HK1077525B - Augmentation of organ function - Google Patents

Description

Enhancement of organ function

Reference to related applications

This application claims priority to U.S. provisional application serial No. 60/331500, filed on 16/11/2001. This application is also a continuation-in-part application of U.S. patent application serial No. 09/474525 filed on 29/12/1999. The contents of the two related applications are hereby expressly incorporated by reference.

Background

The technical field of the invention is the enhancement of organ function by transplantation of cultured cell populations. Typically, a significant portion of any organ function may have been lost before the patient has developed a complete organ failure. For example, up to 90-95% of kidney function may have been lost before kidney failure manifests itself. This demonstrates the great ability of rapid self-renewal (Cuppege et al, (1969) Lab. invest.21: 449-. This regenerative capacity allows the kidneys to recover normal function within days or weeks.

The disruption of normal kidney function and capacity may be due to a variety of mechanisms, such as infection, circulatory failure (shock), vascular occlusion, glomerulonephritis, urinary tract occlusion, renal failure associated with trauma, sepsis, post-operative complications, or drugs, particularly antibiotics. For most of the above reasons, reduced renal function results.

Treatment of some of the above-mentioned diseases typically involves dialysis, which removes waste and chemicals from the blood system or transplant. Dialysis is inconvenient for most patients. A typical treatment regimen involves a lengthy period of time during which the patient is connected to the dialysis device and the dialysis process is repeated multiple times within a week. In many instances, many patients experience side effects such as muscle spasms and hypotension associated with rapid changes in the patient's fluid.

For kidney transplantation, the main risk is kidney rejection, even with a good histocompatibility match. Immunosuppressive drugs such as cyclosporin and FK506 are commonly administered to patients in order to inhibit rejection. However, the immunosuppressive drugs have a narrow therapeutic window between adequate immunosuppression and toxicity. Prolonged immunosuppression may weaken the immune system, which may lead to the threat of infection. In some cases, even immunosuppression may not be sufficient to prevent renal rejection.

In order to avoid the above problems, various methods have been reported in which patient's own kidney cells have been cultured in vitro. For example, U.S. patent No. 5,429,938 to Humes discloses a method of reconstructing renal tubules using cultured renal cells. The reconstructed renal tubules may be transplanted into a patient.

Naughton et al disclose a three-dimensional tissue culture system in which stromal cells are layered on a polymeric support system (see U.S.5,863,531) and parenchymal cells are cultured on the substrate. Vacanti et al also disclose methods for culturing cells on three-dimensional matrices prepared with biodegradable polymers. The above method depends on shaping the support structure to the desired shape of the entire organ and allows such an artificial organ to be transplanted as a substitute into a body cavity. However, there are many occasions when it is not necessary to replace the entire organ, since only a part of the organ is damaged.

Thus, there is a need for better methods and compositions for enhancing organ function that do not require the growth or replacement of the entire organ. It is desirable to use smaller, simpler support structures that mimic the structure of the native organ.

Summary of The Invention

The present invention provides methods and compositions for enhancing organ function using a small-scale matrix graft prepared by seeding tissue-specific or undifferentiated cells onto a matrix material (e.g., a wafer, sponge or hydrogel). The seeded matrix composition may then be cultured in vitro to form a three-dimensional biomatrix in which the cells have grown to produce a tissue layer capable of developing a new morphology (neomorphic) of organ reinforcement. Once transplanted, the three-dimensional biomatrix develops and proliferates at one or more target sites within the organ so as to enhance organ function at the sites.

The present invention is based in part on the following findings: the seeded mini-matrix is capable of sustaining active proliferation of other cell populations. This may be due, in part, to the increased surface area of the matrix structure, which allows new cells to remain actively proliferating for a longer period of time. Prolonged proliferation allows the cells to develop new forms of organ-enhancing structures which may themselves develop into organ-enhancing units, or which can provide support for the growth and development of other cell populations that develop into organ-enhancing structures. In addition, the matrix allows to perform a spatial distribution that mimics the conditions in vivo, thus enabling the formation of a microenvironment that induces cell maturation and migration. The correct spatial distance thus provided enables cell-cell interactions to occur. Cell growth in the presence of such a matrix can be further enhanced by the addition of proteins, glycoproteins, glycosaminoglycans, and cell matrix.

