HK1233552A1 - Biomatrix scaffolds - Google Patents

Description

Biological matrix scaffold

The present application is a divisional application of PCT application having an application date of 2011, 7/1, application No. 201180042689.9 and an invention name of "bio-matrix scaffold".

Priority declaration

Priority of U.S. provisional application serial No. 61/360,939 filed 2010, 7/2/2010, at 35u.s.c. § 119(e), which is incorporated herein by reference in its entirety.

Statement of government support

Aspects of the invention are made with government support under grant numbers AA014243 and IP30-DK065933 of the National Institutes of Health (NIH), DK34987 of the national institute of diabetes, digestive and renal disease (NIDDK), CA016086 of the national institute of cancer (NCI) and DE019569 of the national institute of dental and craniofacial research. The united states government has certain rights in this invention.

Technical Field

The present invention relates to biomatrix scaffolds and methods of producing biomatrix scaffolds, and their use in different applications, either as intact scaffolds or as scaffolds that are variously sliced or comminuted and dispersed for specific experimental and clinical uses.

Background

The ability to use differentiated cells in ex vivo (ex vivo) or in clinical projects such as cell therapy depends on the ability to maintain cells that have an adult phenotype and are fully functional or can lineage restrict stem or progenitor cells ("stem/progenitor cells") to achieve that adult phenotype. The ongoing revolution in stem cell research has made possible the identification and isolation of stem/progenitor cell populations, including those from embryonic, fetal, and postnatal tissues¹. The ability to identify and isolate stem/progenitor cells for all adult cell types, and the ability to expand and differentiate them, greatly increases their potential to be exploited for pharmaceutical and other industrial research projects, for academic research, and for clinical projects such as cell-based therapies and tissue engineering²。

Current methods for maintaining differentiated cells ex vivo or limiting stem cell lineages to adult fates are partially successful and involve plating or embedding cells into a matrix of one or more extracellular matrix components and into a culture fluid consisting of specific hormones, growth factors, and nutrients tailored to the desired adult phenotype. For very primitive stem cells such as Embryonic Stem (ES) cells or induced pluripotent stem cells (iPS), or postnatally derived stem cells that can be converted to a variety of adult fates, such as Mesenchymal Stem Cells (MSC) or amniotic fluid derived stem cells (AFSC), these stem cells are subjected to a mixture of soluble signals and/or extracellular matrix components and must be processed with multiple sets of such signals over a period of weeks. Typically, the adult phenotype achieved is different with each preparation and has over-or under-expression of certain adult-specific genes and/or dysregulation of one or more adult tissue-specific genes.

Extracellular matrix is secreted by cells, is adjacent to them on the surface of one or more cells, and has long been recognized as a structural support for cells⁷. It is an extremely complex scaffold consisting of a variety of bioactive molecules that are highly regulated and critical for determining the morphology, growth and differentiation of attached cells⁸. Tissue-specific gene expression of cultured cells is improved by culturing the cells on a matrix extract or purified matrix composition⁹. However, individual matrix components, alone or in combination, are not capable of generalizing the complex matrix chemistry and structure of tissue. This is related to the following facts: the matrix composition is in a gradient associated with the native tissue region as well as with histological structures such as blood vessels. This complexity of the tissue matrix is more easily achieved by extraction methods that decellularize the tissue and leave the matrix as a residue^10、11. However, current decellularization protocols result in substantial loss of some matrix components due to the use of matrix degrading enzymes or buffers that solubilize the matrix components.

The present invention provides biomatrix scaffolds and methods of making and using such biomatrix scaffolds. The methods of the invention result in the production of tissue-specific extracts rich in most collagens of tissues and having a matrix-binding component and matrix-binding hormones, growth factors and cytokines that collectively produce more reproducible and more effective differentiation effects on lineage restriction of mature cell and stem/progenitor cell populations.

Disclosure of Invention

In one aspect, the present invention provides a method of producing a biomatrix scaffold from a biological tissue comprising the steps of: a) perfusing or homogenizing a biological tissue with a first culture fluid, wherein the osmolality of the first culture fluid is about 250mOsm/kg to about 350mOsm/kg, and the first culture fluid is serum-free and at a neutral pH; then b) perfusing the biological tissue of step (a) or extracting the homogenate of step (a) in the first culture broth with a degreasing buffer comprising a lipase and/or a detergent; then c) perfusing the tissue of step (b) or extracting the homogenate of step (b) with a buffer at neutral pH and comprising a salt concentration of about 2.0M NaCl to about 5.0M, the concentration being selected to keep the collagen identified in the biological tissue insoluble; then d) perfusing the tissue of step (c) or extracting the homogenate of step (c) with ribonuclease (RNase) and deoxyribonuclease (DNase) in a buffer; and then e) rinsing the tissue of step (d) or the homogenate of step (d) with a second culture medium at neutral pH, serum-free, and having an osmolality of about 250 to about 350mOsm/kg, thereby producing an intact or homogenized biomatrix scaffold from the biological tissue, the biomatrix scaffold comprising at least 95% of the collagen and a majority of the collagen-binding matrix components and matrix-bound growth factors, hormones, and cytokines of the biological tissue.

In addition, the present invention provides a biomatrix scaffold comprising collagen, fibronectin, laminin, endothelin/nidogen, elastin, proteoglycans, glycosaminoglycans, growth factors, cytokines, and any combination thereof, all of which are part of a biomatrix scaffold.

In a further aspect, the invention provides a method of producing a cell culture comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of step (a) with a culture solution of a cell culture in a culture device; and c) seeding the bio-matrix scaffold of step (b) with cells, thereby producing a cell culture.

Also provided herein are methods of producing a cell culture, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) freezing the bio-matrix scaffold of step (a); c) preparing frozen sections from the bio-matrix scaffold of step (b) as cell culture medium; d) contacting the cell culture medium of step (c) with a culture medium of a cell culture in a culture device; and e) seeding the cell culture medium of step (d) with cells, thereby producing a cell culture.

In addition, the present invention provides a method of producing a cell culture comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) grinding the bio-matrix scaffold of step (a) into a powder (e.g., in some embodiments, after freezing the bio-matrix scaffold of step (a)); c) coating at least a portion of the culture device with the powder of step (b) to produce a cell culture medium; d) contacting the cell culture medium of (c) with a culture medium of a cell culture in a culture device; and e) seeding the cell culture medium of step (d) with cells, thereby producing a cell culture. In a particular embodiment of the method, the grinding of the bio-matrix scaffold may be performed in a cryomill, for example at or near the temperature of liquid nitrogen.

Also provided herein are uses of the tissue-specific biomatrix scaffolds of the invention to promote differentiation of embryonic stem cells or induced pluripotent cells towards a specific fate, and uses of the tissue-specific biomatrix scaffolds of the invention to promote differentiation of amniotic fluid derived stem cells or mesenchymal stem cells or any determinate stem cells (e.g., lung, intestine, biliary system, kidney, skin, heart) from bone marrow, adipose tissue, or any fetal tissue or birth biopsy, towards a specific adult fate.

In other embodiments, the invention provides methods of enhancing and accelerating the differentiation of stem and/or progenitor cells into mature cells comprising producing a cell culture according to the methods of the invention, wherein the cells are stem cells and the culture broth of the cell culture is formulated against the mature cells, thereby enhancing and accelerating the differentiation of the stem and/or progenitor cells into mature cells.

The invention also provides a method of delivering cells to a subject comprising seeding a bio-matrix scaffold of the invention with cells and then transplanting the bio-matrix scaffold seeded with cells into a subject.

Additionally, provided herein are methods of identifying the metastatic potential of a tumor cell in a tissue type, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) inoculating the biomatrix scaffold of (b) with tumor cells; d) maintaining the bio-matrix scaffold of (c) under culture conditions; and e) monitoring the growth of the tumor cells on the biomatrix scaffold of (d), wherein the growth of the tumor cells on the biomatrix scaffold identifies that the tumor cells can form colonies in vivo in the tissue type from which the biomatrix scaffold was derived, thereby identifying the metastatic potential of the tumor cells in said tissue type.

In addition, the present invention provides a method of identifying a response of a tumor cell to an anti-tumor therapy, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) inoculating the biomatrix scaffold of (b) with tumor cells; d) maintaining the bio-matrix scaffold of (c) under culture conditions; e) applying an anti-tumor therapy to tumor cells on the bio-matrix scaffold; and f) monitoring the growth of tumor cells on the biomatrix scaffold of (e), wherein the absence of growth and/or death of tumor cells on the biomatrix scaffold of (e) identifies the response of the tumor cells to the anti-cancer therapy.

The present invention also provides a method of producing a tumor graft for transplantation into a host animal, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) inoculating the biomatrix scaffold of (b) with tumor cells; d) maintaining the bio-matrix scaffold of (c) under culture conditions; and e) establishing a population of tumor cells on the biomatrix scaffold of (d), thereby producing a tumor graft for transplantation into a host animal. In some embodiments, the invention may further comprise the step of transplanting the tumor graft into a host animal.

In other embodiments, the invention provides methods of producing a virus particle of a lineage-dependent virus, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) seeding the bio-matrix scaffold of (b) with cells of the type and lineage stage that can be infected by the lineage-dependent virus; d) infecting the cells of (c) with a lineage-dependent virus; e) maintaining the infected cells on the bio-matrix scaffold under culture conditions; and f) collecting viral particles produced in the infected cells, thereby producing viral particles of the lineage-dependent virus.

The present invention also provides a method of preparing organoids formed by recellularization of a biomatrix scaffold comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) seeding the biomatrix scaffold of (b) with cells of the same tissue type as the biological tissue used to prepare the biomatrix scaffold; d) maintaining the cells on the biomatrix scaffold of (c) under culture conditions, whereby organoids are formed from the cells, thereby producing organoids formed by recellularization of the biological tissue scaffold.

Also provided herein are methods of producing a protein of interest in cells cultured on a biomatrix scaffold comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) seeding the biomatrix scaffold of (b) with cells that produce the protein of interest; d) maintaining the cells of (c) on a bio-matrix scaffold under culture conditions; and f) collecting the protein of interest produced by the cells of (d) and producing the protein of interest in the cells cultured on the bio-matrix scaffold.

The foregoing and other aspects of the present invention will now be described in more detail with respect to other embodiments described herein. It should be appreciated that the present invention may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

Brief description of the drawings

FIGS. 1A-E1: preparing a rat liver biological matrix scaffold. (A) A four-step decellularization process including perfusion wash, defatting with PLA2 and SDC, high salt wash, and nuclease treatment for nucleic acid removal. (B-D) four stages in the preparation of rat biomatrix scaffolds. (B) After 10 minutes of perfusion washing with basal medium, the liver became pale; (C) during defatting, the liver became partially transparent under GC; (D) the final intact scaffold appeared transparent after 40 minutes of perfusion; (E) biomatrix scaffolds shown at low magnification. (E1) The stent infused with rhodamine-labeled dextran particles visually showed increasing flow along the channel from the large vessel to the branch of the small vessel, and no leakage, indicating a proprietary vessel in the stent. Corresponding hematoxylin and eosin (H & E) staining of the biomatrix scaffold at different stages showed that the histological structures encapsulating parenchymal cells, such as blood vessels and the cellular matrix, were retained, while the cells were removed. The normal rat hepatic portal triple tube structure consists of Portal Vein (PV), Hepatic Artery (HA) and Bile Duct (BD) (B1); during the decellularization process, the matrix fibers become transparent as the cells are gradually removed (C1); comparisons (B1) and (D1), decellularized triple hepatic portal areas; d2 and D3 show that all cells were removed from the matrix scaffold, but the network was retained, such as blood vessels, GC, and a mesh-like matrix surrounding parenchymal cell walls.

FIGS. 2A-H: TEM (A-C) and SEM (D-H) images of rat liver biomatrix scaffolds. (A) Low magnification of Blood Vessels (BV) with thick walls (W). Type I collagen (large arrows) is numerous and contains cross sections of fibers that do not take up heavy metal dyes (white dots, small arrows). (B) Higher magnification of the vessel wall shows basement membrane (large arrow), amorphous elastin (, x) and associated elastic fibers, small amount of membrane vesicle residues (small arrow), type I collagen ribbon fibers (arrow) and small fibers (small arrow). The fibrils may be fibrillar proteins (type VI collagen), which are closely associated with and help organize type I collagen. (C) High magnification of type I collagen with 64nm banding pattern (arrow). (D) Low magnification of the walls of blood vessels with thin walls (BV) and larger blood vessels (W). (E) At higher magnification, the great vessel wall (W) is scalloped, consistent with the hepatic artery of the portal triplet, see (a). Below the wall are numerous collagen type I bundles (large arrows) connected by long branched thin, reticular (type III) collagen fibers (small arrows). (F) The large bundles of type I collagen have characteristic parallel fibers (large arrows) that are associated with various smaller fibers (arrows) and nodes or beaded fibers (arrows). (G) A 3D network of interconnected large/small fibers in a plane that form a boundary, for example, the hepatic sinus. (H) Higher magnification of the network shows various fibers (arrows): type III collagen (larger diameter straight line), elastic fibers, or type VI collagen.

FIGS. 3A-B: chemical analysis of collagen in biomatrix scaffolds and expression of extracellular matrix (ECM) components. (A) The content of all three amino acids found in collagen: hydroxyproline (Hyp), hydroxylysine (Hyl) and glycine (Gly). The numbers indicate the residue of each amino acid/1000 amino acids. This data shows a significant increase in collagen content during decellularization, from < 0.2% in the liver to greater than 15% in the biomatrix scaffold. (B) Immunohistochemical staining of matrix molecules in the biomatrix scaffolds revealed the distribution of Laminin (LAM), Heparin Sulfate (HS), collagen type III (COL3), and Fibronectin (FN) in the liver biomatrix scaffolds as well as the typical basement membrane proteins associated with vascular wall residues. At higher magnification, one skilled in the art can observe the major members of the basement membrane, including collagen type IV (COLA), entactin (Ent, also known as endoproteins), Laminin (LAM), and perlecan (Per), which is the form of HS-PG in the scaffold portion near the portal triplet.

FIGS. 4A-D: pattern of ECM components from portal triplet to central vein in the biomatrix scaffold. Histological comparison of normal liver (a) with liver biomatrix scaffold (B) from the portal triplet (region 1) to the central vein (region 3); both are hematoxylin/eosin stained sections. (C) The moldKnown lineage stages in human liver begin and mature around the portal vein in region 1 (around the portal triplet), and end with apoptotic cells in region 3 known matrix chemicals identified in the liver stem cell niche include hyaluronic acid, type III collagen, laminin in form bound to α 6 β 4 integrin, and CS-PG in weakly sulfated form^43、44Type IV collagen, commonly sulfated CS-PG and HS-PG, and laminin in the form of a binding to αβ 1 integrin, were found only outside the stem cell niche HP-PG has been shown to be uniquely located around the center^45、46(D) study of the matrix composition and their location in the liver compared to the matrix composition and its location in a biological matrix scaffold, data summarized from immunohistochemical findings (N/D untested. only near the central vein as found by others.) most of the components of the cytoskeleton were lost during washing, with some but not all of the residues of cytoskeletal proteins present.

