsburgh, PA; Gatan, Inc., Pleasanton, CA; Illumina, San Diego, CA; Ingenuity, Redwood City, CA; Life Technologies Corp., Grand Island, NY; LifeSpan Biosciences, Inc., Seattle, WA; Molecular Devices, Sunnyvale, CA; Olympus Scientific Solutions Americas Corp., Waltham, MA; Polysciences, Inc., Warrington, PA; Research Triangle Labs (TRL), Research Triangle Park, NC; R&D Systems, Minneapolis, MN; RayBiotech, Norcross, GA; Santa Cruz Biotechnology, Inc., Dallas, TX; Sigma Aldrich , St. Louis, MO; Tousimis Research Corp., Rockville, MD; Qiagen, Germantown, MD; Umetrics, Umea, Sweden.

Example 1 - Human liver cell collection and processing

Human fetal liver is obtained via selective termination of pregnancy and is provided by an officially recognized institution, ABR. The tissue used in the experiment was a fetus from 17-19 weeks. The study was reviewed and approved by the Review Board (IRB) of the Human Research Institute at the University of North Carolina at Chapel Hill. A method of preparing a human fetal liver cell suspension is described in a prior publication. Briefly, the liver was first mechanically homogenized and then enzymatically dispersed to a type IV collagenase supplemented with 0.1% bovine serum albumin (BSA), 1 nM selenium, 300 U/ml, and 0.3 mg/ml. Oxygen ribonuclease and antibiotics in a cell suspension of RPMI-1640. Digestion was completed by stirring at 32 ° C for 30 to 60 minutes. Most groups Weaving requires two rounds of digestion and then centrifugation at 1100 rpm at 4 °C. The cell pellets were combined and resuspended in cell wash (RPMI-1640, which contained 0.1% BSA, 1 nM selenium, and antibiotics). The cell suspension was centrifuged at 300 rpm for 5 minutes at 4 ° C to remove red blood cells. The cell pellet was resuspended in the cell wash and filtered through a 70 μm nylon cell strainer (Becton Dickenson). An aliquot of 1 x 106 cells was isolated and processed for RNA and used as a control group for assays using qRT-PCR (t = 0).

Adult human tissue (n=3) obtained from the Triangle Research Laboratory (TRL) was used as a rapidly frozen tissue and used as a control group for mRNA expression via RNA sequencing. RNA was processed using Qiagen RNeasy Mini Kit (Qiagen) and according to the manufacturer's instructions. The results for the three donors were averaged by comparisons between fetal liver/progenitor cells (t=0) and bioreactors (t=14 days). A suspension of freshly isolated adult hepatocytes is obtained from TRL with the aim of using a traditionally used hepatocyte culture medium with a hormone-defined, serum-free medium designed for liver differentiation in a bioreactor experiment (BIO-LIV-HDM) HDM) for comparison. Three 6-well sandwich sandwich plates were cultured under two different media conditions, one plate for each human donor and cultured for 7 days. Three portions of the culture under each condition were prepared from each donor.

Example 2 - Preparation and Analysis of Biological Tissue Stents

Decellularization of rat hepatocytes. Wistar rats (body weight 250-300 grams) were from Charles River Laboratories and housed in animal facilities treated by the UNC Laboratory Animal Management Department. They are arbitrarily fed until they are used experiment. All experimental work was performed through the approval of the UNC Institutional Animal Use and Care Committee guidelines and in accordance with the guidelines of the UNC Institutional Animal Use and Care Committee.

The procedure for decellularizing the liver to produce a biomatrix scaffold has been previously described. Wang Y., et al. (2011) Hepatology 53: 293-305; Gessner, R.C. et al. (2013) Biomaterials 34: 9341-9351. Male rats were anesthetized with Ketamine-Xylazine and then opened in the abdominal cavity. The portal vein is cannulated with a 20 gauge catheter to provide a fill portal for the vascular system of the liver and to traverse the vena cava and hepatic artery to provide a fill outlet. The liver is removed from the abdominal cavity and placed in a flow-through bioreactor. The liver was removed by washing the liver with 300 ml of serum-free DMEM/F12 (Gibco). It is then filled with high-salt buffer (NaCl) for 90 minutes; the solubility constants of known collagen types in the liver, such as 3.4 M NaCl, are sufficient to keep them all in an insoluble state, in combination with any matrix composition and collagen or collagen-binding matrix components. Binding cytokines/growth factors. The liver was rinsed with serum-free DMEM/F12 for 15 minutes to remove the defatting buffer, and then filled with 100 ml of DNase (1 mg/100 mL; Fisher) and RNase (5 mg/100 mL; Sigma) to remove any residual nucleic acid contaminants. The final step was to rinse the stent with serum-free DMEM/F12 for 1 hour to remove any residual salts or nucleases. The image is provided in Figure 11. The biomatrix scaffolds were injected via a Masterflex peristaltic pump (Cole-Parmer) at 1.3 ml/min for two hours and the cells were seeded with Kubota medium supplemented with 10% fetal bovine serum (FBS). Fetal liver cells are inoculated immediately after being induced. If the cell suspension has been adequately treated for removal of the cell suspension The enzyme can eliminate the step of using SSM to rinse the stent.

Collagen analysis. The amount of collagen in the biomatrix scaffold was evaluated based on the content of hydroxyproline (hyp). Samples of fresh liver (n=5) and biomatrix scaffold (n=6) were snap frozen and pulverized into powder. High performance liquid chromatography (HPLC) was used to quantify the collagen content of each total protein, and total collagen was estimated from the 300 residual/collagen hydroxyproline values. The measured values were measured separately using a Cytofluor Spectramax 250 multiwell plate reader (Molecular Devices). The hydroxyproline content was used to assess the extent of collagen retention after decellularization. These analyses were performed using HPLC to compare the amount of collagen from fresh tissue with the amount of collagen from biomatrix scaffolds (decellularized tissue). The result is expressed as the mass of hydroxyproline (collagen-specific amino acid). It was determined that about 99% of collagen was present after decellularization of rat liver (Fig. 1p).