In one aspect of the invention, an artificial organ construct for enhancing organ function is disclosed, comprising: a three-dimensional biomatrix formed by perfusing a matrix material with at least one cultured cell population such that the cells attach to the matrix material and produce a tissue layer capable of enhancing organ function, e.g., enhancing an organ such as the heart, kidney, liver, pancreas, spleen, bladder, ureter or urethra.

The constructs can be made by seeding cells on small matrix materials such as decellularized tissue, hydrogels or synthetic or natural polymers. The substrate is preferably a "mini-substrate" having a maximum dimension of less than about 50 mm. In a preferred embodiment, the substrate is a substantially flat structure having a ratio of its largest dimension to its thickness of greater than 5: 1.

In another aspect of the present invention, the organ-enhancing structure is a structure for enhancing kidney function, which comprises a three-dimensional biological matrix formed by perfusing a matrix material with a population of kidney cells, so that the kidney cells attach to the matrix and a tissue layer is generated that is differentiated into a nephron structure or a portion thereof, thereby enhancing kidney function.

In another aspect of the invention, the matrix is initially perfused with a population of endothelial cells such that the endothelial cells attach to the matrix material, producing an endothelial cell layer comprising the vasculature, and then seeded with a second cell population such that the second cell population attaches to an endothelial tissue layer comprising the vasculature and differentiates to enhance organ function.

In one aspect, the present invention is designed to enhance organ function without replacing, or reconstructing, the entire organ. For example, to enhance kidney function, small biological biopsies can be taken from the kidney and then kidney cells expanded in vitro. Cells are sorted to remove damaged cells, then normal cells are placed on a matrix (e.g., EGA wafer, sponge, hydrogel) and cultured. The matrix is then cultured until the cells produce a layer of renal tissue capable of differentiating into a new morphology of organ structure, thereby producing a biological matrix. The biomatrix is then re-implanted into the patient, at a desired site in the kidney, or near the urethra (e.g., near the ureter or bladder).

All types of renal cells are isolatable, i.e., proximal tubules, glomeruli, distal tubules and collection tubes. The cells may be seeded separately or simultaneously. When the cells are seeded simultaneously, it may be desirable to seed different types of cells onto the substrate sequentially, or in other cases, to seed various types of cells simultaneously. After transplantation, the cell mixture will regenerate into renal tissue within weeks. In one embodiment of the invention, the kidney cells are placed on a wafer of polymeric material, such as Ethyl Glycol Acetate (EGA) or decellularized tissue. The wafer can be placed in the positioning area in any shape suitable for implantation, e.g., it can be rolled, it can be flat, etc. In one embodiment, the wafer may be placed in a region of the organ, e.g., kidney. In another embodiment, multiple wafers may be placed at different locations of the organ. In a preferred embodiment, the matrix is miniaturized to facilitate implantation. In many cases, a small substrate is required, the largest dimension of which is on the order of 50 mm or less, preferably 25 mm or less, most preferably 10 mm or less. For example, the polymer wafers may be about 2-5 mm in size, preferably about 2-3 mm in size. The wafer is preferably thin enough to enable blood vessel growth to occur between the cells on the wafer and the cells surrounding it. In one embodiment, the matrix may be treated with a growth factor, e.g., VEGF.

During growth in vitro, the cells develop and produce a layer of tissue that can coat the matrix material. The tissue layer is capable of developing new forms of organ enhancement structures and supporting the growth and development of other cultured cell populations. In one embodiment, the tissue layer may be from a kidney cell. In another embodiment, the tissue layer may be from endothelial cells that develop to create the original vasculature. This primitive vascular system can continue to grow and develop and further support the growth of other parenchymal cells.