FIGS. 5A-I: characterization of hhpscs on liver biomatrix scaffolds compared to type I collagen. Phase contrast images (a-D) show morphological changes of hHpSC colonies derived from the same liver and cultured in serum-free Kubota' medium and on tissue culture plastic (a), a condition for self-replication, in contrast to differentiation conditions in serum-free differentiation medium for liver and on type I collagen (B), in contrast to (C-E) on bovine liver biomatrix scaffolds. Functional and fully viable cultures survived no more than-2 weeks on collagen type I. By contrast, cells on a liver biomatrix scaffold were viable and healthy, and had all constituents of function, and lasted for at least one month. The culture was transformed by 7-12 days into cells with increased cytoplasmic/nuclear ratio and labeled glycogen expression (C) and then into cells with typical polygonal cell morphology interspersed through clear bile canaliculi (D), which continued thereafter as shown by the representative culture at day 24 (E). RT-PCR assays showed changes in gene expression of hHpSC under self-replicating conditions on cultured plastic at day 7 (F) compared to rat liver biomatrix scaffolds (G). We compared the expression, hHpSC markers, including CXCR4 and EpCAM; early hepatocyte genes including CK19(KRT19), HNF6, FOXA2, AFP and low levels of albumin; mature hepatocyte markers including high levels of Albumin (ALB), Transferrin (TF), CYP450-3a4, Tyrosine Aminotransferase (TAT), and glucose-6-phosphatase (G6PC), as well as biliary epithelial genes including CFTR, gamma glutamyl transpeptidase (GGT1), cation exchange 2(AE2), and apical sodium-dependent bile acid transporter (ASBT). Biochemical assays measure urea (H) synthesized on type I collagen with cultures on rat liver biomatrix scaffolds, and measure CYP450-3A4 activity (I) on type I collagen with cultures on biomatrix scaffolds prepared from rat or bovine livers. Table 7 provides a summary of quantitative measurements comparing the attachment, viability, growth, culture life cycle, and tissue-specific gene expression of freshly isolated hhpscs under culture conditions for self-replication (type III collagen), or under conditions for differentiation of type I collagen compared to on liver biomatrix scaffolds.

FIGS. 6A-D: immunofluorescence staining of cells restricted by hHpSC lineage on a biomatrix scaffold. (A) Staining with the liver-specific marker albumin (Alb, light grey) and with the hepatic stem cell surface marker EpCAM (white). Note that cells plated on the biomatrix scaffold did not express EpCAM. Scale bar 200 μm. (B) Staining with early hepatic marker alpha fetoprotein (AFP, light grey) and antibodies against the human bile duct epithelial cell marker cytokeratin 19(CK19, white) revealed mature bile duct epithelial cells at this expression level. Antibody assays against CK19 were human specific and did not stain residues at rat CK19 in scaffolds without cell seeding. AFP expression was low but still evident at day 7. Scale bar 200 μm. (C) Staining with Alb (light grey) and hepatic stellate cell markers, alpha-smooth muscle actin (ASMA, white). The expression of albumin and ASMA strongly indicates the presence of both maturing hepatocytes and astrocytes. Scale bar 100 μm. (D) Staining with the functional hepatic protein CYP450-3a4 (light grey) and the bile duct epithelial cell specific marker secretin receptor (SR, white) shows that maturing hepatocytes and bile duct epithelial cells are functional and express typical markers for both cell types. Scale bar 200 μm.

FIGS. 7A-D: stability of fully functional, mature human hepatocytes on a biomatrix scaffold. Adult hepatocytes were plated in differentiation medium, and on type I collagen (A, B) in contrast to bovine liver biomatrix scaffolds (C), which were cryogenically ground, dispersed in medium and allowed to deposit on the plates. Cells on collagen type I are fully viable and at their peak of differentiation on days 7-12 (a shows day 7); they begin to deteriorate after-2 weeks and by 20 days (B) they die, dying and nonfunctional. By contrast, those cells (C) plated on liver biomatrix scaffolds were functional for at least 8 weeks (not evaluated for longer time yet); shown here after 21 days in culture on crushed liver biomatrix scaffolds. CYP450-3a4 assays for the culture of two independent preparations of frozen adult liver cells plated on a bio-matrix scaffold compared to type I collagen, and was determined on day 12 (D). Sample ZHep-007 represents cryopreserved samples with good adhesion after thawing; sample ZL-013 represents those batches with poor or no adhesion after thawing. Thus, even these poorer quality samples are able to attach to the bio-matrix scaffold and remain viable for long periods of time. In both samples tested, the level of P450s was higher when cultured on liver biomatrix scaffolds. With time on the bio-matrix scaffold, poorer quality cryopreserved batches will improve.

FIG. 8: lysolecithin is produced by activation of phospholipase a2 by sodium deoxycholate. The principle of the scheme is as follows: phospholipase a2(PLA2), activated by sodium deoxycholate, will degrade phosphoglycerides located on the cytoplasmic and mitochondrial membranes to lysolecithin, which is a powerful surfactant that induces cell necrosis.

FIG. 9: collagen composition analysis of rat liver compared to rat liver biomatrix scaffolds. The amino acid composition of the biological matrix (black) and the whole liver (light grey) is presented in the form of a rose. Three letter abbreviations are used for each amino acid analyzed. Tryptophan and cysteine were not analyzed. The numbers show amino acid residues/1000.

FIGS. 10A-C: nucleic acid analysis of rat liver biomatrix scaffolds. Phase contrast map (a) and fluorescent DAPI staining (B) of liver biomatrix slides, and quantitative determination of total DNA and RNA from rat fresh liver tissue compared to biomatrix scaffolds (C).

FIGS. 11A-C: staining of the biomatrix scaffolds after plating of hhpscs onto the biomatrix scaffolds. Living (calcein-AM, white)/dead (ethidium bromide or EtD-Br)₁Light grey) assay showed that hHpSC colonies were viable on biomatrix scaffold sections, but did not take up dye in the middle of the colony (A, B) for the first few days, due to elimination of vital dye by known pumps in stem cells (e.g., MDR 1). In (B), the fluorescence image is combined with the phase image to show that the colony contains cells in the middle. By 7 days, the cells of the whole colony had differentiated and taken up the vital dye (C) in almost all cells of the whole colony.

FIGS. 12A-F: rat hepatocytes cultured on type I collagen compared to rat hepatocytes cultured on rat liver biomatrix scaffolds. Adult rat hepatocytes cultured on type I collagen and on a biomatrix scaffold on days 3 (A, C) and 10 (B, D). They attached to the liver biomatrix scaffold within minutes and survived as long as the test for more than 8 weeks (C, D); no longer time was tested. The culture is very three-dimensional on the bio-matrix scaffold. Urea synthesis (E) and cell viability assay (F) at days 1, 3, 5, 7, 10, 14, 21 and 28, with n-3.

FIGS. 13A-D: comparison of human pancreas with human pancreas biomatrix scaffolds. Comparison of human pancreas (A) embedded in paraffin, sectioned and stained with hematoxylin and eosin (H & E) with human pancreas biomatrix scaffolds (B-D). The islet structure has been depicted in B. The acinar regions of the pancreatic biomatrix scaffold are shown in C and D.

FIG. 14: representative stromal components and a cytoskeletal component vimentin found in human pancreatic tissue compared to rat pancreatic biomatrix scaffolds. No detectable amounts of other cytoskeletal components (desmin, tubulin, actin) or traces of cytoskeletal components (cytokeratin) were found. The dotted line surrounds the islets, noting that both syndecan 1 and type VI collagen are strongly positive in pancreatic tissue and in islets of the biomatrix scaffold. Syndecan 1 is found only in islets (dashed line) and not in acinar cells (arrow); type III collagen is more abundant in acinar cells and perivascular (arrows), but not in islets.

FIGS. 15A-D: histological and immunohistochemical staining of human duodenal biomatrix scaffolds. (A) The lateral and luminal sides of a human duodenal biomatrix scaffold. The multilayered structure between normal tissue (B) and the biomatrix scaffold (C) was compared in H & E stained sections and the results showed that the scaffold retained villi and blood vessels in the mucosal and submucosal layers. (D) Immunohistochemical staining of human duodenal biomatrix scaffolds showed retention of variable amounts of extracellular matrix components in the scaffolds.

FIGS. 16A-D: comparison of human gallbladder tissue with human gallbladder biological matrix scaffolds. Comparison of human gallbladder tissue (A, B) with a biological matrix scaffold (C, D) prepared therefrom. Tissue and biomatrix scaffolds were embedded in paraffin, sectioned and stained with hematoxylin and eosin.

FIGS. 17A-B: the colon tumor cell lines HT29(a) and SW480(E) were seeded on a bio-matrix scaffold and formed cell colonies, which consisted of hundreds to thousands of cells, and they were very three-dimensional.

Detailed Description

The present invention will now be described more fully hereinafter. This invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this specification and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of this application and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

Also, as used herein, "and/or" means and includes any and all possible combinations of one or more of the associated listed items, and no combinations when interpreted as alternatives ("or").

Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted. For purposes of illustration, if the specification states that the complex includes components A, B and C, it is particularly desirable that either one of A, B or C, or a combination thereof, may be omitted or disclaimed, either alone or in any combination.

As used herein, the transitional word "consisting essentially of" (and grammatical variants) should be interpreted as including the materials or steps described, "as well as those materials or steps that do not materially affect one or more of the basic and novel features of the claimed invention. See Inre Herz, 537 F.2d 549, 551-52,190U.S.P.Q.461, 463(CCPA1976) (highlighted herein); see also MPEP § 2111.03. Thus, the term "consisting essentially of, as used herein, should not be construed as being equivalent to" comprising ".

The term "about" as used herein, when referring to a measurable value such as an amount or concentration (e.g., the percentage of collagen in the total protein in a biomatrix scaffold), etc., is intended to include a change of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

The present invention relates to the discovery and development of a biomatrix scaffold that has unexpected improvements and advantages over currently known decellularized tissue scaffolds, some examples of which are the effective maintenance of mature cells and/or lineage restriction and/or differentiation of stem cells into mature fates and/or the maintenance of the function of the mature cells over a long period of time using the biomatrix scaffold of the present invention. As a further example, the use of the biomatrix scaffold of the present invention reduces the time to generate cells of mature fate from about 3-6 weeks or more to about 1-2 weeks. The biomatrix scaffold of the present invention is produced using a specific protocol that utilizes an appropriate balance of salt concentration and ionic strength (different collagens have different solubility constants (23)) for a given tissue such that native collagen present in the tissue in an insoluble form is retained, resulting in a biomatrix scaffold retaining a high percentage of native collagen that provides signals to drive lineage restriction and differentiation. In contrast, decellularized scaffolds produced according to known protocols do not utilize such a balance of salt concentration and ionic strength that a high percentage of these native collagens are retained, while most of these native collagens are lost when using these known protocols. In addition, the bio-matrix scaffolds of the present invention allow for the generation of lineage-dependent (e.g., differentiation-dependent) viruses and/or pathogens in sufficient quantities for experimental and/or therapeutic uses (e.g., for vaccine production).

Thus, in one embodiment, the present invention provides a method of producing a biomatrix scaffold from a biological tissue comprising the steps of: a) perfusing or homogenizing a biological tissue with a first culture fluid, wherein the osmolality of the first culture fluid is about 250mOsm/kg to about 350mOsm/kg, and the first culture fluid is serum-free and at a neutral pH; then b) perfusing the biological tissue of step (a) or extracting the homogenate of step (a) in the first culture broth with a degreasing buffer comprising a lipase and/or a detergent; then c) perfusing the tissue of step (b) or extracting the homogenate of step (b) with a buffer at neutral pH and comprising a salt concentration of about 2.0M NaCl to about 5.0M, the concentration being selected to keep the collagen identified in the biological tissue insoluble; then d) perfusing the tissue of step (c) or extracting the homogenate of step (c) with ribonuclease (RNase) and deoxyribonuclease (DNase) in a buffer; and then e) rinsing the tissue or homogenate of step (d) with a second culture fluid at neutral pH, serum-free, and having an osmolality of about 250 to about 350mOsm/kg, thereby producing an intact or homogenized biological matrix scaffold from the biological tissue, the biological tissue scaffold comprising at least 95% (e.g., 80%, 85%, 90%, 95%, 98%, 99%, 100%) of collagen and a majority of collagen-binding matrix components and matrix-bound growth factors, hormones, and cytokines of the untreated biological tissue. Also provided herein are biomatrix scaffolds produced by any of the methods of the invention.

As used herein, "biomatrix scaffold" refers to an isolated tissue extract enriched in extracellular matrix that retains much or most of the collagen and collagen binding factors naturally occurring in biological tissues, as described herein. In some embodiments, substantially all of the collagen and collagen binding factors are retained, and in other embodiments, the biomatrix scaffold comprises all of the collagen known to be in the tissue. The biological matrix scaffold can comprise collagen, collagen-binding matrix components, and/or matrix-binding growth factors, hormones, and/or cytokines in any combination in at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% of any combination in native biological tissue. In some embodiments, the biomatrix scaffold comprises at least 95% collagen in biological tissue and a majority of collagen-binding matrix components in biological tissue, and matrix-binding growth factors, hormones, and/or cytokines. As used herein, "a majority of collagen-binding matrix components, and matrix-binding growth factors, hormones, and/or cytokines in a biological tissue" refers to about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% of the collagen-binding matrix components, and matrix-binding growth factors, hormones, and/or cytokines present in native (e.g., untreated) biological tissue retained by the biological matrix scaffold.

Exemplary collagens include all types of collagen, such as, but not limited to, types I through XXIX collagen. The biological matrix scaffold can comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more of one or more collagens found in native biological tissue and/or can have one or more collagens present at a concentration of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more of that found in native biological tissue. The amount of collagen in the biomatrix scaffold may be determined by various methods known in the art and as described herein, such as, but not limited to, determining hydroxyproline content.