Immunohistochemistry of the Biomatrix The biomatrix scaffold was embedded in the OCT and snap frozen for cryosection. The frozen sections were thawed at room temperature for 1 hour and then fixed in 10% buffered formaldehyde. After fixation, the sections were washed 3 times in 1x phosphate buffered saline (PBS) and then subjected to endogenous peroxidation for 15 minutes at room temperature with 3% hydrogen peroxide. After washing with 1 x PBS, sections were again blocked with 2.5% horse serum in PBS for 1 hour at room temperature. Primary antibodies diluted in 2.5% horse serum in PBS were added and incubated overnight at 4 °C. The next morning, the sections were rinsed 3 times with PBS and incubated with secondary antibody for 30 minutes at room temperature. A Nova Red substrate (Vector) was used as a developer which was prepared according to the manufacturer's instructions. Images were taken using an Olympus IX70 microscope (Olympus). Hematoxylin and eosin staining of the biomatrix scaffold showed no cells remaining after decellularization (data not shown). The DNA/RNA content of the decellularized biomatrix scaffold was further analyzed and the DNA/RNA levels were determined to be negligible.

Histology indicates that the presence of collagens I, III, IV, V, and VI is present and that it spans the traditional location of the acinus (Figs. 1f to j). The high osmotic pressure that is maintained during decellularization is such that collagen is in an insoluble state so that they can be present in the biomatrix scaffold. Alcian blue staining also qualitative results also indicate that proteoglycans (the major components of the extracellular matrix) are also present (Fig. 1k, l); they are called chemical scaffolds for growth factors and cytokines, and affect the availability of these factors and active. After decellularization (Fig. 1m to o), the basement membrane cell adhesion molecules (elastin, fibronectin and laminin) were identified at appropriate regional locations. Both elastin and laminin were found in the area surrounding the portal vein in the presence of hHpSCs and other liver precursors. Fibronectin was identified throughout the matrix across all regions.

Growth factor. The presence and concentration of matrix-bound growth factors and cytokines in samples of rat liver (fresh tissue) and rat liver biomatrix scaffold (decellularized tissue) were analyzed. The sample was rapidly frozen in liquid nitrogen, pulverized into powder at the temperature of liquid nitrogen and sent to RayBiotech for analysis. Semi-quantitative growth factor assays were performed using the RayBiotech® Human Growth Factor Array G1 series (Raybiotech) and the results are reported in units of fluorescence intensity (FIUs). After comparison with a non-specifically bound negative control and FIUs levels have been normalized to protein concentration, a decrease in FIUs levels was found. In fresh, non-decellularized rat liver tissue (n=3) 40 growth factors were determined and compared to those in the biomatrix liver scaffold (n=3). Average data from multiple replicates. Although the assay was developed for human growth factors, there is sufficient overlap in the cross-reactivity with rat growth factors, allowing for the use of rat tissue. Three samples of both fresh tissue and biomatrix scaffolds were analyzed for 40 growth factors (Fig. Ia). Analysis shows that all growth factors found in tissues in vivo still exist in the biomatrix scaffold extract; although most of them are below a certain level in vivo, they are still at physiologically relevant levels. Of particular importance is the presence of growth factors associated with angiogenesis, such as: multiple forms of FGF, PDGF, and VEGF; and those important for cell proliferation and differentiation, such as: EGFs, heparin-binding EGF, HGF, IGFI, and II, and combinations thereof Protein and TGF. The availability of these growth factors is important for many different biological functions (mitosis and tissue-specific gene expression).

Scanning electron microscope (SEM). Imaging of the decellularized liver biomatrix revealed retention of the vascular structure of the native liver, including the complete portal triple tube (Fig. 1b). As shown in Figure 1b, the bile duct, hepatic artery, and portal vein are all evident. In addition, the honeycomb structure normally containing the liver parenchyma remains intact but has no cells (Fig. 1c). Matrix molecules such as elastin, collagen I and III can also be identified via SEM (Fig. Id, e).

Example 3 - Medium

All media were sterile filtered (0.22 μm filter) and kept dark at 4 °C prior to use. Base medium and fetal bovine serum (FBS) were purchased from GIBCO/Invitrogen. All growth factors are purchased from R&D Systems. All other reagents, except those indicated, were obtained from Sigma. Traditional Hepatocyte Maintenance Medium (HMM) for media comparison studies was purchased from Triangle Research Laboratories (TRL) and supplemented with HEPES, GlutaMax, ITS+ (insulin, transferrin and selenium), dexamethasone, and penicillin-streptococcus William's E medium.

Inoculate the medium. Kubota's medium is a well-defined serum-free medium designed for colony formation, self-replication and amplification of endodermal stem/progenitor cells. It is an organoid culture for serum-free monolayer cultures or fetal liver cells. Kubota's medium has been shown to be effective in the selection of cultures for murine, rodent and human liver stem/progenitor cells. The medium consists of copper free, low calcium (0.3 mM), 1 nM selenium, 0.1% bovine serum albumin (purified, free of fatty acids; fragment V), 4.5 mM nicotinamide, 0.1 nM zinc sulfate heptahydrate, 5 μg /ml of transferrin/Fe, 5 μg/ml insulin, 10 μg/ml high-density lipoprotein, and a mixture of purified free fatty acids consisting of RPMI-1640. The preparation of the work is described in detail in the review of the method. Wauthier, E. et al. Hepatic stem cells and hepatoblasts: identification, isolation and ex vivo maintenance Methods for Cell Biology (Methods for Stem Cells) 86, 137-225 (2008). When used to establish a bioengineered liver, the Kubota medium is temporarily supplemented with 10% FBS to overcome the enzyme used to prepare the hepatocyte suspension, which is then switched to serum-free hormone-defining medium, which is defined by serum-free hormones. The medium is tailored to optimize differentiation of parenchymal and non-parenchymal cells and is referred to as BIO-LIV-HDM.