In one embodiment, the enhancement target is an organ selected from the group consisting of: heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. In another embodiment, the enhancement target is a portion of an organ selected from the group consisting of: heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra. In a preferred embodiment, the target organ is a kidney.

In another aspect, the invention relates to a method of treating a subject having an organ disease, the method comprising: implanting a biological matrix formed by seeding a matrix material with a population of cells such that the cells attach to the matrix, thereby producing proto-tissue (proto-tissue) including primitive vasculature capable of differentiating into a new morphology of organ structure; and monitoring the subject for modulation of the organ disease.

In another aspect, the invention relates to an artificial organ construct comprising: a matrix produced by seeding a matrix material with a population of cells such that the cells attach to the matrix, producing tissue comprising the original vasculature capable of differentiating into a new morphology of organ structure.

In another aspect, the present invention relates to a method for reconstituting an artificial kidney construct, the method comprising: seeding tissue-specific or undifferentiated cells onto a matrix material (such as a wafer, sponge or hydrogel) so that the cells attach to the matrix; culturing said cells in and on said matrix until said cells give rise to a tissue structure; and transplanting the inoculated matrix to a target site for organ enhancement in vivo.

In another aspect, the invention relates to a method of treating a subject having kidney disease, the method comprising: a matrix formed by seeding a matrix material with a population of cells such that the cells attach to the matrix, resulting in a tissue comprising the original vasculature capable of differentiating into a new form of kidney structure; and monitoring the subject for modulation of the kidney disease.

In another aspect, the invention relates to an artificial kidney construct comprising: a matrix formed by seeding a matrix material with a population of cells such that the cells attach to the matrix, resulting in a native tissue including the original vasculature capable of differentiating into a new morphology of organ structure.

Brief Description of Drawings

FIG. 1 is a schematic representation of the enhancement of a kidney using a biological matrix.

Detailed Description

The practice of the present invention employs, unless otherwise indicated, conventional microbiological, molecular biological and recombinant DNA techniques well known to those skilled in the art. The technique is described in detail in the literature (see, e.g., Sambrook, et al, Molecular Cloning: organic Manual (Current Edition); DNA Cloning: A practical application, Vol.I & II (D.Glover, ed.); Oligoreotide Synthesis (N.Gate, ed., Current Edition); Nucleic Acid Hybridization (B.Hames & S.Higgins, eds.), transformation and transformation (B.Hames & S.Higgins, eds., Current Edition), CRC Handbook partitions, Vol.I & II (P.J.J., ed.; amino visual, 2. D.M.J.M.. The present invention also employs techniques disclosed in the tissue engineering literature (see, for example, Principles of tissue engineering, Lanza et al (Current Edition)). In order that the invention may be better understood, certain terms are first defined:

the phrase "enhancing organ function" or "enhancing organ function" as used herein means to increase, enhance, improve the function of an organ that operates under less than optimal conditions. The term is used to denote increasing function so that the organ operates at the physiologically acceptable capacity of the subject. For example, the physiologically acceptable capacity of an organ such as the kidney or heart from a child is different from that of an adult or elderly patient. The entire organ, or a portion of the organ, may be enhanced. The enhancement preferably results in an organ having the same physiological response as the native organ. In a preferred embodiment, the capacity of an organ is enhanced when the organ functions at least 10% of its natural capacity.