Exemplary collagen binding matrix components include, but are not limited to, adhesion molecules; an adhesion protein; l-and P-selectin; heparin binding growth related molecule (HB-GAM); thrombospondin type I repeat (TSR); amyloid p (ap); laminin; endothelin/nestin; fibronectin; elastin; vimentin; proteoglycans (PGs); chondroitin sulfate-PG (CS-PG); dermatan sulfate-PG (DS-PG); SLRP family members such as biglycan and decorin; heparin-PG (HP-PG); heparin sulfate-PG (HS-PG), such as phosphatidylinositolglycan, syndecan, and perlecan; and Glycosaminoglycans (GAGs) such as hyaluronic acid, heparin sulfate, chondroitin sulfate, keratan sulfate, and heparin. In some embodiments, the biomatrix scaffold comprises, consists of, or consists essentially of (in any combination) collagen, fibronectin, laminin, endothelin/nidogen, elastin, proteoglycans, glycosaminoglycans, growth factors, hormones, and cytokines bound to various matrix components. The biological matrix scaffold may comprise at least about 50%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more of one or more collagen-binding matrix components, hormones, and/or cytokines found in native biological tissue, and/or may have one or more of these components present at a concentration of at least about 50%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% or more found in native biological tissue. In some embodiments, the biomatrix scaffold comprises all or most of the collagen-binding matrix components, hormones, and/or cytokines known to be in tissue. In other embodiments, the biological matrix scaffold comprises, consists essentially of, or consists of one or more collagen-binding matrix components, hormones, and/or cytokines at concentrations that are close to those found in native biological tissue (e.g., about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% of the concentrations found in native tissue).

Exemplary growth factors include, but are not limited to, Fibroblast Growth Factor (FGF), Nerve Growth Factor (NGF), Epidermal Growth Factor (EGF), transforming growth factor, Hepatocyte Growth Factor (HGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), IGF binding protein, basic fibroblast growth factor, and Vascular Endothelial Growth Factor (VEGF). Exemplary cytokines include, but are not limited to, interleukins, lymphokines, monokines, colony stimulating factors, chemokines, interferons, and Tumor Necrosis Factor (TNF). The biological matrix scaffold may comprise at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 100% or more (in any combination) of one or more matrix-bound growth factors and/or cytokines found in native biological tissue, and/or may have one or more of these growth factors and/or cytokines (in any combination) present at a concentration of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, 100% or more of that found in native biological tissue. In some embodiments, the biological matrix scaffold comprises a physiological or near physiological level of many or most of the matrix-bound growth factors, hormones, and/or cytokines known to be detected in native tissue and/or in tissue, and in other embodiments, the biological matrix scaffold comprises one or more matrix-bound growth factors, hormones, and/or cytokines at concentrations close to those physiological concentrations found in native biological tissue (e.g., no more than about 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% contrast). The amount or concentration of growth factors or cytokines present in the biomatrix scaffold may 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, "biological tissue" refers to any tissue of or derived from a living or dead organism. The term "native biological tissue" and variations thereof as used herein refers to a biological tissue when it is present in its native or unmodified state in an organism. The biomatrix scaffold of the present invention may be prepared from any biological tissue. A biological tissue may include any single tissue (e.g., a collection of cells that may be interconnected) or a group of tissues that make up an organ or portion or region of an organism. The tissue may comprise homogeneous cellular material or it may be a composite structure such as found in a region of the body including the breast, which may include, for example, lung tissue, skeletal tissue, and/or muscle tissue.

Exemplary biological tissues of the invention include, but are not limited to, liver, lung, thyroid, skin, pancreas, blood vessels, bladder, kidney, brain, biliary system, duodenum, abdominal aorta, iliac vein, heart, and intestine, including any combination thereof. The organism (i.e., subject) to which the biological tissue is related or derived can be any animal, including mammals and non-mammals such as invertebrates.

Exemplary subjects include, but are not limited to, mammals, such as, but not limited to: human, mouse, rat, ferret, hamster, rabbit, guinea pig, pig (pigs), pig (porcine), dog, cat, horse, cow, sheep, monkey, and chimpanzee, as well as non-mammalian animals such as, but not limited to: birds, reptiles, and invertebrates. The subject may be of any age and/or size. The biological tissue may be healthy, diseased, and/or have a genetic mutation. In some embodiments, the biomatrix scaffolds of the present invention are tissue-specific in their chemical and functional aspects, i.e., the biomatrix scaffolds are representative of, or comparable to, the biological tissue from which they were produced in their chemical and functional aspects.

In some embodiments, native tissue and proprietary vessels (patent vastus) are retained in the biomatrix scaffold. This may include identifiable residues of major tissue entities of biological tissue, such as but not limited to blood vessels and other vessels for any tissue; bile ducts and the Grignard Capsule (GC) of the liver; pancreatic ducts, pancreatic islets and acini of the pancreas; the bronchi, trachea and alveoli of the lungs, etc. In other embodiments, the chemistry of the biomatrix scaffold is histologically matched (e.g., the matrix surrounding the blood vessels is different than the matrix surrounding the hepatocytes). In some embodiments, the chemistry of the biomatrix scaffold is in a histologically relevant gradient. For example, where the biological tissue is the liver, the biological matrix scaffold may retain a gradient of matrix chemistry associated with hepatic acinar regions 1-3 from the portal triplet to the central vein and with histological entities such as vascular channels and the Glehns Capsule (GC). Other examples include, but are not limited to, blood vessels, where the chemistry of the perivascular matrix is saturated with high levels of network collagen (e.g., type IV and VI), elastin, various forms of HS-PG; around the hepatocytes of the periportal region (zone 1), where laminin has a high concentration along with a mixture of CS-PG and HS-PG, and around the central peripheral region (zone 3), are hepatocytes surrounded by a mixture of HS-PG and HP-PG; associated with the bile duct, there are high levels of collagen type I, fibronectin, and various forms of CS-PG and DS-PG. There is a parallel gradient of matrix chemicals in each tissue.

There are a variety of wash media, such as first and second media, and buffers that can be used in the present invention. In particular, any wash medium or buffer that maintains collagen and binding factors (e.g., matrix components, growth factors, and cytokines) in an insoluble state can be used. In selecting a culture or buffer, the salt concentration, pH, and ionic strength should be suitable to maintain the collagen and/or most or many of the collagen binding matrix components and other factors in an insoluble state (e.g., by virtue of their chemical attachment directly or indirectly to the collagen). Table 1 provides molar concentration ranges of sodium chloride for each type of collagen to assist one of ordinary skill in the art in providing a culture or buffer that ensures that the collagen, collagen-binding matrix components, and matrix-binding growth factors and cytokines remain insoluble. Deyl et al ("preliminary products and purityassessment of collagen proteins" Journal of Chromatography B790 (2003)) 245-275 additionally provide information about collagen chemicals that may facilitate the identification of optimal conditions for maintaining the insoluble state of collagen and binding factors, and are incorporated herein by reference in their entirety.

Table 1 shows that pH is a variable that works with salt concentration to define solubility. By having a high salt concentration, the pH can be neutral. In some embodiments of the invention, the selected salt concentration is the concentration of all tissue collagens that maintain an insoluble state, rather than the concentration of only one tissue collagen that maintains an insoluble state. For example, collagen known in fetal liver is insoluble in salt concentrations of about 4.5M NaCl, while those in somatic liver tissue are insoluble in salt concentrations of about 3.4M-3.5M NaCl.

The osmolality of any of the flushing media and/or buffers can be, for example, 200 to about 400mOsm/kg, about 250 to about 350mOsm/kg, about 275 to about 325mOsm/kg, or about 300 to about 325mOsm/kg, including but not limited to any values within these ranges not expressly stated. Distilled water and dilution buffers (e.g., having osmolality < 100mOsm/kg) will result in the loss of significant amounts of collagen, collagen-bound matrix components, and matrix-bound growth factors and cytokines. Thus, in some embodiments of the methods of the invention, distilled water and dilution buffer are not included.

As one of ordinary skill in the art will appreciate, osmolality is the expression of the osmolality per mass of solute, and osmolality is the concentration of solute per volume. Thus, the conversion from osmolality to osmolality can be obtained by multiplying by the mass density. Osmolality can be measured using an osmometer that measures colligative properties such as freezing point depression, vapor pressure, and boiling point elevation.

Osmolarity is a measure of solute concentration, defined as the number of osmolality moles (Osm) of solute per liter (L) of solution (osmol/L or Osm/L). The osmolarity of the solution is usually expressed as Osm/L. While molarity measures the number of moles of solute per unit volume of solution, osmolarity measures the number of moles of solute particles per unit volume of solution. Osmolality is a measure of the osmolality (osmol/kg or Osm/kg) of solute per kilogram of solvent.

Molarity and osmolarity are not commonly used in osmometry because they are temperature dependent. This is because water changes its volume with temperature. However, if the solute concentration is very low, the osmolality and the osmolality are considered equal.

The osmolarity of a solution can be calculated by the expression:

whereinIs the permeability coefficient, which accounts for the degree of non-ideality of the solution; n is the number of particles (e.g., ions) from which the molecules dissociate; c is the molarity of the solute; and the index i represents the identity of a particular solute. In the simplest case of the above-mentioned case,is the degree of dissociation of the solute. Then, the user can use the device to perform the operation,between 0 and 1, where 1 represents 100% dissociation. However,and may also be greater than 1 (e.g., for sucrose). For salts, electrostatic effects result even when 100% dissociation occursAlso less than 1.

Perfusion of the biological tissue with any culture fluid or buffer may be accomplished by forcing the culture fluid or buffer through the relevant vessels of the biological tissue. For example, if the biological tissue is liver, culture fluid or buffer may be perfused through the portal vein of the liver. Alternatively, the culture fluid or buffer may be poured onto the biological tissue and/or allowed to diffuse through the biological tissue. For example, the biological tissue may be submerged and/or dialyzed in a culture fluid or buffer to allow the culture fluid or buffer to diffuse through the biological tissue. The solution and biological tissue may be agitated, such as on a rocker and/or while submerged and/or dialyzed in the culture fluid or buffer. In some embodiments, the culture fluid and buffer fluid are perfused through the associated vessel of the biological tissue.

Alternatively, the tissue may be homogenized in the initial culture and buffer and then the culture used for homogenization. A homogenized form of the biomatrix scaffold is prepared from large organs (e.g., from bovine or porcine tissue) and then pulverized into a powder at liquid nitrogen temperature and the powder is used on dishes for culture studies.

In some embodiments, the first broth and/or the second broth is a basal broth, such as, but not limited to, RPMI1640, DME/F12, DME, F12, BME, DMEM, Waymouth broth, or William broth. Other exemplary basal media are known in the art and are commercially available. The first culture fluid and/or the second culture fluid can comprise, consist essentially of, or consist of a composition that is combined to keep a majority of collagen insoluble and as a native molecule as described herein (e.g., by a particular combination of osmolality and ionic strength and absence of serum). The first culture fluid and/or the second culture fluid may comprise, consist of, or consist essentially of components that are present or similar to or mimic those components present in tissue fluid, such as, but not limited to: water; salts, such as but not limited to inorganic salts; a vitamin; a mineral; amino acids such as, but not limited to, glycine, serine, threonine, cysteine, asparagine, and/or glutamine; a sugar; a fatty acid; a coenzyme; a hormone; and neurotransmitters. In certain embodiments, wherein the first culture fluid and/or the second culture fluid comprises components that are present or similar or mimic those components present in tissue fluid, these components can produce osmolality approximately equal to that of a commercially available basal culture fluid, or produce osmolality of about 250mOsm/kg to about 350 mOsm/kg. In some embodiments, the first culture fluid and/or the second culture fluid comprises serum-free, comprises components present in a tissue fluid, and/or has an osmolality of about 250mOsm/kg to about 350 mOsm/kg. The culture fluid may also be at neutral pH. The specific composition of the first culture fluid and/or the second culture fluid is determined in certain embodiments by the insolubility constant of the biological tissue collagen used to prepare the biomatrix scaffold, as is known to those of ordinary skill in the art.

The degreasing buffer of the invention should be efficient and also gentle. The degreasing buffer may comprise, consist of, or consist essentially of a detergent or surfactant, a basal medium, a salt, and/or a lipase. In selecting ingredients for the degreasing buffer, harsh, powerful detergents (e.g., sodium dodecyl sulfate; TritonX-100) should be avoided to minimize loss of matrix components. Exemplary detergents of the invention include, but are not limited to, anionic detergents such as deoxycholate, 1-heptane sulfonic acid, N-lauroylsarcosine, lauryl sulfate, 1-octane sulfonic acid, and salts of taurocholic acid; cationic detergents such as benzalkonium chloride, cetylpyridinium chloride, methylbenzyloxyammonium chloride, and decahydrocarbonium bromide; zwitterionic detergents such as alkyl betaines, alkylamidoalkyl betaines, N-dodecyl-N, N-dimethyl-3-amino-1-propanesulfonate, and phosphatidylcholine; and non-ionic detergents such as n-decyl alpha-D-glucopyranoside, n-decyl beta-D-maltopyranoside, n-dodecyl beta-D-maltopyranoside, n-octyl beta-D-glucopyranoside, sorbitan ester, n-tetradecyl beta-D-maltopyranoside, triton, Nonidet-P40, Poloxamer 188, and any detergent Tween group; sodium lauryl sulfate; and sodium deoxycholate. In some embodiments, the degreasing buffer comprises sodium deoxycholate.

Exemplary lipases include, but are not limited to: phospholipases such as phospholipase A2, human pancreatic lipase, sphingomyelinase, lysosomal lipase, endothelial lipase, and hepatic lipase. In some embodiments, the degreasing buffer comprises phospholipase a 2. In other embodiments, the degreasing buffer comprises sodium deoxycholate and phospholipase a 2. In some embodiments, the combination can comprise about 20 to about 50 units/L of phospholipase a2 and about 1% sodium deoxycholate, prepared in a neutral pH and serum-free basal medium, which can be, for example, the first medium. The combination of sodium deoxycholate and phospholipase a2 rapidly degrades phosphoglycerides located on the plasma and mitochondrial membranes to lysolecithin, a potent surfactant that induces necrosis and cell lysis. As one of ordinary skill in the art can appreciate, the amount and type of lipase and/or detergent can depend on the biological tissue.

In some embodiments, the step of perfusing the biological tissue with a degreasing buffer is performed until the tissue becomes transparent. In other embodiments, the step of perfusing the biological tissue with a degreasing buffer is performed until the exudate becomes transparent. In some embodiments, the defatting step is performed until the tissue becomes transparent and the exudate becomes transparent.

In some embodiments, prolonged exposure of the biomatrix scaffold to enzymes from ruptured cells is avoided because it can greatly reduce elastin content and content of glycosaminoglycans such as heparin sulfate, chondroitin sulfate, dermatan sulfate, and heparin, which are sites for cytokine and growth factor binding. Exposure to enzymes from ruptured cells may be avoided, for example, during defatting and/or subsequent washing after defatting. In some embodiments, the use of protease inhibitors and/or careful control of pH, temperature, and/or time may be used to limit the activity of proteases and/or other enzymes from ruptured cells.