A differentiation medium (BIO-LIV-HDM) of human liver tissue was generated. Supplemented with dexamethasone (0.04mg/L), prolactin (10IU/L), glycoside (1mg/L), nicotinamide (10mM), triiodothyronine (T3, 67ng/L) , epidermal growth factor (EGF, 20 ng/ml), high-density lipoprotein (HDL, 10 mg/L), hepatocyte growth factor (HGF, 20 ng/ml), human growth hormone (hGH, 3.33 ng/ml), vascular endothelium Growth factor (VEG-F, 20 ng/ml), insulin-like growth factor (IGF, 20 ng/ml), cyclic adenosine monophosphate (2.45 mg/L), basic fibroblast growth factor (bFGF, 20 ng/ml), In the HDM containing Kubota medium, a mixture of galactose (0.16g), angiogenin (0.2mg/ml) and free fatty acid, L-glutamine and antibiotics, the cells were cultured in the inoculation medium for 36 hours after the initial incubation. The culture was carried out for 14 days in a bioreactor. The HDM started 3 days after inoculation and then changed every 2 days thereafter. All reagents were from R & D Systems. BIO-LIV-HDM has been shown to be more successful in albumin production and in response to urea secretion with the addition of 2 mM ammonia (Figure 12) compared to conventional hepatocyte maintenance medium (HMM). However, compared to the other two donors, the results of albumin did not differ significantly due to the very high albumin levels exhibited by one donor sample, resulting in a high standard deviation. In the future, statistical significance will be clarified through additional preparation by adult liver donors to minimize the standard deviation of this report. All three donors performed equally well in terms of urea secretion. Samples in BIO-LIV-HDM were significantly higher on days 1, 4, 6 and 7 (p < 0.05).

Generating liver tissue engineered biological Example 4

Human liver/precursor cells were isolated and stored at 4 °C for 4 hours and in Kubota medium until inoculation. By peristaltic pump through the portal vein The matrix residues were introduced into these cells in a filled manner and these cells were seeded in Kubota medium supplemented with 10% FBS (inoculation medium). Approximately 90 x 106 total cells were filled into the scaffold at intervals of 20 minutes. During each interval, 30 x 106 cells were filled at 15 ml/min for 10 minutes and then allowed to stand for 10 minutes (0 ml/min). Repeat this step 3 times. Once all cells were introduced into the matrix scaffold, the flow rate was reduced to 1.3 ml/min and the scaffold was filled with the inoculation medium for 36 hours. After inoculation, the inoculation medium was collected and any cells remaining in the medium were counted in a hemocytometer. The medium was then changed to a differentiation medium (BIO-LIV-HDM) that was changed every 2 days. The re-inoculated matrix scaffold was cultured in a bioreactor for up to 14 days. After 14 days, the leaves of the re-inoculated matrix scaffold were frozen for histology and immunohistochemistry, which were used for scanning electron microscopy (SEM) and transmission electron microscopy (TEM), or for rapid freezing for RNA sequencing ( t=14 days). The analysis of these bioreactors was presented as Bio_FL724, Bio_FL728 or Bio_FL732, representing the bioreactor inoculated with the respective cells. After 36 hours of inoculation, ~99% of the cells had adhered to the stroma, as evidenced by the lack of cells found in the inoculation medium collected and counted by the hemocytometer (data not shown). After staining with hematoxylin and eosin (H&E), a large number of cells were found around the blood vessels and throughout the parenchyma (data not shown). SEM imaging after 14 days of culture showed endothelial cells lining the vasculature (Fig. 3i and Fig. 7b).

Histology. (Fig. 3) shows the location and expression of proteins identified by immunocytochemistry and immunofluorescence. Expression of the mature marker indicates differentiation and recombination of fetal liver cells after 14 days of culture. In area 1, the portal vein area, The cells express EpCAM and CK19, and the biomarkers are co-expressed in hepatic stem cells and hepatoblasts, and are found around the bile duct. This region also contains cells expressing AFP, which are biomarkers of hepatoblasts. This figure shows hepatic cords that have begun to develop, as well as markers of hepatocyte polarity, ie, expression of E-cadherin, which is located at the site where hepatocytes form cell-cell junctions. Identification of the biliary transport marker MRP2 on the luminal side of hepatocytes helps to identify cell polarity. It seems that these cells surround the bile duct and indicate the function of potential bile such as bile secretion. As identified by Periodic Acid-Schiff (PAS) staining, it is apparent that glycogen is stored in parenchymal cells. Glycogen can be found in hepatocytes throughout the acinus, but glycogen stores are highest in the area surrounding the portal vein. Along the zone gradient, cells are found in parenchymal cells that express markers that represent the central region (region 3), such as Cyp3A4 and albumin (found in all regions). In general, most cells acquire a differentiated state consistent with cells found in the area surrounding the portal vein and mature cells found in the central acinar region and the central peripheral region.

In addition to cells of the hepatic parenchymal cell lineage, stellate cells identified by their expression of myofectin were found, and sinusoidal endothelial cells, lyve-1+ cells were found to be located in a position corresponding to the scaffold located in the living body. Stellate cells are often co-localized with their epithelial partners and are required for paracrine signaling involving mitosis and specific cellular functions. The shape of these cells in the histological image is slender as the cells are squeezed during the process of wrapping around the cells (positive control panels are shown as references). Cells expressing α-smooth muscle actin (α SMA) were found around the vascular structure. α SMA Positive cells may be pericytes that can be activated to proliferate with endothelial cells, CD31+ cells found to be vascularized. They are evident via immunohistochemistry and SEM (Fig. 3i). Ki67 staining showed significant hyperplasia (data not shown) and was found primarily in cells surrounding the blood vessels. Larger cells were not positive for Ki67 staining and were therefore assumed to be in a non-proliferating, fully mature state.

RNA sequencing. We performed paired-end high-throughput RNA sequencing of samples from three different bioreactors, with an average of approximately 200 million paired-end reads per sample, with approximately 87% of the average mapped to the human genome. Many aspects of function and differentiation phases have been identified by analyzing RNA sequencing data. First, it is clear that the cells in the bioreactor are designed to remodel the matrix by increasing the expression of MMP-2 and MMP-9 (matrix degrading enzymes, Figure 4a) and increasing collagen, laminin, fibronectin and The expression of the peripherin (Fig. 4b to e) was identified. The mRNA expression levels of these genes in the bioreactor were significantly higher than in the fetal and adult liver samples (p<0.05), with the exception of peripherin, laminin 10 and 11 for peripherin, in bioreactors. The mRNA expression level was only significantly higher in adult hepatocytes, and there was no significant difference between the samples for laminin 10 and 11. There are also indications that fetal liver-derived stem/progenitor cells in bioreactors have differentiated to represent all mature parenchymal cell lineage stages, which are more pronounced by lowering fetal gene expression and upregulating more mature genes (Fig. 5 and 6). The expression levels of fetal genes such as LGR5 and EpCAM (known liver stem cell markers) in bioreactor samples were significantly reduced compared to fetal cells, with 3.4 and 1.2 fold changes, respectively (Figure 5). Similarly, compared to bioreactor samples and adult liver tissue, AFP is 11 times higher in fetal tissues than the most well-known gene for identifying hepatocytes; levels between bioreactors and adult liver tissues There are no significant differences.