The phrase "three-dimensional biological matrix" or "enhancing construct" or "new morphology of organ enhancing structures" as used herein means a small matrix that has been perfused with the cells and cultured until the cells form a tissue layer. The tissue layers may be a single monolayer, or multiple layers. Tissue-specific cells mean cells derived from a particular organ in need of enhancement, for example, cells from a kidney organ to be organ enhanced, and cells from a heart to be heart organ enhanced. The cells in the three-dimensional biomatrix establish a "tissue-like" histology, can regenerate tissue-like structures, and develop into primitive organoids with complex, multilayered structures that ultimately develop into actual organs or parts of organs. The three-dimensional biomatrix is an artificial organ, or a portion of an organ is a "functional equivalent" of a native organ, i.e., functions in the same or similar manner as a native organ, e.g., an artificial kidney enhancement construct has the same functional characteristics as a kidney in vivo. For example, the kidney enhancement structure may be a structure having a layer of tissue capable of developing into a nephron structure or a portion of a nephron structure. For renal enhancement, the tissue-specific cells may be an isolated population of cells selected from the group consisting of: distal tubule cells, proximal tubule cells, glomerular cells, Bowman's bursa cells, and loop of henle cells. Alternatively, the tissue-specific cells can be a mixed population of cells including cells from the distal tubule, proximal tubule, glomerular, Bowman's bursa, and loop of Henle. Various three-dimensional biomatrix can be prepared for a particular disease or condition. For example, the three-dimensional biomatrix may be specially prepared to alleviate glomerular-related disorders, including perfusion of the matrix material with a uniform population of glomerular cells. In addition, the three-dimensional biomatrix may be a common construct prepared with a mixed cell population of kidney cells.

When the three-dimensional biomatrix is contacted with host tissue of a target site in the organ, it is capable of growing and propagating within the target site and supplementing or enhancing the diminished activity of the organ at that site. The enhancing construct may be added at a single site within the organ. In addition, multiple enhancing constructs may be prepared and added to multiple sites within the organ.

The phrase "renal cell" as used herein refers to a cell derived from any region of the kidney, such as a cell derived from a distal tubule cell, a proximal tubule, a glomerular cell, a Bowman's bursa cell, or a loop of henle cell. The term is used to denote a mixture of cells that includes all cells from the kidney. The term is also used to refer to an isolated subpopulation of cells derived from a region of the nephron, e.g., a single population of glomerular cells only. Cells from the kidney can be obtained by harvesting a biopsy from the subject. Healthy cells can be isolated from diseased cells using cell sorting techniques. Cell sorting techniques can also be used to isolate subpopulations of cells.

The term "nephron structure" as used herein, refers to the complete functional unit of the kidney, which is capable of clearing waste and excess material from the blood in order to produce urine. Each of the millions of nephrons in each kidney is a tubule having a length of 1.2-2.2 inches (30-55 millimeters). One end of the tubule is closed, dilated, and folded into a double-walled cup-shaped structure, called a "Bowman's capsule," surrounding a cluster of capillaries called a "glomerulus. Fluid flowing from the blood is forced through the capillary walls of the glomeruli into the Bowman's capsule, into the adjacent tubules, where water and nutrients are selectively reabsorbed from the fluid into the blood and electrolytes such as sodium and potassium are equilibrated in the loop of henle cells and proximal tubules. The final concentrated product was collected in a collection tube as urine.

The term "part of the nephron structure" as used herein denotes any part of the nephron in question. For example, the cell population can be sorted to produce an isolated cell population consisting only of glomerular cells. This isolated population of glomerular cells can be used to inoculate a mini-matrix material and cultured to produce a three-dimensional mini-biological matrix with a layer of glomerular tissue that can differentiate into glomeruli. The same method can be applied to the preparation of three-dimensional small biological matrices from specific cells originating from different regions of the nephron, which differentiate into a part of the nephron, such as the distal tubule part. Other kidney diseases associated with specific regions of the nephron include pathological conditions associated with tubular cells.

The term "target site" as used herein means the region in the organ where enhancement is desired. The target site may be a single region within the organ, or may be multiple regions within the organ. The entire organ, or a portion of the organ, may be enhanced. The enhancement preferably results in an organ having the same physiological response as a normal organ. The entire organ may be enhanced by placing multiple biomatrix at appropriate distances over the entire organ, for example, along the entire longitudinal section of the kidney. In addition, a portion of the organ may be enhanced by placing at least one biological matrix at a target site of the organ, such as the top of the kidney.