Exemplary protease inhibitors include, but are not limited to: serine protease inhibitors such as, but not limited to, antinociceptin, aprotinin, chymotrypsin inhibitor, elastase inhibitor, phenylmethanesulfonyl fluoride (PMSF), APMSF, TLCK, TPCK, leupeptin, and soybean trypsin inhibitor; cysteine proteases such as, but not limited to, IAA (indoleacetic acid) and E-64; aspartic protease inhibitors such as, but not limited to, pepstatin and VdLPFFVdL; metalloproteinases such as, but not limited to, EDTA, 1, 10-phenanthroline and amiloride phosphate (phosphamodon); exopeptidases such as, but not limited to, aminopeptidase inhibitors, bestatin, aprotinin a and aprotinin B; a thiol protease; alpha-2-macroglobulin, soybean or lima bean trypsin inhibitor; (ii) a trypsin inhibitor; egg white egg fixed protein; an egg white cysteine protease inhibitor; and combinations of protease inhibitors, commonly referred to by the inhibitor supplier as "protease inhibitor cocktails".

The pH of the bio-matrix scaffold, buffer, and/or culture solution can be maintained at about 6.0 to about 9.0, about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.5 to about 8.0. In some embodiments, the pH of the biological matrix scaffold, buffer, and/or culture fluid is maintained at about 7.5 to about 8.0 or at about 7.3 to about 7.5, including but not limited to any values included within these ranges but not explicitly recited herein. In other embodiments, the bio-matrix scaffold, buffer, and/or culture fluid maintains a neutral pH. The temperature of the bio-matrix scaffold (e.g., during and/or after preparation), buffer, and/or culture fluid can be about 0 ℃ to about 30 ℃, about 5 ℃ to about 25 ℃, or about 10 ℃ to about 20 ℃, including, but not limited to, any value included within these ranges but not explicitly recited herein. In some embodiments, the temperature is maintained at about 20 ℃. The time for perfusing the biological tissue with any culture fluid or buffer can be about 5 hours or less, about 3 hours or less, about 1 hour or less, about 30 minutes or less, or about 15 minutes or less. In some embodiments, the step of perfusing the biological tissue with the degreasing buffer is about 30 minutes or less. In some embodiments in which an acidic pH is used, the salt concentration used to maintain insolubility of the collagen and collagen-related components may be different; this concentration is determined by the existing literature on collagen chemistry by choosing a concentration of salts that maintain the insolubility of collagen.

Exemplary buffers include, but are not limited to, sodium chloride, sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate, sodium gluconate, citrate buffer, bis \ tris buffer, phosphate buffer, potassium phosphate, citrate/glucose, sodium bicarbonate, ammonium chloride, 3- { [ tris (hydroxymethyl) methyl ] amino } propanesulfonic acid, tris (hydroxymethyl) methylamine, N-tris (hydroxymethyl) methylglycine, 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, and 3- (N-morpholino) propanesulfonic acid.

In some embodiments, the buffer of the invention (e.g., for use in the steps described herein)Buffer(s) may comprise a salt concentration of about 2.0M or higher. For example, in some embodiments, the salt concentration may be from about 2.0M to about 5.0M, from about 2.5M to about 5.0M, from about 3.0M to about 4.5M, or from about 3.0M to about 4.0M, including but not limited to any values included within these ranges but not explicitly stated herein. For example, in some embodiments, the buffer used in the methods of the invention may comprise a salt, such as sodium chloride, at a concentration of about 2.0M NaCl to about 4.5M NaCl. In other embodiments, such as those for adult liver, the buffer used may comprise from about 3.4M to about 3.5M naci. In embodiments such as for fetal liver, the buffer used may comprise a salt such as sodium chloride at a concentration of about 4.0M to about 4.50M. In some embodiments, perfusing biological tissue with saline solution, such as in step c) of the exemplary methods described herein, is performed until the perfusate (i.e., the liquid used for perfusion, such as the liquid that has been forced through the vessel) is negative for proteins by an Optical Density (OD) of 280 nm.

Any of the culture fluids and/or buffers of the present invention may comprise a protease inhibitor. Exemplary protease inhibitors are described above. In some embodiments, a buffer, such as the buffer in step c) of the exemplary methods described herein, comprises a protease inhibitor, such as a soybean trypsin inhibitor. In other embodiments, the buffer of step d) comprises one or more protease inhibitors, such as soybean trypsin inhibitor.

The culture and/or buffer of the invention may comprise one or more nucleases, which in some embodiments may be prepared in standard buffers recommended by the supplier of these enzymes. For example, in some embodiments, the buffer of step d) comprises one or more nucleases, such as, but not limited to, rnases and dnases. Perfusion with nuclease eliminated the residual nucleic acid. In other embodiments, the buffer of step d) comprises an RNase, a DNase, and one or more protease inhibitors. In some embodiments, perfusion of biological tissue with one or more nucleases is performed until the perfusate (i.e., the liquid used for perfusion, such as the liquid that has been forced through the vessels) is negative for nucleic acids by an Optical Density (OD) of 260 nm. In some embodiments, the nuclease eliminates 75%, 80%, 85%, 90%, 95%, 98%, or 100% of nucleic acids in the biological tissue.

The second medium (e.g., the final rinse medium) can be any medium that ensures that the collagen and binding factors (e.g., matrix components, growth factors, and cytokines) will remain insoluble as described above. Exemplary final rinse media are as described above for the first media, and are serum-free, at neutral pH, and have an osmolality of 250-350 mOsm/kg. For example, in some embodiments, the second culture fluid comprises a basal culture fluid. In some embodiments, the second culture liquid is a serum-free basal culture liquid. In other embodiments, the second culture liquid is a serum-free, hormone-defined culture liquid (HDM) comprising hormones, growth factors, lipids, and serum albumin, and which is tailored to the needs of the cells to be cultured. An exemplary second medium is Kubota's medium (Kubota and Reid, PNAS 97: 12132-. In certain embodiments, the second culture broth may or may not comprise a supplement of serum or serum-derived factors, such as, but not limited to, human serum albumin. In some embodiments, rinsing the tissue with the second culture fluid eliminates residual degreasing buffer and nucleic acids. In other embodiments, washing with the second culture fluid and/or any subsequent buffer or culture fluid allows equilibration of the bio-matrix scaffold with the culture fluid or buffer. In some embodiments, the first culture fluid and the second culture fluid may be the same, while in some embodiments, the first culture fluid and the second culture fluid may be different, thereby producing a biological matrix scaffold from a biological tissue.

In some embodiments, none (i.e., no detectable amount) of the one or more culture fluids and/or buffers used in the preparation of the biomatrix scaffold contains one or more enzymes that degrade extracellular matrix components. In other embodiments, all of the culture and buffer solutions used in the preparation of the biomatrix scaffold are free of (i.e., do not contain detectable amounts of) one or more enzymes that degrade extracellular matrix components. Exemplary enzymes include, but are not limited to, collagenase; a protease; glycosidases, such as heparinase, heparanase, chondroitinase, and hyaluronidase; and elastase.

The sterilization of the biological tissue, homogenate, and/or biomatrix scaffold of the present invention may be accomplished by any method known in the art with the caveat that the use of factors that bind to the biomatrix scaffold (e.g., ethylene oxide) should be avoided. Exemplary sterilization methods include, but are not limited to, gamma radiation, Radio Frequency Glow Discharge (RFGD) plasma sterilization, electron beam sterilization, and supercritical carbon dioxide sterilization. In some embodiments, sterilization of the tissue, homogenate, and/or biomatrix scaffold is accomplished using about 5,000rads of gamma radiation. Sterilization may not be required if the scaffold is used immediately for recellularization, and if sterile procedures are used during decellularization (particularly after high salt extraction).

Preservation of the bio-matrix scaffold may be accomplished by methods known in the art. In some embodiments (e.g., when the scaffold is to be used intact), the bio-matrix scaffold may be stored at about 4 ℃, and in other embodiments (e.g., when the scaffold is to be dispersed into small pieces), the bio-matrix scaffold is frozen at, for example, about-80 ℃.

In some embodiments, the biomatrix scaffold comprises, consists of, or consists essentially of collagen, fibronectin, laminin, endothelin/nidogen, elastin, proteoglycans, glycosaminoglycans, and any combination thereof, all of which are part of (e.g., bound to) the biomatrix scaffold. In some embodiments, the biomatrix scaffold lacks detectable amounts of collagen, fibronectin, laminin, endothelin/nidogen, elastin, proteoglycans, glycosaminoglycans, and any combination thereof.

The bio-matrix scaffolds of the present invention have been demonstrated to be potent differentiation groups of cells and can be used for many cell types, such as but not limited to any mature cell or for various stem cell populations. These include, for example, Embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, embryonic stem cells (e.g., definitive endodermal stem cells), definitive stem cells (e.g., hepatic, pulmonary, pancreatic, or intestinal stem cells), human hepatic stem cells (hHpSC), perinatal stem cells (e.g., amniotic fluid derived stem cells (AFSC)), Mesenchymal Stem Cells (MSC) such as bone marrow-derived or adipose tissue-derived, committed progenitors, adult cells of any type, diseased cells, tumor cells, mature cells, parenchymal cells, stellate cells, biliary epithelial cells, biliary lineage cells such as cells other than biliary epithelial cells, hepatocytes, kidney cells, urothelial cells, mesenchymal cells, smooth or skeletal muscle cells, muscle cells (muscle stem cells), fibroblasts, chondrocytes, adipocytes, fibroblastoblasts (fibroblastoses), endothelial cells, mesenchymal stem cells, pancreatic cells, endothelial cells, and adipose cells, Ectodermal cells include tough and skin cells, nerve cells, islet cells, cells present in the intestine, osteoblasts, other cells that form bone or cartilage, and any combination thereof. These cells may be normal or diseased.

In some embodiments, the biomatrix scaffold is used in biological, pharmaceutical, genetic, molecular, and/or virological studies of cells, whether freshly isolated from tissue or isolated from lineage-restricted stem cells. In other embodiments, the bio-matrix scaffold is used in an implantable, vascularized engineered organ, such as, but not limited to, a liver. Other exemplary uses for the bio-matrix scaffold include, but are not limited to, protein manufacture, drug toxicology testing, drug development, antibody screening, and/or virus production for vaccine preparation of a virus. Virus production of lineage-dependent viruses (e.g., papillomavirus and hepatitis c) can be achieved by plating stem cell populations on tissue-specific bio-matrix scaffolds, followed by culture in culture broth that cooperates with the bio-matrix scaffolds to completely induce differentiation of the cells. These mature virions will be produced when the cell is fully mature. Mature cells infected with the virus can be maintained for at least eight weeks as long as the virus itself does not affect cell viability, thereby providing a means for producing large quantities of virus using a stable culture system.

The bio-matrix scaffold may be used intact, such as but not limited to for 2-D and/or 3-D culture of cells. In some embodiments, the bio-matrix scaffold may be used in combination with specific culture fluids for differentiation in 2-D and/or 3-D culture of cell lines, such as, but not limited to, normal or diseased cells from stem cells to late cells from any mature lineage stage.

Alternatively, the bio-matrix scaffold may be frozen. These frozen sections can be prepared and used as a matrix. The bio-matrix scaffold can be flash frozen on dry ice and the frozen sections prepared with a cryostat, placed on a culture device (e.g., dish, flask, fabric, transfer well, etc.), sterilized and rehydrated in culture, and then seeded with cells. In some embodiments, the frozen biomatrix scaffold of the present invention may be sectioned.

In some embodiments, a cell culture is produced comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of step (a) with a culture solution of a cell culture in a culture device; and c) seeding the bio-matrix scaffold of step (b) with cells, thereby producing a cell culture.

In some embodiments, a cell culture is produced comprising: a) producing a biomatrix scaffold of the invention; b) freezing the bio-matrix scaffold of step (a); c) preparing frozen sections from the bio-matrix scaffold from step (b) as cell culture medium; d) contacting the cell culture medium of step (c) with a culture medium of the cell culture in a culture device; and e) seeding the cell culture medium of step (d) with cells, thereby producing a cell culture.

In other embodiments, the bio-matrix scaffold may be ground into a powder. One method of grinding the bio-matrix scaffold into powder comprises grinding the bio-matrix scaffold into powder in a cryomill at or near liquid nitrogen temperature. Other means for grinding (e.g., freezing with dry ice) at liquid nitrogen or equivalent temperatures are known in the art. The powder can be brought to room temperature, where it obtains the consistency of a paint that can be coated on the culture device using a sterile paint brush or equivalent device. The powder or plate may be sterile.

Thus, in some embodiments, a cell culture is produced comprising: a) producing a biomatrix scaffold of the invention; b) grinding the bio-matrix scaffold of step (a) into a powder; c) coating a culture device with the powder of step (b) to produce a cell culture medium; d) contacting the cell culture medium of (c) with a culture medium of a cell culture in a culture device; and e) inoculating the cell culture medium of (d) with the cells, thereby producing a cell culture. In some embodiments of the method, the grinding of the biological substrate is performed in a cryomill (e.g., cryomilling) at or near liquid nitrogen temperature.

In some embodiments, a portion of the culture fluid is added to the culture device before seeding the cells for cell culture, as the cells can attach within seconds. In some embodiments, for normal adult cells, the cells attach within seconds to minutes, and for various types of stem cells, the cells attach within minutes to hours. In some embodiments, the attachment of the cells may depend on how the bio-matrix scaffold is dispersed for use in culture. The cell culture fluid may be any fluid suitable for producing a cell culture. In some embodiments, a culture fluid of a cell culture comprises at least one component present in a tissue fluid, wherein the osmolality of the culture fluid is from about 250mOsm/kg to about 350mOsm/kg, wherein the culture fluid is serum-free and wherein the pH is neutral. In other embodiments, the culture medium for cell culture can be a basal medium such as, but not limited to, RPMI-1640, DME/F12, Ham broth, Kubota broth, and the like.

In some embodiments, the cell culture produced with the biomatrix scaffold comprises, consists essentially of, or consists of cells of the same type as the biological tissue cells used to prepare the biomatrix scaffold. Non-limiting examples of cells of the invention include: embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, definitive stem cells, perinatal stem cells, amniotic fluid derived stem cells (AFSCs), Mesenchymal Stem Cells (MSCs) from any source, committed progenitor or adult cells of any tissue type, mature cells, normal cells, diseased cells, tumor cells, and any combination thereof. Additional non-limiting examples include liver cells, parenchymal cells, stellate cells, endothelial cells, hepatocytes, biliary epithelial cells, biliary lineage cells that are not biliary epithelial cells, and pancreatic cells.