In contrast, the gene expression level of the mature liver marker steadily increased in less than one week in the bioreactor sample compared to fetal tissue, indicating the maturation of the hepatic parenchymal cell lineage. In Region 1, the mature bile markers CK7, SLC4A2, JAG1, HNF1B, and SCTR (Figure 6) were both up-regulated compared to fetal and adult liver samples. CK7, JAG1, and SCTR were significantly increased compared to fetal liver cells, which were higher than 98, 1.75, and 1.85 times, respectively. The expression level of the region 3 marker of metabolic function up-regulated in the bioreactor sample includes the mature form of the P450 gene (CYP1A1, CYP-1B1, and CYP-2C8); all genes are increased by at least 3-fold relative to fetal cells; uridine The diphosphate-glucuronyltransferase UTG1A1, which is approximately 10-fold more abundant than fetal cells; and the genes involved in lipid and cholesterol metabolism (ACOX3, APOL6, LDLR), are only significantly higher in LDLR. Compared to fetal liver samples (data not shown), the expression of CYP3A7 (fetal form P450) was reduced by more than 4-fold in bioreactor tissue, indicating this maturity. Another marker for mature hepatocytes, C/EBP, also increased in the bioreactor, although not significant, and the expression of HNF4a did not change compared to fetal liver.

The level of gene expression measured in a bioreactor, although primarily at a level above maturity in fetal liver cells, is in most cases still different from that in adult tissues. This suggests that further maturation is an additional time under culture conditions that require culture or modification (eg further Reduce serum usage and adjust oxygenation to a greater extent). With this in mind, the gene expression levels of Yap, related targeting genes, and Hippo indicate that the regeneration process is active. The gene expression level of MST1 (a Hippo kinase) in the bioreactor was significantly lower than that of the fetal and adult livers; in parallel, the Yap signaling gene was significantly increased in the bioreactor compared to the fetal and adult livers ( Figure 7a). Gene expression of angiogenic markers indicates that bioreactor tissue is undergoing angiogenesis and angiogenesis (Fig. 7b). Compared with fetal liver cells, the expression levels of VEGF, VEGF-B and CD133 in bioreactor samples were elevated, especially in VEGF and CD133 (p<0.05).

According to RNA sequencing data, it means hematopoietic differentiation (Fig. 7c). Markers of early hematopoietic stem cells (Gata-2, SCF, and IL-7R) are down-regulated in bioreactor samples, and levels found in fetal tissues are converted to levels that match those in adult liver. At the same time, the genetic archives of mature hematopoietic cells in the lymphoid and myeloid lineages differ between fetal liver, bioreactor, and adult liver. Bioreactor samples have CD3 gene expression levels similar to those found in adult liver; Rag1 expression is elevated (two genes, Rag1 and CD3 are associated with T cells), and CSF expression (by bone marrow cells) Expression) is significantly higher compared to fetal and adult livers. These markers represent a possible hematopoietic effect, but require a broader analysis to allow for an accurate interpretation.

Cell viability. The aminotransferases ALT and AST for assessing hepatocyte health were evaluated on days 2, 4, 6, 8, 10, 12 and 14. During this experiment, the level of ALT did not exceed the lower limit of detection (data not shown). Therefore, it was determined that it is not a sensitive biomarker for this ex vivo model system. Bio_FL724 is the only bioreactor that has a detectable AST level above the lower detection limit (4 U/L) throughout the incubation time (data not shown). The LDH level (Fig. 8a) for each bioreactor was initially high but decreased over time. However, after the first day of culture, the LDH measurements at each time point were significantly lower than the initial measurements (p < 0.05). The interpretation of this data is that in the first few days, there are cells with higher renewability due to the pressure of the separation process and/or the inoculation process. After this recovery period, the cells produce phenotypic characteristics, meaning rapid organ development in the liver.

Full-length K18 (FL-K18) levels in the culture medium (and therefore secreted or released from the cells) are specific for cell necrosis; values in all bioreactors are above the baseline (25.3 U/L). The trend for FL-K18 levels was similar in all three bioreactors. The level on day 2 (Fig. 8a) was significantly higher and was assumed to be due to cellular stress or cell damage from the isolation procedure. After day 2, the level was significantly reduced, and days 4 and 6 were significantly lower than the initial reading on day 2 (p < 0.05). The initial decrease in FL-K18 means that fewer necrotic cells in culture may be responding to complete cell death, and, especially in the case of Bio_FL728 and Bio_FL732, the remaining cells indicate the presence of healthier cells. . However, during the 8-12 period, the FL-K18 increased and then the level dropped again.

The level of ccK18 (Fig. 8a) detected followed a similar trend in FL-K18 levels, with levels rising on day 6, peaking on day 8, and then decreasing. Although these data may indicate conditions for high apoptosis, there is no significant difference in levels throughout the culture period, meaning that over time There was no significant increase in apoptosis between the passages.

Overall, data describing cell survival and health indicate that when cells damaged during isolation and vaccination are eliminated, they undergo a transition period of 2 to 3 days, then the remaining cells are stabilized and differentiated.

The increase in FL-K18 and ccK18 also corresponds to an increase in albumin secreted by cells in all three bioreactors. It is hypothesized that this increase is due to terminally differentiated polyploid hepatocytes that undergo apoptosis as part of the normal cell cycle process. Immediately after this peak of apoptosis, ccK18 levels decreased, indicating that the precursor cells are maturing to replace the lost central peripheral liver cells.

AFP (Figure 8b). Each bioreactor showed a different initial level of AFP on day 2, the first day of sample collection, corresponding to differences in gene expression between fetal liver cells at t=0. Regardless of the initial value of the AFP, the yield drops sharply over time.