The term "attached" as used herein means cells that are directly attached to the substrate or cells that themselves are attached to other cells.

The phrase "small matrix material" as used herein denotes a supporting framework to which cells can attach. The "mini-matrix" preferably has a maximum dimension of less than about 50 mm. In a preferred embodiment, the substrate is a substantially flat structure having a ratio of its largest dimension to its thickness of greater than 5: 1. The microparticulate matrix material includes any material and/or shape to which cells can attach or to which cells can attach (or which can be modified so that cells can attach or to which cells can attach) and which allows cells to grow into at least one monolayer or at least one tissue layer. The cultured population of cells can then be grown on or within the substrate. In one embodiment, the matrix material is a polymer matrix that provides the gap distance required for cell-cell interaction. In another embodiment, the matrix material is a hydrogel composed of a crosslinked polymer network that is generally insoluble or poorly soluble in water, but is capable of swelling to an equilibrium size in the presence of excess water. Various types of hydrogels have been synthesized and characterized due to their unique properties and their potential use in areas such as drug delivery.

The size of the mini-matrix material may also vary depending on the area of the organ to be enhanced. The size is typically smaller than a complete organ. The volume of the matrix is preferably about 1 cubic millimeter to the size of the organ. The volume is most preferably from about 0.01 cubic millimeters to about 30 cubic millimeters, more preferably from about 0.1 cubic millimeters to about 20 cubic millimeters, even more preferably about 1 cubic millimeter, 2 cubic millimeters, 3 cubic millimeters, 4 cubic millimeters, 5 cubic millimeters, 6 cubic millimeters, 7 cubic millimeters, 8 cubic millimeters, 9 cubic millimeters, and 10 cubic millimeters in size. In many cases, an elongated or flat substrate is preferred. The length of the largest dimension of the substrate is preferably greater than 0.2 mm and less than 100mm, more preferably in the range of about 0.50 mm to about 30 mm. In a preferred embodiment, the mini-matrix is substantially flat in shape and has a ratio of its largest dimension to its thickness of greater than 5: 1, more preferably greater than 10: 1.

The shape and size of the mini-stroma material is determined by the organ to be enhanced, and the type of mini-stroma material used to prepare the mini-biomatrix. For example, if a polymer matrix is used for renal enhancement, the dimensions of the polymer matrix may vary in terms of the width and length of the polymer matrix, e.g., the dimensions may be about 1mm wide by 1mm long by 1mm high to about 10 mm wide by 20 mm long by 1mm high. The skilled person will appreciate that the size and dimensions of the polymer matrix may be determined in dependence on the region of the organ to be amplified, as well as the actual organ to be amplified.

In addition, if the matrix material used to reinforce the kidney is a hydrogel, the volume of the hydrogel can be determined based on the size of the area in the kidney to be reinforced. For example, the cell population can be cultured in the range of about 1 cubic millimeter, 2 cubic millimeters, 3 cubic millimeters, 4 cubic millimeters, 5 cubic millimeters, 6 cubic millimeters, 7 cubic millimeters, 8 cubic millimeters, 9 cubic millimeters, and 10 cubic millimeters. In one embodiment, the hydrogel may be injected into one or more target sites of the organ. The volume of the hydrogel can be varied depending on the organ and region of the organ to be enhanced. For example, if the organ is a heart and the infarct zone in the heart is to be enhanced, the volume of the hydrogel can be from a volume less than the size of the infarct to the actual size of the infarct.

The term "biological structure" as used herein refers to a portion of an organ that has been decellularized by removing intact cells and tissue components from the portion of the organ.