In some embodiments, the naive stem cells (whether ES, iPS, MSC or AFSC) will, at least in part, limit cell lineage to the tissue type used to prepare the biomatrix scaffold. Where scaffolds are made from tissue derived from a given germ layer, the definitive stem cells of that germ layer limit the lineage to the type of tissue used to make the biomatrix scaffold, and may partially differentiate into adult fates if on scaffolds derived from tissue of a different germ layer. Thus, the ability of adult cells to fully differentiate can be determined by the tissue type of the biomatrix scaffold. In parallel, the fate of the stem cells may be determined in part or in whole by the tissue type of the bio-matrix scaffold, or the fate of the stem cells may be determined in whole by the tissue type of the bio-matrix scaffold. In some embodiments, the cells of the cell culture are of a different type than the cells of the biological tissue used to prepare the biomatrix scaffold. As described in detail above, exemplary cell types that may be used to generate the cell culture include, but are not limited to, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, amniotic fluid derived stem cells, definitive stem cells, mature cells, normal cells, diseased cells, tumor cells, and any combination thereof. These cells may be from any of the biological tissues described herein.

In some embodiments, the biomatrix scaffold induces slow growth or growth arrest associated with normal cell differentiation, whether stem cells or mature cells. In some embodiments, mature cells are fully differentiated within hours and thereafter remain stably differentiated for at least eight weeks. In some embodiments, adult cells (i.e., fully mature cells) attach to the scaffold within minutes and thereafter retain their full differentiation for more than eight weeks. In some embodiments, the stem cells undergo several divisions and then enter growth arrest and fully differentiate. Stem cells remain stable, viable and fully differentiated in growth arrest for at least 8 weeks. In some embodiments, the stem cells seeded on the biomatrix scaffold enter growth arrest or slow growth, lose stem cell markers and differentiate into mature, functional cells within about one week, remain stable phenotype and viability for at least eight weeks or more (e.g., for extended periods of time (e.g., for at least one week, at least two weeks, at least three weeks, at least four weeks, at least five weeks, at least six weeks, at least seven weeks, at least eight weeks, at least nine weeks, at least ten weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least 11 months, at least one year, etc.)) of 40%, 50%, 60%, or more, 70%, 80%, 90%, 95% or 100% viability).

In other embodiments, the biomatrix scaffold is used to differentiate Embryonic Stem (ES) cells and/or Induced Pluripotent Stem (IPS) cells into specific fates. For example, in some embodiments, tissue-specific biomatrix scaffolds are used to promote differentiation of embryonic stem cells or induced pluripotent stem cells to a particular fate.

In certain embodiments of the invention, the biomatrix scaffold is used to differentiate amniotic fluid derived stem cells (AFSCs), or Mesenchymal Stem Cells (MSCs) from bone marrow or from adipose tissue or from any fetal or postnatal tissue, or any determinative stem cells (e.g., lung, intestine, biliary lineage, kidney, skin, heart, etc.) towards a specific adult fate. In some embodiments, the biomatrix scaffold of the present invention is used to enhance and accelerate the differentiation of stem cells into mature cells by generating cell cultures. In other embodiments, the invention provides methods of enhancing and/or accelerating differentiation of stem and/or progenitor cells into mature cells comprising producing a cell culture according to the methods of the invention, wherein the cells are stem cells, and a culture broth for the cell culture is formulated against the mature cells, thereby enhancing and/or accelerating differentiation of the stem and/or progenitor cells into mature cells. The culture solution for cell culture may be any culture solution formulated for mature cells. The components in the culture broth are different for each cell type. Differentiation means that these conditions cause the cells to mature into adult cell types that produce adult-specific gene products. The cells used in these methods may be any type of adult or stem or progenitor cell, non-limiting examples of which include embryonic stem cells, induced pluripotent stem cells, germ layer stem cells, definitive stem cells, perinatal stem cells, amniotic fluid derived stem cells, mesenchymal stem cells, transient expanded cells, or committed progenitors of any tissue type.

Cells seeded on a bio-matrix scaffold (e.g., an intact bio-matrix scaffold, a scaffold slice, or a powdered bio-matrix scaffold mixed in/on other implantable materials) can be transplanted into an animal or human as a method of transplanting cells in vivo. In some embodiments, there is provided a method of delivering cells to a subject comprising contacting the subject with a biomatrix scaffold of the present invention, wherein the biomatrix scaffold comprises cells. In other embodiments, methods of delivering cells to a subject are provided, comprising seeding a bio-matrix scaffold of the invention with cells, and then transplanting the bio-matrix scaffold seeded with cells into a subject. In some embodiments, a biological matrix scaffold that has not been seeded with any cells may be transplanted into a subject.

In some embodiments, the biomatrix scaffold may be used as a graft that may be used to regenerate a tissue or organ in a subject.

The biomatrix scaffold of the present invention may be used to create bioartificial organs that are useful in analytical and/or clinical projects. The bio-matrix scaffold can also be used to identify aspects of a particular gene product or disease state. In some embodiments, the biomatrix scaffold is prepared from the tissue of a mutant animal and is subsequently used to define one or more association factors associated with one or more mutations. In other embodiments, the biomatrix scaffold is prepared from diseased tissue and used to define changes in the stroma associated with a disease.

Other non-limiting examples of uses of the bio-matrix scaffold of the present invention include: 1) using the scaffold for culturing malignant cells to define metastatic potential (the ability of tumor cells to form cell growth colonies on a given type of bio-matrix scaffold predicts the ability of the cells to transfer to the tissue used to prepare the scaffold); 2) placing a tissue graft on a scaffold for transplantation into a subject; 3) generating a organoid formed by recellularisation of the scaffold for use as an auxiliary device, such as, for example, a liver organoid, which is then connected to a subject with liver failure; 4) use of scaffolds for protein production (cells on the scaffold produce factors that can be isolated from culture fluid and/or from cells and then purified); and 5) use of the scaffold for generation of lineage-dependent viruses; for example for the production of viruses that require differentiated cells to produce sufficient particles for use as vaccines.

Accordingly, the present invention provides a method of identifying the metastatic potential of a tumor cell in a tissue type, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) inoculating the biomatrix scaffold of (b) with tumor cells; d) maintaining the bio-matrix scaffold of (c) under culture conditions; and e) monitoring the growth of the tumor cells on the biomatrix scaffold of (d), wherein the growth of the tumor cells on the biomatrix scaffold identifies that the tumor cells can form colonies in vivo in the tissue type from which the biomatrix scaffold was derived, thereby identifying the metastatic potential of the tumor cells in said tissue type.

Also provided herein are methods of identifying a response of a tumor cell to an anti-tumor therapy, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) inoculating the biomatrix scaffold of (b) with tumor cells; d) maintaining the bio-matrix scaffold of (c) under culture conditions; e) applying an anti-tumor therapy to tumor cells on the bio-matrix scaffold; and f) monitoring the growth of tumor cells on the biomatrix scaffold of (e), wherein the absence of growth and/or death of tumor cells on the biomatrix scaffold of (e) identifies the response of the tumor cells to an anti-tumor therapy. Non-limiting examples of anti-tumor treatments include: chemotherapeutic agents, antibodies, radiation therapy, immunotherapy, hormonal therapy, and the like, as are well known in the art. In some embodiments, tumor cells from a subject can be seeded onto different bio-matrix scaffolds of the invention and exposed to respective anti-tumor treatments. Based on the results of the respective analysis of the different anti-tumor treatments, an anti-tumor treatment effective against the tumor cells of the subject can be selected, and the anti-tumor treatment can be administered to the subject to treat the tumor of the subject.

In other embodiments, the invention also provides methods of producing a tumor graft for transplantation into a host animal, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of step (a) with a culture solution of a cell culture in a culture device; c) inoculating the biomatrix scaffold of (b) with tumor cells; d) maintaining the bio-matrix scaffold of (c) under culture conditions; and e) establishing a population of tumor cells on the biomatrix scaffold of (d), thereby producing a tumor graft for transplantation into a host animal. In some embodiments, the method may further comprise the step of transplanting the tumor graft into a host animal. In various embodiments, the tumor graft can be syngeneic, allogeneic, or xenogeneic with respect to the host animal.

Also provided herein are methods of producing virus particles of lineage-dependent viruses, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) seeding the bio-matrix scaffold of (b) with cells of the type and lineage stage that can be infected by the lineage-dependent virus; d) infecting the cells of (c) with a lineage-dependent virus; e) maintaining the infected cells on the bio-matrix scaffold under culture conditions; and f) collecting viral particles produced in the infected cells, thereby producing viral particles of the lineage-dependent virus.

Non-limiting examples of lineage-dependent viruses of the invention include hepatitis c virus, hepatitis b virus, norovirus, human papilloma virus, and any other virus now known or later identified as lineage-dependent. Lineage dependent means that the cell in which the virus is present must mature or differentiate to a particular stage before the virus can successfully replicate within the cell and produce viral particles, as is known in the art.

Further, the present invention provides a method of producing organoids formed by recellularization of a biological tissue scaffold, comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) seeding the biomatrix scaffold of (b) with cells of the same tissue type as the biological tissue used to prepare the biomatrix scaffold; and d) maintaining the cells on the biomatrix scaffold under culture conditions, whereby organoids are formed from the cells, thereby producing organoids formed by recellularization of the biological tissue scaffold. The method may further comprise the step of contacting the organoids produced in steps (a) to (d) with a subject, for use as an auxiliary device, as is known in the art. Any cell type that can be used to produce a bio-matrix scaffold of the invention can be used in the present method. In some embodiments, the cell is a liver cell.

The invention further provides a method of producing a protein of interest in cells cultured on a biomatrix scaffold comprising: a) producing a bio-matrix scaffold according to the method of the invention; b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device; c) seeding the biomatrix scaffold of (b) with cells that produce the protein of interest; d) maintaining the cells of (c) on a bio-matrix scaffold under culture conditions; and e) collecting the protein of interest produced by the cells of (d) and producing the protein of interest in the cells cultured on the bio-matrix scaffold. The method may comprise a further step of purifying the protein of interest collected in step (f). The protein of interest of the present invention may be any protein produced by cells in an amount that can be collected from cultured cells and/or a culture broth of the culture, the protein being produced from an endogenous gene and/or as a recombinant protein. Many examples of such proteins of interest are known in the art.

The invention will be explained in more detail in the following non-limiting examples.

Examples

Example 1: efficient human hepatic stem cell lineage restriction to mature fate by tissue-specific biomatrix scaffolds

And (3) abstract: current protocols for stem cell differentiation use multiple treatments of soluble signals and/or matrix factors and typically result in partial differentiation into mature cells through underexpression or overexpression of adult tissue-specific genes. In the present invention, a strategy for rapid and efficient differentiation of stem cells was developed using a matrix of biological matrix scaffolds, tissue-specific extracts enriched in extracellular matrix, and associated growth factors and cytokines, in combination with serum-free, hormone-defined medium (HDM) tailored to the adult cell type of interest. The studies described herein demonstrate the efficacy of the biomatrix scaffold of the present invention in differentiating human hepatic stem cells (hhpscs) into mature fates and maintaining mature parenchymal cells in full function for extended periods of time. Biological tissue scaffolds were prepared by a new four-step perfusion decellularization protocol using conditions designed to keep all collagen types insoluble. The scaffold retains native histology, the proprietary vessels and about 1% of tissue protein but > 95% of its collagen, most of the tissue collagen binding matrix components, and physiological levels of matrix binding growth factors and cytokines. Collagen increased from barely detectable levels to > 15% of scaffold proteins with residues, including laminin, fibronectin, elastin, endothelin/nidogen, proteoglycans, and matrix-bound cytokines and growth factors in histologically relevant patterns. Human hepatic stem cells (hhpscs), seeded on liver biomatrix scaffolds and in HAM tailored for human adult liver cells, lost stem cell markers and differentiated into mature, functional parenchymal cells in approximately one week, which remained viable and had a stable mature cell phenotype for more than eight weeks. Thus, the biomatrix scaffold of the present invention may be used for lineage-restricted stem cell biological and pharmaceutical research, for mature cell maintenance, and for implantable, vascularized engineered tissues or organs.

Procedure for decellularization: after anesthesia with ketamine-xylazine, the abdominal cavity of the rat was opened and a cannula-bearing sleeve was inserted into the portal vein to perfuse the entire liver. (1) Perfusion was performed for 10 min with RPMI 1640; then (2) defatting with lipase (e.g., 20-50 units of phospholipase A2-PLA2) in combination with a mild detergent such as 1% Sodium Deoxycholate (SDC) for about 30-60 minutes until the tissue becomes transparent and the exudate becomes clear; (3) perfused with high-salt wash (4.5M NaCl for fetal liver, and 3.4M-3.5M NaCl for adult liver) until the Optical Density (OD) of the perfusate at 280nm is negative for protein; (4) perfusing with nucleases (DNase, RNase) in RPMI1640 until the perfusate is negative for nucleic acids at OD 260; and (5) a final rinse with RPMI1640 for 2 hours or more.

The biomatrix scaffolds were flash frozen on dry ice and frozen sections were prepared with a cryostat, placed on 24-well cell culture plates, sterilized by gamma radiation (5000rads) and rehydrated in culture broth (KM) for 30 minutes before seeding the cells. The biological matrix scaffold slices cover-95% of the well surface in a 24-well culture plate.

An alternative method for distributing the bio-matrix scaffold on the culture dish comprises crushing the bio-matrix scaffold into powder using a cryomill filled with liquid nitrogen. The pulverized powder, when brought to room temperature, attains the consistency of a coating and can be coated on any surface such as a dish, slide, fabric, filter or other surface for attachment of cells and/or cell culture. Crushing the scaffold eliminates the gradient of matrix components and signal, but the mixture of components present still causes a strong differentiation effect. Scaffolds can also be used intact and reseeded with cells in the preparation of engineered organs for in vivo transplantation or for 3-D culture.

Alternative methods were developed for use with porcine and bovine livers. Porcine and bovine livers were obtained from USDA certified meat processing plants (CT). For an overview of the representative protocol, see example 3. Each liver was USDA checked and underwent USDA stamping before shipment. The liver was transported in ESP-Gro medium (Gigacyte, Branford, CT; catalog No. 1101-250). The livers received in the laboratory were weighed, photographically recorded and prepared for perfusion. After milling, the mixture was thawed and diluted to 1: 48 broth: the ratio of the biological matrix. The bio-matrix slurry is then used to coat the plate. After drying, the biological matrix is washed three times and then the cells are applied. Adult liver cells attached to the plate within 10 minutes. Stem/progenitor cells can be used for a longer period of time (several hours). However, for stem/progenitor cells and adult liver cells, essentially 100% of the viable cells attach.

Culture solution and solution: all cultures were sterile-filtered (0.22- μm filter) and kept in the dark at 4 ℃ before use. In order to maintain collagen stabilization in the biomatrix, the pH of the perfusion medium used for the preparation of the biomatrix scaffold was maintained at 7.5-8.0. RPMI-1640(Gibco/Invitrogen, Carlsbad, Calif.) was used as the basal medium for the preparation of the biological matrix scaffolds and for the culture of hepatocytes or hepatic stem cells. All reagents, except those mentioned, were obtained from Sigma (st.