Albumin (Fig. 8b). The level of albumin production in all three bioreactors was initially low, but increased over time, with a significant increase in albumin production levels between 6 and 10 days (p < 0.05). The actual amount of albumin produced by a single bioreactor is different, but the general trend of production growth for all bioreactors is consistent. The peak reached the level on the 8th day and began to decrease on the 10th day. Hypothesis is assumed to correspond to cell differentiation in the late lineage stage, as indicated by the high yield of albumin in hepatocytes. They then undergo apoptosis, which results in a decrease in albumin production as the precursor cells continue the regeneration process. This interpretation of this data is also supported by the ccK18 level measured at each time point.

Urea (Fig. 8b) differs from the production of AFP and albumin in that urea levels did not change significantly within 14 days. All three bioreactors had the largest amount of urea secretion on day 2 and then decreased slightly thereafter. By day 10, the urea level was significantly lower than the initial value of day 2 (p < 0.05), although overall, the secretions appeared to remain stable over time.

The cellular metabolite function of the cells was assessed via metabolic activity and measured by nuclear magnetic resonance (NMR) spectroscopy (Figure 9). Principal component analysis (PCA, Figure 9b) was performed, indicating that the two Bio_FL724 and Bio_FL732 reactions in the bioreactor were more similar in the medium than the third bioreactor Bio_FL732. In all bioreactors, cells depleted and metabolized glucose, glutamine, pyruvate and acetate provided in the medium and converted it to the production of lactate (Figure 9a). These actions determine that the cell is undergoing glycolytic action and entering the Krebs cycle. Bio_FL724 was active immediately on day 2 and Bio_FL728 had a similar trend on day 6. The third bioreactor, Bio_FL732, became metabolically active on day 8, although its level was lower than the other two bioreactors. This indicates the presence of two bioreactors, Bio_FL728 and Bio_FL732, which require recovery from the possible pressure of the vaccination process, or when isolated, the cells are not as healthy as Bio_FL724 and require more time to become Metabolically active state. The VIP map (Figure 9c) shows the metabolites that contribute to the separation. VIP>=1.0 is considered important.

Transmission electron microscopy (TEM). The tissue of the cells in the respective bioreactors was further evaluated by TEM. In order to function, epithelial cells must form cell signals in cell polarity, communicate with neighboring cells, and Cell-cell junctions that play a role in matrix interactions. The composition of the ligation complex (Fig. 10e, f) can be observed via TEM imaging because the hepatocytes cluster together to form a sheet or plate with bile ducts (Figs. 10a to c) between them for transport secretion. The necessary configuration of bile. A sinusoidal space was observed between these hepatocyte-like cells (Fig. 10a) and possible secretory vesicles were seen around the bile duct (Fig. 10b). In addition to hepatocytes, there were several cells having physical characteristics of endothelial cells, stellate (Ito) cells, and stem cells during differentiation, which were identified by TEM (data not shown). Inoculation of cells is uneven throughout the biomatrix scaffold, resulting in sites in different stages of the cell in the organogenesis process represented in the TEM image. Lipid droplets are seen in images that are not associated with cells (data not shown), which can be expressed as signs of cell disintegration that occur during sample preparation for imaging or possibly during cell senescence in cell culture, similar Represented by data on cell necrosis and apoptosis.

Experiment 5: Characteristics of collagen in the matrix

The tissue is washed to minimize the amount of blood and interstitial fluid. Most fibrillar collagen cannot be extracted with the typical initial rinse solution that people use: phosphate buffered saline (PBS). However, uncrosslinked collagen and related matrix components can be extracted with PBS, including procollagen, collagen monomers (before fibrils) and non-fibrillar collagen types (eg, type IV, type VI). Therefore, the initial rinsing is carried out using a basal medium (a mixture of amino acids, nutrients, lipids, vitamins, trace elements, etc.). And has an ionic strength that does not cause collagen to dissolve.

Degreasing steps used by others and tissue delipidation Time (sometimes hours or even days (!!). SDS binds very tightly to the substrate and makes it toxic. Triton X and other such coarse detergents dissolve various matrix compositions. One procedure uses SDS And then use Triton-X, a procedure that causes "very clean" matrix scaffolds, but in fact they look "clean" because many have been lost. Therefore, low concentrations of bile salts, sodium deoxycholate and The combination with phospholipase results in rapid and very mild degreasing. Destruction takes place within 20 to 30 minutes.

Extraction with low ionic strength buffer (1M NaCl or less) resulted in significant loss of uncrosslinked collagen; with 1M NaCl, some collagen (mostly type I collagen) can be preserved, but not all (not Network collagen). Thus, the method does not lose any collagen (fiber or network; cross-linked or uncrosslinked), and thus retains everything that is combined therewith. In contrast, the use of distilled water may lose all of the highly cross-linked collagen and its combined composition, which is dissolved in water.

Nucleic acids are removed according to standard methods in the art.

The difference was obtained by isolating the biomatrix scaffold by collecting the supernatant, dialyzing it, lyophilizing it, and measuring the collagen content among them via amino acid, cross-linking, Western blotting and growth factor analysis. And as its characteristics. This will determine the collagen retained by this method. Parallel extraction is performed using a) in PBS; b) in low ionic strength buffer; c) through various degreasing methods; d) in distilled water. The supernatant of each of these steps was collected and subjected to amino acid analysis to assess whether collagen was lost and lost. When the amount of collagen is determined to be sufficient, the collagen in the supernatant is treated with [3H]-borohydride, hydrolyzed and subjected to crosslinking. Joint analysis. In addition, Western blotting using antibodies was performed to identify the extent of cross-linking and the type of collagen present. In addition, growth factor analysis was performed to show the characteristics of the resulting scaffold.

Exemplary embodiment

Non-limiting exemplary embodiments are provided below:

[1] A container for generating bioengineered tissue, wherein the generating comprises introducing epithelial and mesenchymal cells into or onto a biological matrix scaffold, wherein the biomatrix scaffold comprises collagen.

[2] The container according to [1], wherein the epithelial and mesenchymal cells are mature lineage partners

[3] The container according to [1] or [2], wherein the epithelial and mesenchymal cells are in a seeding medium, and after an initial culture period, the seeding medium is replaced with a differentiation medium.