The term "decellularized" or "decellularized" as used herein means a biological structure (e.g., an organ or portion of an organ) from which cellular and tissue content has been removed, leaving an intact acellular infrastructure. Organs such as the kidney are composed of various specialized tissues. The specialized tissue structure or parenchyma of an organ provides a specific function associated with the organ. The supportive fiber mesh of the organ is the matrix. Most organs have a stromal framework consisting of undecified connective tissue, which supports the specialized tissue. The decellularization process removes the specialized tissue, leaving a complex three-dimensional network of connective tissue. The basic structure of connective tissue, mainly composed of collagen. The decellularized structure provides a matrix material upon which various cell populations can be infused. The decellularized biological structure can be rigid or semi-rigid, with the ability to change its shape. Examples of decellularized organs useful in the invention include, but are not limited to, heart, kidney, liver, pancreas, spleen, bladder, ureter, and urethra.

The phrase "three-dimensional scaffold" as used herein, refers to the residual infrastructure formed after decellularization of a native biological structure, such as an organ. This complex three-dimensional scaffold provides the supportive framework to which cells can attach and grow on. The cultured cell population can then be grown on the three-dimensional scaffold, which provides the exact gap distance required for cell-cell interaction. Thereby providing a reconstructed organ that resembles a natural in vivo organ. Such a three-dimensional scaffold is perfused with a cultured population of endothelial cells that grow and develop to provide a layer of endothelial tissue including the native vasculature that is capable of developing into a mature vasculature. The endothelial tissue layer and the primitive vascular system are also capable of supporting the growth and development of at least one other cultured cell population.

The term "primitive vasculature" as used herein, refers to the early stages of development of the vasculature, which includes the vessels that deliver blood to the tissue structures.

The term "subject", as used herein, means any living organism capable of eliciting an immune response. The term subject includes, but is not limited to, humans, non-human primates, such as chimpanzees and other apes and monkey species; livestock such as cattle, sheep, pigs, goats, and horses; domestic mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats and guinea pigs, and the like. The term does not denote a particular age or gender. Thus, the term is intended to include adult and newborn subjects, as well as fetuses, whether they are male or female.

I. Isolation and culture of cells

Cells can be isolated from a variety of sources, for example, from biopsy tissue, or autopsy tissue, stem cell populations programmed to differentiate into desired organ cells, heterologous cell populations, xenogeneic cells, and allogeneic cells that have been coated to render them non-immunogenic. Also included within the scope of the invention are methods of transfecting a population of cells with factors such as growth factors that improve tissue formation.

The isolated cells are preferably allogeneic, autologous cells obtained from the subject by biopsy. For example, the kidney cells may also be derived from a malfunctioning kidney of the subject and cultured in vitro. The biopsy may be obtained by using a biopsy needle, or a quick action needle that enables this procedure to be quick and simple. The biopsy area may be treated as a local anesthetic by injecting a small amount of lidocaine subcutaneously. Small biopsy cores of organs such as the kidney can then be propagated and cultured in vitro as disclosed in the following references: atala, et al, (1992) j.urol.148, 658-62; atala, et al (1993) J.Urol.150: 608-12. Cells from relatives or other donors of the same species may also be used for appropriate immunosuppression.

Methods for cell isolation and culture are disclosed in the following references: fauza et al (1998) J.ped.Surg.33, 7-12 and Freshney, animal cell culture, A Manual of basic technology, 2d Ed., A.R.Liss, Inc., New York, 1987, Ch.9, pp.107-126, which are incorporated herein by reference. The cells can be isolated using techniques well known to those skilled in the art. For example, the tissue or organ may be dissociated by mechanical means and/or treated with digestive enzymes and/or chelators to weaken the linkage between adjacent cells, allowing it to disperse the tissue into a suspension of single cells without significant cell damage. Enzymatic dissociation may be accompanied by disruption of the tissue, and the disrupted tissue is treated with any one of a variety of digestive enzymes, alone or in combination. Such digestive enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase, pronase, and dispase. Alternatively, mechanical disruption may be employed, and this may be accomplished by a variety of methods including, but not limited to, scraping the surface of the organ, using a grinder, stirrer, screen, homogenizer, pressure chamber, or sonication. For an overview of tissue spreading techniques, see Freshney (1987), animal cell culture, A Manual of Basic Technique, 2dEd., A.R. Liss, Inc., New York, Ch.9, pp.107-126.