Perfusion medium for bio-matrix scaffold preparation:

(1) perfusion cleaning and perfusion rinsing: serum-free basal medium (e.g., RPMI-1640);

(2) and (3) perfusion with a detergent: 36 units/L PLA2 plus 1% SDC;

(3) and perfusion with high salt: 3.4M NaCl with 0.1mg/ml soybean trypsin inhibitor;

(4) and perfusion with nuclease: 5mg/100ml RNase, 1mg/100ml DNase and 0.1mg/ml soybean trypsin inhibitor (e.g., prepared in RPMI 1640).

Kubota culture solution: KM was originally designed for hepatoblasts⁴⁷And has now been found for human hepatic progenitors⁴⁸And for other endoderm progenitors, including cells from the biliary system (Wang et al "Multi patent Stem/Progenitecells in human biliary trees to hepatocytes, cholestenocytes and pancreatic islets" Hepatology, 2011, published) and pancreas (Wang, Y and Reid L, unpublished data). It consists of any basic culture medium (hereRPMI 1640), said basal medium being copper-free, having low calcium (0.3mM), 10^-9M selenium, 0.1% BSA, 4.5mM nicotinamide, 0.1nM zinc sulfate heptahydrate (from Specpure, Johnson Matthey Chemicals, Royston, England), 10nM^-8M hydrocortisone, 5. mu.g/ml transferrin/Fe, 5. mu.g/ml insulin, 10. mu.g/ml high density lipoprotein, and a mixture to which free fatty acids bound to purified human serum albumin are added.

To differentiate cells to adult fates, serum-free, hormone-defined media (HDM) tailored to the adult cell type can be used. For example, we used HDM for adult liver fate, consisting of KM, which was further supplemented with calcium to reach 0.6mM concentration, 10^-12M copper, 1nM thyronine triiodide (T3), 7ng/ml glucagon, 20ng/ml FGF, 2g/L galactose, 10ng/ml oncostatin M (OSM), 10ng/ml Epidermal Growth Factor (EGF), 20ng/ml Hepatocyte Growth Factor (HGF), and 10ng/ml^-8M hydrocortisone.

Seeding cells in the HDM-free serum if plated on scaffolds; in the case of treating cells or tissues with enzymes, we then supplemented HDM with 5% FBS (HyClone, Waltham, MA) for several hours, then switched to serum-free HDM thereafter. In parallel control experiments, cultures were maintained in HDM with 5% FBS at all times, but we found that the presence of serum caused cells to lose differentiation function over time. Given that so many factors bind to the bio-matrix scaffold, less soluble factors are required than would normally be the case for culturing on other given matrices. Given that so many factors bind to the bio-matrix scaffold, less soluble factors are required than would normally be the case for culturing on other given matrices.

Characterization of the complete vascular system (vascular trees) in liver biomatrix scaffolds: the vessels in the rat liver biomatrix scaffolds including branched (bridging) and reticulated (ramifying) matrix residues of the capillary network were visualized by light microscopy and fluorescence microscopy, respectively. Rhodamine-labeled 250kDa dextran particles were injected through the portal vein residue into the liver biomatrix scaffold to check the integrity of the matrix residue of the vasculature in the biomatrix scaffold. Movies were prepared using a Leica MZ16FA fluorescence dissection microscope (motorized).

Human fetal liver treatment: fetal liver tissue was provided by a certification authority (Advanced Biological Resources, san francisco, CA) from 16-20 weeks gestational age fetuses obtained by selecting pregnancy termination. The study protocol was reviewed and approved by the institutional review board at the university of north carolina, chapel mountain for human studies. Suspension of human fetal liver cells was prepared as previously described^48、49. Briefly, treatment was performed in RPMI1640 supplemented with 0.1% bovine serum albumin, 1nM selenium and antibiotics. The enzyme treatment buffer contained 300U/ml collagenase type IV and 0.3mg/ml DNase, which was frequently stirred at 32 ℃ for 15-20 minutes. The enriched buffer was pressed through a 75 gauge mesh and spun at 1200RPM for 5 minutes, then resuspended. Cell viability assessed by trypan blue exclusion method was typically above 95%.

Enrichment and culture of hhpscs on biomatrix scaffolds: we used two methods for purification or enrichment of hhpscs:

1) culture selection about 3 × 10⁵Cells were plated in 10cm tissue culture dishes and KM. The culture medium was changed every three days. Colonies formed within 5-7 days and were observed for up to 3 months. We used an inverted microscope (1X-FLAIII; Olympus, Japan and Melville, NY) to pick up colonies manually after 14-18 days.

2) Magnetic immunoselection of multipotent liver progenitor cell subsets (multipotent liver promoter subparticles) was achieved using the Miltenyi Biotech MACS System (Bergisch Gladbach, Germany) following the manufacturer's instructions by selecting cells positive for epithelial cell adhesion molecules (hHpSC and hHB) using the magnetic bead immunoselection technique⁵⁰. Briefly, dissociated cells were incubated with EpCAM antibody bound to magnetic microbeads for 30 minutes at 4 ℃ and separated using a magnetic column separation system from Miltenyi following the manufacturer's recommended procedure.

Colonies with 250hHpSC, or 5 × 10⁵Enriched hHpSC or 2.5 × 10⁵Inoculating the culture with the primary adult hepatocytes. The culture broth was changed daily and the collected broth was stored at-20 ℃ for further analysis. Cells cultured on 24-well type I collagen-coated culture plates served as controls.

Separating adult rat liver cells: freshly isolated suspensions of rat hepatocytes were obtained from 3-month-old adult male Lewis rats weighing 200-. Using the modified two-step perfusion method as described previously⁴⁹Used for separating and purifying rat liver cells. The liver was perfused with calcium-free buffer containing EGTA for 10-15 minutes and then with calcium-containing buffer containing collagenase for 10-15 minutes. The liver was then dissociated mechanically by squeezing the digested liver through gauze and then continuing to pass the cell suspension through a narrow mesh size screen. Cells were washed twice and then centrifuged at 50 g. Viability was defined by counting cells after trypan blue staining. Typically, 200-300 million cells were isolated per rat with a viability of 89-95% and a purity of > 99%.

Isolation and culture of adult hepatocytes fresh human liver cell suspensions were obtained from CellzDirect (now part of Invitrogen, RTP, NC.) Each CellzDirect method treated the suspension and then resuspended in HeptoMAIN broth (catalog No. 1103-⁵Cells/cm²Plating was done in a multi-well culture plate coated with liver biomatrix scaffolds or on type I collagen (1. mu.g/ml, Meridian catalog number A33704H).

Chemical analysis of collagen: the amount of collagen in the biomatrix scaffold was estimated based on hydroxyproline (hyp) content. Samples of whole liver and samples of bio-matrix scaffolds were crushed, washed and lyophilized. Aliquots were then hydrolyzed and subjected to amino acid analysis⁵¹And assessing the amount of collagen in each total protein based on the hyp value of 300 residues/collagen.

Quantitative analysis of DNA and RNA content: is composed ofTo assess total DNA remaining in the decellularized liver biomatrix, fresh rat liver tissue and decellularized biomatrix were weighed, cut and digested with proteinase K, and total cellular DNA was isolated⁵². To assess total RNA remaining in the decellularized liver biomatrix, fresh rat liver tissue and decellularized rat liver biomatrix were weighed, then homogenized in TRIzol solution (Invitrogen), and total cellular RNA was isolated.

And (3) growth factor determination: samples of rat liver, rat liver biomatrix scaffolds, human biliary tissue, and human biliary biomatrix scaffolds (two samples each) were sent to RayBiotech, Inc (Norcross, Georgia) for growth factor analysis. These samples were homogenized, prepared as lysates, and then assayed with 1mg/ml protein, yielding fluorescence, defined in Fluorescence Intensity Units (FIU). Use ofSemi-quantitative Growth Factor assays were performed in Human Growth Factor Arrays, G Series 1. FIU from the negative control for non-specific binding was subtracted from FIU and normalized to protein concentration. Data from duplicate replicates were averaged. Four sets of arrays were used to achieve measurements for-40 growth factors. Although this assay was developed for human growth factor, it had sufficient overlap in cross-reactivity with rat growth factor to allow for use in both rat and human samples.

Transmission electron microscopy and scanning electron microscopy (TEM and SEM): for TEM, the biomatrix scaffolds were washed with Phosphate Buffered Saline (PBS) and fixed overnight in 3% glutaraldehyde/0.1 sodium cacodylate at pH 7.4. After three washes with sodium cacodylate buffer, the biomatrix scaffolds were post-fixed in 1% osmium tetroxide/0.1 sodium cacodylate for 1 hour. After rinsing in deionized water, it was dehydrated and embedded in Polybed812 epoxy (Polysciences, Niles, IL). The bio-matrix scaffold was sliced perpendicular to the substrate at 70nm using a diamond knife. Ultrathin sections were collected on 200 mesh copper mesh and stained with 4% aqueous uranyl acetate for 15 minutes, followed by 7 minutes with Reynolds' lead citrate. The samples were observed using a LEO EM910 transmission Electron microscope (LEO Electron Microcopy, Oberkochen, Germany) operating at 80 kV. Digital images were acquired using a Gatan Orius SCl000CCD Digital camera and a Digital Micrograph 3.11.0(Gatan, Pleasanton, CA).

For SEM, after fixation and washing, the biomatrix scaffolds were dehydrated and transferred in 100% ethanol to a BalzersCPD-020 critical point dryer (Bal-Tec AG, Balzers, Switzerland) using carbon dioxide as a transition solvent for drying. The substrate was mounted on an aluminum sample support using a carbon adhesive label and coated with a 10nm thick gold palladium alloy (60: 40 alloy) using a Hummer X spray coater (antatech, Worcester MA). The samples were examined using a Zeiss Supra55FESEM at 5kV acceleration voltage and digital images were collected using Zeiss SmartSEM software (Carl Zeiss SMT, Germany and Thornwood, NY).

Immunocytochemistry and immunohistology: for fluorescent staining of cells cultured on the bio-matrix scaffold, cells were fixed with 4% Paraformaldehyde (PFA) for 20 minutes at room temperature, washed with HBSS, blocked with 10% goat serum in HBSS for 2 hours, and washed. The fixed cells were incubated overnight at 4 ℃ with primary antibody, washed, incubated with labeled isotype-specific secondary antibody for 1 hour, washed, counterstained with 4', 6-diamidino-2-phenylindole (DAPI) for visualization of the nucleus, and viewed using a Leica DMIRB inverted microscope (Leica, Houston, TX).

For immunohistochemistry, the biomatrix scaffolds were fixed overnight in 4% PFA and stored in 70% ethanol. They were embedded in paraffin and cut into 5 μm sections. Deparaffinizing the sections, and repairing the antigen. Endogenous peroxidase passage at 0.3% H₂O₂The solution was incubated for 30 minutes and blocked. After blocking with 10% horse serum, the primary antibody was applied overnight at 4 ℃; secondary antibody and ABC staining were performed using the RTU Vectastain kit (Vector Laboratories, Burlingame, CA). Vector Nova RED was used as substrate. Sections were dehydrated, fixed and embedded in Eukitt mounting medium (Electron Microcopy S)ciens, Hatfield, PA) and analyzed using an inverted microscope. Antibodies used for liver sections and for culture are listed in table 4.

Reverse transcription polymerase chain reaction (RT-PCR) analysis: the hhpscs were cultured on cell culture plates and the colonies were transferred to a bio-matrix scaffold. After further culture for 7 days, the colonies were lysed for RT-PCR. Total RNA was extracted using RNeasy PlusMini kit (Qiagen GmbH, Valencia CA) according to the manufacturer's instructions. Reverse transcription was performed for RT-PCR (Invitrogen, Carlsbad, Calif.) using the SuperScriptfirst-Strand Synthesis System. HotStarTaq Master Mix kit (Qiagen) was used for PCR. The PCR primers are listed in Table 5.

Live/dead assay and cell survival assay: living/dead viability assay kits (molecular probes/Invitrogen, Carlsbad, Calif.) were used for the adhesion and proliferation assays. hhpscs or hepatocytes were incubated with two probes calcein-AM (live, light grey) and ethidium bromide homodimer-1 (EtdD-1, dead) for intracellular lactonase activity and plasma membrane integrity, respectively. The samples were observed under a fluorescent Olympus SZX12 stereomicroscope (OLYMPUS, Japan and Melville, NY). The resazurin cell viability assay kit (Biotium, Hayward, CA) was used following the manufacturer's manual. Briefly, a 10% resazurin solution was added to the cultured broth and incubated overnight at 37 ℃. OD was obtained using a Biotek synergy HT multiple detection microplate reader (Winooski, VT)₅₇₀-OD₆₀₀Absorbance and viability curves were plotted. All experiments were performed in triplicate using a minimum of 3 samples under each experimental condition.

Liver-specific functional assay: using P450-Glo^TMThe screening system (Promega, Madison, WI) detects CYP4503a4 activity. Briefly, cultured cells were incubated with a medium containing a luminescent CYP 3a4 substrate (PPXE, fluorescein for CYP) for 4 hours at 37 ℃. Fluorescein detection and analysis was performed using a Wallace Victor2Multilabel Counter (now part of Perkins/Elmer by Waltham, MA) according to the manufacturer's instructions. Quantification of albumin using human albumin ELISA quantification kit (Bethy Laboratories, Montgomery, TX)And (4) secreting. For urea synthesis analysis, cells were incubated with 2mM ammonium for 24 hours, and supernatants were collected and assayed using the Quantichrom urea assay kit (bioassays systems, Hayward, CA). Supernatants from one sample for each culture condition were assayed in triplicate and the experiment was repeated 3 times.

Statistical analysis: experiments were repeated at least 2-3 times for each condition using duplicate or triplicate samples. Data from representative experiments are presented, while similar trends were observed in multiple experiments. All error bars represent s.e.m.

The biomatrix scaffold was prepared using a new four-step protocol: the bio-matrix scaffolds were prepared using a protocol including defatting followed by high-salt extraction and using a perfusion method (fig. 1). The method gives a detailed description of the scheme. This is achieved by a new four-step scheme: 1) carrying out mild degreasing; 2) the composition is prepared by mixing the mixture having a molar mass of about 2.0M to about 5.0M (e.g., 2.0M-2.5M, 2.6M-3.0M; 3.1M-3.5M, 3.6M-4.0M, 4.1M-4.5M; 4.6M-5.0M) salt concentration known to maintain collagen in an insoluble state²³(the exact concentration of buffer and pH is determined by the type of collagen in the tissue), which is known to maintain collagen in an insoluble state²³(ii) a 3) Nuclease treatment to eliminate residual nucleic acids; and 4) washing with basal medium to eliminate detergent, salt and nuclease residues, and equilibrating the matrix composition with the medium (FIG. 1A).