[4] The container according to [3], wherein the differentiation medium comprises: a. a basal medium; b. lipid, insulin, transferrin, antioxidant; c. copper; d. calcium; e. for epithelium One or more signal agents that the cells propagate or maintain; and/or f. one or more signal agents for the proliferation or maintenance of mesenchymal cells.

[5] The container of [3] or [4], wherein the inoculation medium is serum-free or supplemented with about 2% to 10% fetal serum for a duration of several hours.

[6] The container according to [3] to [5], wherein the inoculating medium comprises: a. a basal medium; b. lipid; c. insulin; d. transferrin; e. antioxidant.

[7] The container according to any one of [3] to [6] wherein the epithelial and mesenchymal cells in the inoculation medium are cultured in an inoculation medium at 4 ° C before introduction of the biological matrix scaffold 4 Up to 6 hours.

[8] The container according to any one of [1] to [7] wherein the biological matrix scaffold is a three-dimensional one.

[9] The container of any one of [1] to [8], wherein the collagen in the biomatrix scaffold comprises: (i) nascent collagen; (ii) collagen molecules aggregated but not cross-linked; Iii) cross-linking collagen.

[10] The container according to any one of [1] to [9] wherein the epithelial and mesenchymal cells in the inoculation medium are introduced at a plurality of intervals, with a pause after each interval Set time.

[11] The container according to [10], wherein the interval is about 10 minutes, and the rest period is about 10 minutes.

[12] The container of [10] or [11], wherein the seeding density is up to about 12 million cells per gram of the wet weight of the biomatrix scaffold, and introduced at one or more intervals.

[13] The container according to any one of [10], wherein the epithelial and mesenchymal or non-parenchymal cells in the inoculation medium are introduced into one or more at a rate of about 15 ml/min. Interval.

[14] The container according to any one of [10] to [13] wherein the epithelial and mesenchymal cells in the inoculation medium are introduced at intervals of 10 minutes, each of which is accompanied by 10 minutes. Resting period.

[15] The container according to any one of [10] to [14] wherein the epithelial and mesenchymal cells in the inoculation medium are introduced at a rate of 1.3 ml/min after three intervals.

[16] The container of any of [1] to [15] wherein the epithelial and mesenchymal cells comprise cells isolated from a fetal or neonatal organ.

[17] The container of any of [1] to [15] wherein the epithelial and mesenchymal cells comprise cells isolated from an adult or a child donor.

[18] The container according to any one of [1] to [17] wherein the epithelial and mesenchymal cells comprise: a. epithelial cells, which are stem cells, committed precursor cells, diploid adult cells, One or more of polyploid adult cells and/or terminally differentiated cells; and/or b. interstitial cells, including hemangioblasts, endothelial precursors, mature endothelium, stellate cell precursors, mature One or more of astrocytes, matrix precursors, mature matrices, smooth muscle cells, hematopoietic precursors, and/or mature hematopoietic cells.

[19] The container according to any one of [1] to [18] wherein the epithelial and mesenchymal cells comprise: a. epithelial cells, which include bile duct trunk cells, gallbladder-derived stem cells, hepatic stem cells, and hepatic cells Cells, committed hepatocytes and biliary precursor cells, axin2+ One or more of precursor cells (eg, axin2+ liver precursor cells), mature parenchymal cells (eg, hepatocytes, biliary cells), pancreatic stem cells, and pancreatic progenitor cells, islet cells, and/or acinar cells; and/ Or b. stromal cells, which include hemangioblasts, stellate cell precursors, stellate cells, mesenchymal stem cells, pericytes, smooth muscle cells, stromal cells, endothelial cell precursors, endothelial cells, hematopoietic precursors, and / or one or more of the hematopoietic cells.

[20] The container according to any one of [1] to [19] wherein the epithelial cells comprise stem cells from the biliary tract, liver, pancreas, hepatopancreatic duct and/or gallbladder and/or mesenchymal cells and One or more of its progeny, including hemangioblasts, endothelial progenitors and stellate cells, mesenchymal stem cells, stellate cells, stroma, smooth muscle cells, endothelial, bone marrow-derived stem cells, hematopoietic precursors And/or one or more of hematopoietic cells.

[21] The container according to any one of [1] to [20] wherein the epithelial and mesenchymal cells are composed of about 80% epithelial and 20% interstitial, respectively.

[22] The container of any of [1] to [21] wherein the epithelial and mesenchymal cells comprise at least 50% stem cells and/or precursor cells.

[23] The container according to any one of [1] to [22] wherein the epithelial and mesenchymal cells do not include any terminally differentiated hepatocytes and/or pancreatic cells.

[24] The container of any one of [1] to [23] wherein the biomatrix scaffold comprises one or more collagen-related matrix components, including laminin, collagen And one or more of elastin, proteoglycan, hyaluronic acid, non-sulfated glycosaminoglycan, sulfated glycosaminoglycan and growth factor and/or cytokine associated with a matrix component.

[25] The container of any of [1] to [24], wherein the biomatrix scaffold comprises more than 20-50% of a matrix binding signal molecule found in vivo.

[26] The container of any of [1] to [25] wherein the biomatrix scaffold comprises a matrix residue of the vascular tree of the tissue and/or wherein the matrix residue is in the bioengineered tissue Provides vascular support for this cell.

[27] A three-dimensional scaffold comprising an extracellular matrix comprising (i) native collagen found in an organ and/or (ii) a matrix residue of a vascular tree found in an organ.

[28] A three-dimensional micro-organ of the container according to any one of [1] to [26].

[29] A bioengineered tissue comprising a region-dependent phenotypic trait characterized by native liver, the phenotypic trait comprising (a) having dry/progenitor cells, diploid adult cells, and/or associated interstitial anterior a periportal region of the somatic cell trait; (b) a central acinar region of cells with mature parenchymal cells and sinusoidal traits of mesenchymal cells; and/or (c) epithelial cells with terminally differentiated and transmucosal endothelium / or axin2+ is a central peripheral region of the trait of apoptotic cells associated with hepatic precursor cells, wherein the axin2+ liver progenitor cells are connected to the endothelium of the central vein.

[30] The bioengineered tissue of [29], wherein the phenotypic trait further comprises a trait associated with a diploid epithelial cell and/or an interstitial cell of the area surrounding the portal vein.