Preferred cell types include, but are not limited to, kidney cells, endothelial cells, heart cells, liver cells, pancreas cells, spleen cells, urothelial cells, mesenchymal cells, smooth or skeletal muscle cells, muscle cells (myostem cells), fibroblasts, chondrocytes, adipocytes, fibromyoblasts, and ectodermal cells, including duct and skin cells, liver cells, pancreatic islet cells, cells present in the gut, and other parenchymal cells, osteoblasts, and other cells that form bone or cartilage. In some instances, it may be desirable to also include nerve cells. In a preferred embodiment, the kidney cells are isolated. Renal cells from all developmental stages, such as fetal, neonatal, juvenile up to adult, may be used. In another preferred embodiment, endothelial cells are isolated.

Once the tissue is degraded into a suspension of single cells, the suspension can be separated into subpopulations from which the cellular elements can be obtained. This objective can also be achieved using standard techniques for cell separation including, but not limited to, cloning and screening of specific cell types, selective destruction of unwanted cells (negative selection), separation based on differential cell aggregation forces in mixed cell populations, freeze-thaw methods, differential adhesion properties of cells in mixed cell populations, filtration, conventional and zonal centrifugation, centrifugal elutriation (convection centrifugation), unit gravity centrifugation, convection distribution, electrophoresis, and fluorescence activated cell sorting. For a review of the Techniques of clone screening and cell isolation, see Freshney (1987), animal cell culture, A Manual of basic technologies, 2d Ed., A.R. Liss, Inc., New York, Ch.11 and 12, pp.137-168. For example, kidney cells of the kidney can be enriched by fluorescence activated cell sorting. In addition, different regions of kidney cells can be sorted into independent subpopulations. For example, a subpopulation of glomerular cells, Bowman's bursa cells, distal tubule cells, proximal tubule cells, loop of henle cells and collecting duct cells are isolated.

Cell fractionation may also be required in order to sort healthy cells from diseased cells, for example, when the donor is suffering from a disease such as cancer or tumor metastasis. The cell population can be sorted to isolate malignant or other tumor cells from normal non-cancerous cells. The normal non-cancerous cells isolated by one or more sorting techniques may then be used for organ enhancement.

The isolated cells may be cultured in vitro to increase the number of cells available for coating the matrix material. To avoid tissue rejection, preferably allogeneic cells are used, more preferably autologous cells. However, if an immune response occurs in the subject after transplantation of the artificial organ, the subject may be treated with an immunosuppressive agent such as cyclosporin or FK506 in order to reduce the possibility of rejection. In certain embodiments, chimeric cells, or cells from transgenic animals, can be coated on the matrix material. In addition, stem cells may be used.

The stem cells can also be used to prepare the novel forms of the organoenhancement structures of the present invention. The stem cells may be derived from a human donor, e.g., pluripotent hematopoietic stem cells. Embryonic stem cells, adult somatic stem cells, and the like. The stem cells can be cultured in the presence of a combination of polypeptides, recombinant human growth factors, and maturation-promoting factors, such as cytokines, lymphokines, colony stimulating factors, mitogens, growth factors, and maturation factors, to differentiate into a desired cell type, e.g., a kidney cell or a heart cell. For example, methods of differentiating adult Bone Marrow Stem Cells (BMSCs) into kidney and liver cells are disclosed in the following documents: forbes et al (2002), Gene Ther 9: 625-30. Methods have been established for the in vitro differentiation of embryonic stem cells into, for example, cardiomyocytes, which represent all specialized cell types of the heart, such as atrial-like, ventricular-like, sinoatrial-like and Purkinje-like cells (see, e.g., Boheler et al (2002) Circ Res 91: 189-. Pluripotent stem cells from metarenal mesenchyme can give rise to at least three different cell types: glomerular cells, proximal and distal epithelial cells, differentiate into single nephron fragments (see, e.g., Herzlinger et al (1992) Development 114: 565-72).