The choice of the wash medium or buffer for the nuclease can be any of a variety of choices, so long as the salt concentration and ionic strength are such that the matrix components are maintained in an insoluble state. The choice of degreasing method is critical for efficiency and mildness. We chose a combination of Sodium Deoxycholate (SDC) and phospholipase a2(PLA2) to rapidly degrade phosphoglycerides located on the plasma and mitochondrial membranes to lysolecithin, a powerful surfactant that can induce necrosis and cell lysis. FIG. 8 shows the reaction scheme. We avoid powerful detergents such as Sodium Dodecyl Sulfate (SDS) or Triton-X100, which can cleave some matrix components such as glycosaminoglycans (see review by Gilbert et al, "Decellarizationof properties and organs", Biomaterials 27: 3675-.

We avoided long term exposure of scaffolds to enzymes from ruptured cells during defatting and high salt washing, as they could greatly reduce the levels of elastin and glycosaminoglycans (GAG) such as Heparin Sulfate (HS), Chondroitin Sulfate (CS), Dermatan Sulfate (DS), and Heparin (HP), which are sites for cytokine and growth factor binding²⁴. We used soybean trypsin inhibitor and carefully controlled pH (7.5-8.0), temperature (20 ℃), and time (30-60 minutes) to limit the activity of proteases originating from disrupted cells.

We perfuse the entire tissue through the relevant vessels (e.g., the portal vein in the liver), which allows us to rapidly isolate (within a few hours) the bio-matrix scaffold while minimizing loss of matrix components. The rapidity of separation is due to the initial step of degreasing the tissue in about 30-60 minutes (rather than hours or days as in other protocols used). The resulting bio-matrix scaffold was either clear or white (fig. 1). Furthermore, using this perfusion method, we maintain the original vascular access, portal vein and hepatic vein and most of the vascular branches in the liver, which increases the decellularisation efficiency. Fluorescent rhodamine-labeled dextran particles infused through the biomatrix scaffold remained within the vascular remnants, indicating that they were significant (fig. 1E). There is a gradual flow of dye from the large vessel along the channel into the thin vessel branch without leakage. This fact would be helpful in revascularization of stents as a method of preparing engineered tissues for three-dimensional culture and/or for in vitro implantation.

Upon sectioning, the scaffold maintains the histological structure of the original tissue, including identifiable residues in major histological entities, such as blood vessels, bile ducts, and the Grignard Capsule (GC) (fig. 1). Compare fig. 1B1 and 1D1, where a section of liver tissue is compared to a section of a bio-matrix scaffold. The matrix residue of parenchymal cell walls consists of a latticed network (FIGS. 1D2-1D 3).

Collagen, collagen-related proteins and bound cytokines are retained in the biomatrix scaffold: the amount of collagen in the biomatrix scaffold was assessed by amino acid analysis by the method used previously²⁵. Since hydroxyproline (Hyp) is unique to collagen and collagen, the collagen composition relative to total protein is expressed as Hyp residue per 1000 amino acids. The results indicate that the collagen content increased from an almost undetectable level, i.e., less than 0.2 residual hydroxyproline (Hyp)/1000 in the liver, to 13 residual Hyp/1000 in the biomatrix scaffold. This indicates that the defatting and high salt washing as described above did not remove collagen, leaving almost all the collagen in the biomatrix scaffold. The detection of significant levels of another collagen-related amino acid, hydroxylysine (Hyl), and higher levels of glycine (Gly) in the biomatrix scaffold supports our conclusion that collagen is significantly enriched in the biomatrix scaffold (fig. 2A, 9 and table 2).

Using immunohistochemistry and ultrastructural studies, we were able to identify all known forms of collagen found in situ in the liver in scaffolds, including fibrillar collagen (type I, III and type V collagen, 10-30nm diameter for small fibers and 3000nm diameter for assembled fibers) and beaded fibers (probably type VI). Those fibers and fibrils are present in the subconvelous connective tissue layer under the mesothelial layer. Although the typical structure of the basement membrane is not found in the sinuses along the portal triplet to the central vein, we found that type IV collagen and some bound fibrils form a reticulated, porous 3D mesh structure that acts as a scaffold for parenchymal cells (fig. 2). Type I collagen bundles can be considered the main structure of the scaffold to which other types of collagen, glycoproteins, and proteoglycans are attached. In the gap of diels, we find small tracts of type I collagen and fibers of type III and IV and some type V collagen, which are more abundant near the portal triplet and central vein. Representative immunohistochemical data are presented in fig. 3B, and a summary of the matrix components and their location in normal liver tissue compared to their location in the bio-matrix scaffold is presented in fig. 4D. Early studies in the development of protocols directed to the preparation of biomatrix scaffolds showed that a significant amount of cytoskeletal elements were lost in the wash. However, we evaluated scaffolds by immunohistochemistry and we found no evidence of tubulin, desmin or actin, trace cytokeratin 18 and 19, and low levels of vimentin throughout the scaffold.

The stroma associated with the bile ducts and parts of the hepatic vascular system (arterial and venous vessels) consists of a typical basement membrane structure, so the thin layer of stroma is quite different from that associated with the vascular structures found in the blood sinuses. Laminin, nidogen/endoglin, perlecan, and type IV collagen are found in the portal triplet, while only perlecan and some type IV collagen are found in the diels space. There is a large amount of hydrophobic, vimentin; it is cross-linked together and forms sheets and fibers, which are primarily confined to the subcontracted connective tissue, the portal venous area, and the arterial wall. Fibronectin is ubiquitous and throughout the liver matrix and is particularly abundant in the dirichlet space where they form fine fibrils or granular deposits (fig. 2 and 3).

Immunohistochemistry revealed that known proteoglycans in tissues were preserved in the biomatrix scaffold (fig. 3B, 4D). Among the heterogeneous proteoglycans identified, syndecans are intercalated and continuously found along the blood sinuses, whereas perlecan is more intercalated in the dieldrin space. Residues of HS-PG and CS-PG were found in the form of residues throughout the blood sinuses in the biomatrix scaffold, patterns related to known banding in liver tissue.

Proteoglycans and other matrix components are important reservoirs for cytokines and growth factors that bind tightly to their GAGs²⁶. Most growth factors and hormones are found in biological matrix scaffolds, which approach the concentrations found in the original tissue. In table 6, data from a comparison of rat liver lysates and rat liver bio-matrix scaffolds are given, and in table 3, parallel data from a comparison of human bile duct tissue and bile duct bio-matrix scaffolds are provided. Interestingly, there areSeveral examples of the strong enrichment found in liver biomatrix scaffolds compared to liver lysates (e.g., bFGF). The bound growth factors and cytokines differ qualitatively and quantitatively between the comparison of the scaffolds of liver and bile duct tissue, suggesting tissue-or species-specificity. Alternatively, it may, in part, be due to the fact that the biliary stent needs to be prepared by shaking the tissue in buffer on a rocking bar, rather than by perfusion through the vessel.

The chemistry of the biomatrix scaffold is histologically related: an important feature of this new protocol is the retention of stromal chemicals in a pattern that is associated with the portal to the hepatic acinar region 1-3 of the central vein and with histological entities such as vascular access and the Gleason's Capsule (GC), as shown in FIGS. 4A-C. The matrix chemistry around the portal vein in zone 1 is similar to that found in fetal liver and is composed in part of type III collagen, laminin, and forms of CS-PG. It converts to different stromal chemicals in the middle acinus (zone 2) and the central periapical zone (zone 3), which ends up in a very stable stroma with high levels of type IV collagen and HP-PG²⁷。

It is known that various proteins (e.g. growth factors and hormones, coagulation proteins, various enzymes) bind to and remain stable to the matrix by binding to discrete and specific sulfation patterns in GAGs or to other matrix components²⁴. Thus, the matrix chemical is converted from its starting point in the stem cell niche (with unstable matrix chemicals associated with high conversion and minimal sulfation) to a stable matrix chemical with increased amounts of sulfation and proceeds toward the central peripheral region. We expect that preservation of native structures as well as matrix chemicals associated with histological structures will promote recellularization during tissue engineering by directing cells to specific sites on the biomatrix scaffold and/or providing a suitable signal mix to drive expansion and/or differentiation into mature cells.

Biomatrix scaffolds may be prepared from different tissues and species: the bio-matrix scaffold can be readily prepared from any normal or diseased tissue and from any species. In fig. 13-16, we show the bio-matrix scaffolds from human pancreas, biliary system, and duodenum, as well as from rat and pig pancreas. The effect of bovine or rat liver bio-matrix scaffolds on hepatocytes is shown in figures 5-7 and 12. In addition, the biomatrix stents were prepared from the human abdominal aorta, iliac veins, and from the intestines of rats and pigs. Histological, ultrastructural and immunohistochemical studies on biomatrix scaffolds showed significant tissue specificity, but not species specificity, in their structure, chemical composition, and function.

The bio-matrix scaffold induces and/or maintains differentiation of cells: plating hpscs on dishes with liver biomatrix scaffold sections and HDMs tailored for adult liver cells resulted in essentially 100% of live cells attaching to the biomatrix scaffold within a few hours; whether intact or after cryogenic comminution. The cell colonies initially formed on the scaffold sections retained some of their stem cell phenotype, as the cells in the center of the colonies were able to resist staining by dyes (fig. 11) and expressed typical hepatic progenitor markers such as chemokine (C-X-C motif) receptor 4(CXCR4) and epithelial cell adhesion molecule (EpCAM) (fig. 5). They divide 1 or twice and then switch to cell cycle arrest and to three-dimensional (3D) cord-like morphology, which is typical for mature parenchymal cell cultures (fig. 5 and 6 for stem cell differentiation; compare fig. 7 and 12). The HDM used does not require all the usual cytokines or growth factors, as these are present in a form bound to the bio-matrix scaffold. The switch to growth arrest was associated with staining of whole colonies with reactive dyes (figure 12), with a concomitant loss of EpCAM and CXCR4 expression, and a stable increase in expression of adult-specific hepatocyte and cholangiocyte genes such as urea and cytochrome P4503a4 (figure 5).

Normal adult rat and adult hepatocytes were plated on type I collagen or on a bio-matrix scaffold from rat or bovine liver, and for adult cells in HDM. Adult parenchymal cells can be cultured in 10 minutes (even in serum-free culture medium)In) to a scaffold, in contrast to it, which adheres to type I collagen within hours, it remains in growth arrest starting from the point of attachment; and remained viable and fully functional on the scaffold for more than 8 weeks, compared to about-2 weeks on type I collagen (fig. 7 and 12). Functional levels of mature liver cells on a biomatrix scaffold lasting weeks proved identical or similar to findings in other freshly isolated adult liver cells²⁸. The difference that was noted was that the culture on type I collagen deteriorated rapidly after 2 weeks, whereas the culture on the bio-matrix scaffold remained morphologically and functionally stable for the duration of the culture maintenance (up to 8 weeks).

The biomatrix scaffold contains most of the tissue's extracellular matrix components and matrix-bound cytokines and growth factors, provides a complex set of chemical signals that can be used in insoluble, stable scaffolds with the specific ability to induce hhpscs to adult liver fates, and to maintain adult cells fully differentiated for weeks. Comparing the existing types of matrix extracts prepared by researchers with the types of biological matrix scaffolds of the present invention, it is evident that physical, enzymatic and chemical treatments have a substantial impact on composition, mechanical behavior, and host reactions on biological scaffolds derived from natural tissue and organ decellularization, and accordingly, have important implications for their in vitro and in vivo applications. All other existing methods for preparing matrices or scaffolds are by using matrix degrading enzymes¹⁶Or using a buffer dissolving the majority of the matrix¹¹Resulting in the removal of most of the matrix components. Physical methods (e.g., chilling and stirring) can act to remove debris from tissues having a layered structure such as dermis (e.g., SIS, BSM)²⁹A matrix extract is prepared, but cannot be used for organs having a complex tissue structure, such as the liver. By contrast, our approach to biomatrix scaffolds resulted in the loss of most cellular proteins, but preserved substantially all or at least most of the collagen and collagen-related components, including matrix-bound cytokines, hormones, and growth factors.

Extracellular matrix embeddingIn the chimeric lipid bilayer, even in the simplest organisms, it is a complex, heterogeneous and dynamic environment. The degreasing method is a key aspect of the protocol. Commonly used methods for tissue decellularization include ionic detergents such as SDC and Sodium Dodecyl Sulfate (SDS). SDC is relatively milder than SDC, tends to cause less disruption to native tissue structures, and is less effective at lysing cytoplasmic and nuclear membranes³⁰. No report of tissue decellularization using SDC alone was reported. Many studies have used potent non-ionic detergents (e.g., Triton X-100)³¹Or zwitterionic detergents (e.g., 3- [ (3-cholamidopropyl) dimethylamino)]-1-propanesulfonate, CHAPS)³². By contrast, we used the combined method of SDC and PLA2 to degrease tissues quickly and gently.

At least 29 types of collagen (I-XXIX) have been identified in vertebrates to date, which have a functional role in cell adhesion, differentiation, growth, tissue development and structural integrity^33、34. The main structural component in the matrix, collagen, is known to remain insoluble at high salt concentrations and neutral pH^35、36This finding is the basis of our strategy for the preparation of biomatrix scaffolds. This strategy has the additional advantage that the collagen enables preservation of matrix components such as laminin and Fibronectin (FN), small leucine rich Proteoglycans (PG) and GAGs that bind to them, which in turn preserve cytokines, growth factors or cell surface receptors bound to them.

Biomatrix scaffolds are unique in their far reaching ability to induce rapid and stable differentiation of stem/progenitor cells such as hhpscs into adult fates and to maintain their lineage-restricted cells or also to maintain adult cells plated on scaffolds as viable and fully functional cells for many weeks (> 8 weeks).

Differentiation of stem cells such as Embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, or various forms of Mesenchymal Stem Cells (MSCs) into fully mature liver cell types requires storageMultiple sets of signals (soluble and matrix) at each stage, with induction of one set required to elicit responses to a different set, and may require weeks, up to 6 weeks of culture, to generate cells with adult liver fate³⁷. Furthermore, limiting MSC lineage to liver fate produces results that are inconsistent with adult cells with mixed hepatocyte and MSC phenotypes. Differentiation of ES cells, iPS cells and MSCs results in hepatocyte-like cells that express some, but not all, of the major liver-specific genes; has a change in which a gene is observed; and the protein levels for those expressed hepatic genes are generally lower for one hepatic gene⁴¹Or higher and may be ignored for others^41、42. By contrast, differentiation of hhpscs on a biomatrix scaffold resulted in essentially all cells expressing a typical adult phenotype, and in which urea, albumin, and CYP450 activities were at near normal levels after one week in culture.