[31] The bioengineered tissue of [29] or [30], wherein the phenotypic trait further comprises a trait of mature epithelial cells and/or mesenchymal cells found in the central acinar region of the native liver.

[32] The bioengineered tissue of any one of [29] to [31], wherein the phenotypic trait further comprises an epithelial or parenchymal and/or interstitial cell trait of the area surrounding the portal vein.

[33] The bioengineered tissue of any one of [29] to [32], further comprising: (i) polyploid hepatocytes associated with permeabilizing endothelial cells; and/or (ii) linked to Diploid hepatic precursor cells (such as axin2+ cells) of the endothelium of the central vein.

[34] The bioengineered tissue of any one of [29] to [33] wherein the area surrounding the portal vein is rich in hepatic stem cells, hepatoblasts, committed precursor cells, and/or diploid adult liver. The trait of the stem/progenitor cell niche of the cell.

[35] The bioengineered tissue of any one of [29], wherein the epithelial and mesenchymal cells further comprise epithelial cells, wherein the epithelial cells comprise precursors of hepatocytes and/or cholangiocarcinoma cells. And / or mature form.

[36] The bioengineered tissue of any one of [29] to [35], wherein the epithelial and mesenchymal cells further comprise stromal cells, including stellate cells, pericytes, smooth muscle cells a precursor and/or mature form of the matrix, endothelium and/or hematopoietic cells.

[37] A three-dimensional micro-organ of the bioengineered tissue according to any one of [29] to [36].

[38] The three-dimensional micro-organ according to [37], which is the container according to any one of [1] to [26].

[39] A kit for culturing the micro-organ in the container according to any one of [1] to [26] with instructions.

[40] A method of evaluating organ treatment, which is applied to a bioengineered tissue or a three-dimensional micro-organ of any one of [29] to [38].

[41] A differentiation medium for epithelial and mesenchymal cells, comprising: a. a basal medium containing lipids, insulin, transferrin, and an antioxidant; b. copper; c. calcium; d. for epithelial cell proliferation And/or one or more signal agents maintained; and/or e. one or more signal agents for the proliferation and/or maintenance of mesenchymal cells.

[42] The differentiation medium according to [41], wherein the basal medium is Kubota medium.

[43] The differentiation medium according to [41] or [42], further comprising one or more lipid-binding proteins.

[44] The differentiation medium of [43], wherein the one or more lipid binding proteins are high density lipoprotein (HDL).

[45] The differentiation medium of any one of [41] to [44], further comprising one or more purified fatty acids.

[46] The differentiation medium of [45], wherein the one or more purified fatty acids include palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and/or linolenic acid.

[47] The differentiation medium of any one of [41] to [46], further comprising one or more sugars.

[48] The differentiation medium of [47], wherein the one or more sugars comprise galactose, glucose and/or fructose.

[49] The differentiation medium of any one of [41] to [48], further comprising one or more glucocorticoids.

[50] The differentiation medium of [49], wherein the one or more glucocorticoids comprise dexamethasone and/or hydrocortisone.

[51] A bioengineered tissue that is a region-dependent phenotypic trait comprising native pancreatic features and/or includes a trait associated with a pancreatic cell in the pancreatic head and/or with a tail of the pancreas Traits related to pancreatic cells.

Claims (51)