Hepatocyte-like cells derived from any of these precursor cells and differentiated by protocols other than biological matrix scaffolds express some, but not all, of the major liver-specific genes, with changes in the genes observed therein; and the protein levels for the liver genes are generally lower or higher for one liver gene and negligible for the other. For unknown reasons, the results differ from preparation to preparation. It is expected that the use of the biomatrix scaffold of the present invention should result in a more rapid differentiation of these stem cell populations with greater consistency in achieving cells with a stable adult phenotype.

The differentiation of stem cell populations such as hhpscs on biomatrix scaffolds was determined to result in a typical adult repertoire of essentially all cell-expressed genes, and wherein urea, albumin and CYP450 activities were at near normal levels within 1-2 weeks of culture, and wherein the phenotype was stable for several weeks. Thus, the biomatrix scaffold of the present invention has the potential to greatly facilitate the differentiation of a definitive stem cell population into an adult liver phenotype.

The ability to differentiate stem cells on a bio-matrix scaffold to achieve mature and functional cells and tissues provides considerable opportunities for academic, industrial and clinical programs, which allows well-differentiated cell types to be used for each type of analytical study, and most exciting, which allows the generation of implantable, revascularized tissues or even organs that can be used for basic research and clinical programs.

Reference to example 1

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Example 2: use of a biological matrix scaffold for the culture of a tumor cell line or primary culture of a tumor

The biomatrix scaffold of the present invention may be used in the culture of generating tumor cell lines or in the primary culture of tumors. The ability to do this means that the sensitivity of a patient's tumor to various treatments can be assessed in an ex vivo assay.

The biomatrix scaffold of the present invention may also be used as a matrix for tumor (whether allogeneic, or xenogeneic) grafts for transplantation into a host.

The biomatrix scaffolds of the present invention may also be used to assess the metastatic potential of tumors. Tumor cells are seeded at low cell density on the matrix of a biomatrix scaffold from various tissues. Tumor cells will attach and survive on many types of biomatrix scaffolds. They will preferentially grow on some of them and form colonies. Their ability to form colonies on a particular type of bio-matrix scaffold predicts the ability of tumor cells to form colonies on the tissue from which the bio-matrix scaffold is prepared.

Colorectal cancer that has metastasized to the liver is excised and the tissue prepared as a primary culture in Kubota broth and on various matrices. Findings from six patients are shown in table 9. Some patients have tumors that have also metastasized to the lungs. Cells were cultured on tissue culture plastic dishes coated with type I collagen ("collagen"), or on dishes coated with biomatrix scaffolds from rat colon, liver or lung. All wells were seeded at 20,000 cells/well in 96-well plates and fed with Kubota broth. Cells attached to all substrates and survived (see FIGS. 17A-B). However, they are capable of optimal growth and colony formation on certain substrates. The conditions that support the highest number of colonies correlate with the ability of the cells to grow in vivo in those particular tissues from which the scaffolds were prepared. The amount of matrix or matrix components confirms the variation in obtaining all colonies at all. The amount of matrix/well required to observe colonies is shown in table 8. Clearly, the amount required from the site of the primary tumor, colon, is minimal. Significance data is highlighted in bold.

The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is limited only by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, patents, and patents mentioned aboveThe sequences identified by the database and/or SNP accession numbers, as well as the teachings of other references cited herein, in relation to the statements and/or paragraphs in which the references are set forth, are incorporated by reference in their entirety.

Table 1: NaCl molarity Range for type I-V collagen

These data represent insoluble conditions for the collagen type. One skilled in the art must identify the type of collagen in the tissue and then use the highest salt concentrations identified for those collagens in the tissue, as well as the highest salt concentration as a buffer for preparing the biomatrix scaffold. For example, for skin, one skilled in the art would use a buffer with a neutral pH and a salt concentration of 4.0M that would keep the type I and type III collagens insoluble. In contrast, for placenta, one skilled in the art would use a buffer with a neutral pH and a salt concentration of 4.5M to keep the type IV and type V collagen insoluble.

The type of collagen present in a tissue varies at different ages of the host. For example, fetal liver has high levels of type III, IV and V collagen (requiring salt concentrations above 4.0M), while somatic liver has a mixture of type I, III, IV, V, VI and XVIII collagen (requiring lower salt concentrations). Thus, the salt concentration required for the preparation of the biomatrix scaffold is determined by all the components of the collagen that is predominant in the tissue. For further details, see reference 23 of example 1.

Table 3: growth factor analysis bound to biliary tube biomatrix scaffolds

Table 4: antibodies utilized

Table 6: growth factor analysis bound to liver biomatrix scaffolds

Table 8: amount of matrix/well required to observe colonies

Table 9: results from 6 patients

TABLE 9 continuation

Claims (46)

1. A method of producing a biomatrix scaffold from a biological tissue comprising the steps of:

a) perfusing or homogenizing the biological tissue with a first culture fluid, wherein the osmolality of the first culture fluid is about 250 to about 350mOsm/kg, and the first culture fluid is serum-free and at a neutral pH; then the

b) Perfusing the biological tissue of step (a) or extracting the homogenate of step (a) in the first culture fluid with a degreasing buffer comprising a lipase and/or a detergent; then the

c) Perfusing the tissue of step (b) or extracting the homogenate of step (b) with a buffer at neutral pH and comprising a salt concentration of about 2.0M NaCl to about 5.0M NaCl, the concentration selected to keep insoluble collagen identified in the biological tissue; then the

d) Perfusing the tissue of step (c) or extracting the homogenate of step (c) with RNase and DNase in a buffer; and then

e) Washing the tissue or homogenate of step (d) with a second culture medium at neutral pH, serum-free, and having an osmolality of about 250 to about 350mOsm/kg,

thereby producing an intact or homogenized biological matrix scaffold from the biological tissue, the biological tissue scaffold comprising at least 95% collagen and a majority of collagen-binding matrix components and matrix-binding growth factors, hormones and cytokines of the biological tissue.

2. The method of claim 1, wherein the first culture fluid comprises salts, minerals, amino acids, vitamins, and sugars.

3. The method of claim 1, wherein the first culture fluid is a basal culture fluid.

4. The method of claim 3, wherein said basal medium is selected from the group consisting of RPMI1640, DME/F12, DME, F12, Waymouth's medium, and William's medium.

5. The method of claim 1, wherein the second culture fluid comprises at least one of the components present in the tissue fluid.

6. The method of claim 1, wherein the defatting buffer of step (b) comprises about 20 units/L to about 50 units/L of phospholipase a2 and about 1% sodium deoxycholate in the first broth.

7. The method of claim 1, wherein the salt concentration of the buffer of step (c) is from about 3.4M NaCl to about 3.5M NaCl when used to prepare a scaffold from adult liver, and from about 4.0M NaCl to about 4.5M NaCl when used to prepare a scaffold from fetal liver.

8. The method of claim 1, wherein said buffer of step (c) further comprises a protease inhibitor.

9. The method of claim 8, wherein the protease inhibitor is soybean trypsin inhibitor.

10. The method of claim 1, wherein said buffer of step (d) further comprises a protease inhibitor.

11. The method of claim 10, wherein the protease inhibitor is soybean trypsin inhibitor.

12. The method of claim 1, wherein all of the culture and buffer of steps (a) through (e) are free of detectable amounts of enzymes that degrade extracellular matrix components.

13. The method of claim 1, further comprising sterilizing the bio-matrix scaffold.

14. The method of claim 13, wherein the step of sterilizing comprises gamma irradiation of the bio-matrix scaffold.

15. The method of claim 1, wherein the biological tissue is selected from the group consisting of liver tissue, lung tissue, pancreas tissue, thyroid tissue, intestinal tissue, skin tissue, vascular tissue, bladder tissue, heart tissue, and kidney tissue.

16. The method of claim 1, wherein the biological tissue is from a mammal.

17. A bio-matrix scaffold produced by the method of any one of claims 1-16.

18. A biomatrix scaffold comprising collagen, fibronectin, laminin, endothelin/nidogen, elastin, proteoglycans, glycosaminoglycans, growth factors, cytokines, or any combination thereof, all of which are part of the biomatrix scaffold.

19. A method of producing a cell culture, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of step (a) with a culture solution of a cell culture in a culture device; and

c) seeding the bio-matrix scaffold of step (b) with cells, thereby producing a cell culture.

20. A method of producing a cell culture, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) freezing the bio-matrix scaffold of step (a);

c) preparing frozen sections from the bio-matrix scaffold of step (b) as cell culture medium;

d) contacting the cell culture medium of step (c) with a culture medium of a cell culture in a culture device; and

e) inoculating the cell culture medium of step (d) with cells, thereby producing a cell culture.

21. A method of producing a cell culture, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) grinding the bio-matrix scaffold of step (a) into a powder;

c) coating at least a portion of a culture device with the powder of step (b) to produce a cell culture medium;

d) contacting the cell culture medium of (c) with a culture medium of a cell culture in a culture device; and

e) seeding the cell culture medium of (d) with cells, thereby producing a cell culture.

22. The method of claim 21, wherein the grinding of the bio-matrix scaffold is performed in a cryomill at or near liquid nitrogen temperature.

23. The method of any one of claims 19-22, wherein a culture fluid of the cell culture comprises at least one of the components present in a tissue fluid, wherein the osmolality of the culture fluid is from about 250mOsm/kg to about 350mOsm/kg, and wherein the culture fluid is serum-free.

24. The method of claim 23, wherein the culture fluid is RPMI-1640.

25. The method of any one of claims 19-24, wherein the cells are of the same type as the cells of the biological tissue used to prepare the biological matrix scaffold.

26. The method of any one of claims 19-24, wherein the cell is selected from the group consisting of: embryonic Stem (ES) cells, Induced Pluripotent Stem (iPS) cells, definitive stem cells, perinatal stem cells, amniotic fluid derived stem cells (AFSCs), Mesenchymal Stem Cells (MSCs) from any source, committed progenitor or adult cells of any tissue type, mature cells, normal cells, diseased cells, tumor cells, and any combination thereof.

27. The method of any one of claims 19-24, wherein the cell is selected from the group consisting of: liver cells, parenchymal cells, stellate cells, endothelial cells, hepatocytes, biliary epithelial cells, biliary lineage cells other than biliary epithelial cells, and pancreatic cells.

28. Use of a tissue-specific biomatrix scaffold to promote differentiation of embryonic stem cells or induced pluripotent cells towards a specific fate.

29. Use of a tissue specific biomatrix scaffold to promote differentiation of amniotic fluid derived stem cells or mesenchymal stem cells or any determinant stem cells from bone marrow, adipose tissue, or any fetal or postnatal tissue to a specific adult fate.

30. A cell culture produced by the method of any one of claims 19-27.

31. A method of enhancing and accelerating differentiation of stem and/or progenitor cells into mature cells, comprising producing a cell culture according to the method of any one of claims 19-27, wherein the cells are stem cells and the culture broth of the cell culture is formulated for mature cells, thereby enhancing and accelerating differentiation of stem and/or progenitor cells into mature cells.

32. The method of claim 31, wherein the cell is any type of adult cell or a stem or progenitor cell selected from the group consisting of: embryonic stem cells, induced pluripotent stem cells, embryonic stem cells, definitive stem cells, perinatal stem cells, amniotic fluid derived stem cells, mesenchymal stem cells, transient expanded cells, or committed progenitor cells of any tissue type.

33. The method of claim 31, wherein the culture of cells is selected from the group consisting of RPMI1640, DME/F12, DME, F12, Waymouth broth, and William broth.

34. A method of delivering cells to a subject comprising seeding the bio-matrix scaffold of any one of claims 17 or 18 with cells and then transplanting the bio-matrix scaffold seeded with cells into the subject.

35. A method of identifying the metastatic potential of tumor cells in a tissue type, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device;

c) seeding the bio-matrix scaffold of (b) with tumor cells;

d) maintaining the bio-matrix scaffold of (c) under culture conditions; and

e) monitoring the growth of said tumor cells on said biomatrix scaffold of (d),

wherein growth of the tumor cells on the biomatrix scaffold identifies that the tumor cells can form colonies in vivo in a tissue type from which the biomatrix scaffold was derived, thereby identifying the metastatic potential of the tumor cells in the tissue type.

36. A method of identifying a response of a tumor cell to an anti-tumor therapy, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device;

c) seeding the bio-matrix scaffold of (b) with tumor cells;

d) maintaining the bio-matrix scaffold of (c) under culture conditions;

e) applying the anti-tumor therapy to the tumor cells on the bio-matrix scaffold; and

f) monitoring the growth of the tumor cells on the bio-matrix scaffold of (e),

wherein the absence of growth of tumor cells and/or death of tumor cells on said biomatrix scaffold of (e) identifies a response of said tumor cells to said anti-tumor therapy.

37. A method of producing a tumor graft for transplantation into a host animal, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device;

c) seeding the bio-matrix scaffold of (b) with tumor cells;

d) maintaining the bio-matrix scaffold of (c) under culture conditions; and

e) establishing a population of the tumor cells on the bio-matrix scaffold of (d),

thereby producing a tumor graft for transplantation into a host animal.

38. The method of claim 37, further comprising the step of transplanting said tumor graft into said host animal.

39. The method of claim 38, wherein said tumor graft is syngeneic, allogeneic, or xenogeneic with respect to said host animal.

40. A method of producing virus particles of lineage-dependent viruses, comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device;

c) seeding the bio-matrix scaffold of (b) with cells of the type and lineage stage that can be infected by the lineage-dependent virus;

d) infecting said cells of (c) with said lineage-dependent virus;

e) maintaining the infected cells on the bio-matrix scaffold under culture conditions; and

f) collecting the viral particles produced in the infected cells,

thereby producing viral particles of the lineage-dependent virus.

41. The method of claim 40, wherein said lineage-dependent virus is selected from the group consisting of hepatitis C virus, hepatitis B virus, norovirus, and human papilloma virus.

42. A method of producing organoids formed by recellularization of a biological tissue scaffold comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device;

c) seeding the bio-matrix scaffold of (b) with cells of the same tissue type as the biological tissue used to prepare the bio-matrix scaffold; and

d) maintaining the cells on the biomatrix scaffold under culture conditions, thereby forming organoids from the cells,

thereby producing organoids formed by the recellularization of the biological tissue scaffold.

43. The method of claim 42, further comprising the step of contacting the subject with said organoid for use as an adjunct.

44. The method of claim 42, wherein the cell is a liver cell.

45. A method of producing a protein of interest in cells cultured on a biomatrix scaffold comprising:

a) producing a bio-matrix scaffold according to the method of any one of claims 1-16;

b) contacting the bio-matrix scaffold of (a) with a culture solution of a cell culture in a culture device;

c) seeding the bio-matrix scaffold of (b) with cells that produce the protein of interest;

d) maintaining the cells of (c) on the bio-matrix scaffold under culture conditions; and

e) collecting the protein of interest produced by the cells of (d),

thereby producing the target protein in the cells cultured on the biological matrix scaffold.

46. The method of claim 45, further comprising the step of purifying said protein of interest collected in step (e).