A container for generating bioengineered tissue, wherein the generating comprises introducing epithelial and mesenchymal cells into or onto a biological matrix scaffold, wherein the biomatrix scaffold comprises collagen. The container of claim 1, wherein the epithelial and mesenchymal cells are mature lineage partners. The container of claim 1, wherein the epithelial and mesenchymal cells are in a seeding medium, and after an initial culture period, the seeding medium is replaced with a differentiation medium. The container of claim 3, wherein the differentiation medium comprises: a. a basal medium; b. lipid, insulin, transferrin, antioxidant; c. copper; d. calcium; e. for epithelial cell proliferation or One or more signal agents maintained; and/or f. one or more signal agents for the proliferation or maintenance of mesenchymal cells. The container of claim 3, wherein the inoculating medium is serum-free or optionally supplemented with from about 2% to 10% fetal serum over a period of several hours. The container of claim 3, wherein the inoculating medium comprises: a. a basal medium; b. a lipid; c. insulin; d. transferrin; e. antioxidant. The container of claim 3, wherein the epithelial and mesenchymal cells in the inoculation medium are cultured in the inoculation medium at 4 ° C for 4 to 6 hours prior to introduction of the biomatrix scaffold. The container of claim 1 wherein the biomatrix scaffold is a three dimensional one. The container of claim 1, wherein the collagen in the biomatrix scaffold comprises: (i) nascent collagen; (ii) collagen molecules that aggregate but not cross-link; and (iii) cross-linked collagen. The container of claim 10, wherein the epithelial and mesenchymal cells in the inoculation medium are introduced at a plurality of intervals, each interval being accompanied by a resting period. The container of claim 10, wherein the interval is about 10 minutes and the rest period is about 10 minutes. The container of claim 10, wherein the seeding density is up to about 12 million cells per gram of wet weight of the biomatrix scaffold and introduced at one or more intervals. The container of claim 10, wherein the epithelial and mesenchymal or non-parenchymal cells in the inoculation medium are introduced at one or more intervals at a rate of about 15 ml/min. The container of claim 10, wherein the epithelial and mesenchymal cells in the inoculation medium are introduced at intervals of 10 minutes, each of which is accompanied by a 10-minute rest period. The container of claim 10, wherein the epithelial and mesenchymal cells in the inoculation medium are introduced at a rate of 1.3 ml/min after three intervals. The container of claim 1, wherein the epithelial and mesenchymal cells comprise cells isolated from a fetal or neonatal organ. The container of claim 1, wherein the epithelial and mesenchymal cells comprise cells isolated from an adult or a child donor. The container of claim 1, wherein the epithelial and mesenchymal cells comprise: a. epithelial cells, which are stem cells, committed precursor cells, diploid adult cells, polyploid adult cells, and/or terminal differentiation. One or more of the cells; and/or b. interstitial cells, including hemangioblasts, endothelial progenitors, mature endothelium, stellate cell precursors, mature astrocytes, matrix precursors, mature matrices, One or more of smooth muscle cells, hematopoietic cell precursors, and/or mature hematopoietic cells. The container according to claim 1, wherein the epithelial and mesenchymal cells comprise: a. epithelial cells, including bile duct trunk cells, gallbladder-derived stem cells, hepatic stem cells, hepatoblasts, committed hepatocytes, and biliary progenitor cells, One or more of axin2+ precursor cells (eg, axin2+ liver precursor cells), mature parenchymal cells (eg, hepatocytes, biliary cells), pancreatic stem cells, and pancreatic progenitor cells, islet cells, and/or acinar cells; / or b. stromal cells, including hemangioblasts, stellate precursors, stellate cells, mesenchymal stem cells, pericytes, smooth muscle cells, One or more of a stromal cell, an endothelial cell precursor, an endothelial cell, a hematopoietic cell precursor, and/or a hematopoietic cell. The container of claim 1, wherein the epithelial cells comprise one or more of stem cells from the biliary tract, liver, pancreas, hepatopancreatic duct and/or gallbladder and/or mesenchymal cells and progeny thereof, the mesenchyme The cells include one or more of hemangioblasts, endothelial progenitor and stellate cells, mesenchymal stem cells, stellate cells, stroma, smooth muscle cells, endothelium, bone marrow-derived stem cells, hematopoietic precursors and/or hematopoietic cells. . The container of claim 1, wherein the epithelial and mesenchymal cells are composed of about 80% epithelium and 20% interstitial, respectively. The container of claim 1, wherein the epithelial and mesenchymal cells comprise at least 50% stem cells and/or precursor cells. The container of claim 1, wherein the epithelial and mesenchymal cells do not comprise any terminally differentiated hepatocytes and/or pancreatic cells. The container of claim 1, wherein the biomatrix scaffold comprises one or more collagen-related matrix components, including laminin, collagen, elastin, proteoglycan, hyaluronic acid The non-sulfated glycosaminoglycan, the sulfated glycosaminoglycan is one or more of a growth factor and/or a cytokine associated with a matrix component. The container of claim 1, wherein the biomatrix scaffold comprises more than 20-50% of a matrix binding signal molecule found in vivo. The container of claim 1, wherein the biomatrix scaffold comprises a matrix residue of the vascular tree of the tissue and/or wherein the matrix residue provides vascular support of the cell in the bioengineered tissue. A three-dimensional scaffold comprising an extracellular matrix comprising (i) native collagen found in an organ and/or (ii) a matrix residue of a vascular tree found in an organ. A three-dimensional micro-organ generated from the container of claim 1. A bioengineered tissue comprising a region-dependent phenotypic trait characterized by native liver, the phenotypic trait comprising (a) having dry/precursor cells, diploid adult cells, and/or associated mesenchymal precursor cell traits The area surrounding the portal vein; (b) the central acinar region of cells with sinusoidal traits of mature parenchymal cells and mesenchymal cells; and/or (c) epithelial cells with terminally differentiated and permeable endothelium and/or axin2+ A central peripheral region of a trait of an apoptotic cell associated with a liver precursor cell, wherein the axin2+ liver precursor cell is an endothelium that is attached to the central vein. The bioengineered tissue of claim 29, wherein the phenotypic trait further comprises a trait associated with a diploid epithelial cell and/or an interstitial cell of the area surrounding the portal vein. The bioengineered tissue of claim 29, wherein the phenotypic trait further comprises traits of mature epithelial cells and/or mesenchymal cells found in the central acinar region of the native liver. The bioengineered tissue of claim 29, wherein the phenotypic trait further comprises an epithelial or parenchymal and/or stromal cell trait of the area surrounding the portal vein. The bioengineered tissue of claim 29, further comprising: (i) polyploid hepatocytes associated with endothelial cells permeable; and/or (ii) diploid hepatic precursor cells linked to the endothelium of the central vein (such as axin2+ cells). The bioengineered tissue of claim 29, wherein the area surrounding the portal vein is rich in traits of the stem cell/precursor cell niche including hepatic stem cells, hepatocytes, committed precursor cells, and/or diploid adult hepatocytes. . The bioengineered tissue of claim 29, wherein the epithelial and mesenchymal cells further comprise epithelial cells, wherein the epithelial cells comprise precursors and/or mature forms of hepatocytes and/or cholangiocarcinoma cells. The bioengineered tissue of claim 29, wherein the epithelial and mesenchymal cells further comprise mesenchymal cells, including stellate cells, pericytes, smooth muscle cells, stroma, endothelium and/or hematopoietic cells. Body and / or mature form. A three-dimensional micro-organ of a bioengineered tissue as claimed in claim 29. The three-dimensional micro-organ of claim 37, which is produced in the container of claim 1. A kit for culturing the micro-organ in a container as claimed in claim 1 with instructions. A method of assessing organ treatment, which is applied to a bioengineered tissue or a three-dimensional micro-organ of claim 29. A differentiation medium for epithelial and mesenchymal cells, comprising: a. a basal medium containing lipids, insulin, transferrin, and an antioxidant; b. copper; c. calcium; d. one or more signal agents for epithelial cell proliferation and/or maintenance; and/or e. one or more signal agents for stromal cell proliferation and/or maintenance. The differentiation medium of claim 41, wherein the basal medium is Kubota medium. The differentiation medium of claim 41 further comprising one or more lipid binding proteins. The differentiation medium of claim 43, wherein the one or more lipid binding proteins are high density lipoprotein (HDL). The differentiation medium of claim 41, further comprising one or more purified fatty acids. The differentiation medium of claim 45, wherein the one or more purified fatty acids comprise palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, and/or linolenic acid. The differentiation medium of claim 41, further comprising one or more sugars. The differentiation medium of claim 47, wherein the one or more sugars comprise galactose, glucose, and/or fructose. The differentiation medium of claim 41 further comprising one or more glucocorticoids. The differentiation medium of claim 49, wherein the one or more glucocorticoids comprise dexamethasone and/or hydrocortisone. A bioengineered tissue that is a region-dependent phenotypic trait comprising native pancreatic features and/or includes a pancreatic cell associated with the pancreatic head The trait of the region and/or the trait associated with the pancreatic cells in the tail of the pancreas.