The organ cells, such as kidney cells, may be transfected with a specific gene prior to coating the matrix material. The new forms of organ augmentation constructs may carry the host or have the genetic information required for long-term survival of the organ to be augmented.

The isolated cells may be normal or genetically engineered to provide additional or normal function. For example, cells may be transfected with a compound that slows the progression of the disease at a target site within the organ. Cells may also be engineered to attenuate or eliminate an immune response in the host. For example, expression of cell surface antigens such as type I and type II histocompatibility antigens may be inhibited. This may result in transplanted cells having less likelihood of being rejected by the host. In addition, transfection may also be used for gene delivery. Methods of genetically engineering cells with retroviral vectors, polyethylene glycol, or other methods known to those skilled in the art may be used. Among these, the use of nucleic acid vectors that transport and express nucleic acids within the cells is included (see Goeddel; Gene Expression Technology: Methods in enzymology 185, Academic Press, San Diego, Calif. (1990)).

Cells grown on the matrix can be genetically engineered to produce gene products that are beneficial for transplantation, such as anti-inflammatory factors, e.g., anti-GM-CSF, anti-TNF, anti-IL-1, and anti-IL-2. In addition, endothelial cells can be genetically engineered so that "knockdown" promotes expression of natural gene products of inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or MHC "knockdown" expression, so as to reduce the risk of rejection. In addition, cells may be genetically engineered for gene therapy to modulate the level of gene activity in a patient to assist or improve the outcome of tissue transplantation.

Methods of genetically engineering cells with retroviral vectors, polyethylene glycol, or other methods known to those skilled in the art may be used. Including the use of expression vectors for the transport and expression of nucleic acid molecules in such cells (see Geoddel; Gene expression technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)).

Vector DNA is introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in the following references: molecular Cloning, Sambrook et al: a Laboratory Manual, 2nd Edition, Cold spring harbor Laboratory press (1989), and other Laboratory textbooks. The inoculated cells can be engineered with a recombinant DNA construct containing the gene of interest transformed or transfected into the cell. The seeded matrix comprises transfected cells capable of expressing an active gene product, which matrix can be transplanted into an individual lacking the product. For example, in disease states, symptoms of various types of vascular, urogenital, hernia, gastrointestinal disorders, or renal disorders can be inhibited or alleviated. The level of gene activity can be increased by increasing the level of gene product present or by increasing the level of active gene product present in a biological matrix comprising matrix material and tissue, e.g., endothelial tissue layer or renal tissue layer. The biomatrix culture expressing the active target gene product may then be transplanted into a patient lacking the product.

The bio-matrix culture containing the genetically engineered cells can then be transplanted into the subject in order to alleviate symptoms of the disease. The gene expression may be under the control of a non-inducible (i.e., constitutive) or inducible promoter. The level of gene expression and the type of gene regulated can be controlled according to the subsequent pattern of treatment for each patient.

Matrix Material

The methods and compositions of the present invention are prepared by using a matrix material as a substrate, depositing cells on the matrix material, and allowing the cells to grow and adhere thereto. For a particular organ to be enhanced, it is important to reconstitute the cellular microenvironment present in vivo in culture. Maintaining a similar or identical infrastructure to the organs in the body, creates an optimal environment for cell-cell interaction, development and differentiation of cell populations. The extent of cell and tissue layer growth prior to use in vivo may vary depending on the type of organ to be enhanced.

The present invention provides methods for enhancing organ function using matrix materials that support the maturation, development and differentiation of other cells cultured in vitro to form components of adult tissue that resemble their in vivo counterparts. The matrix allows for optimal cell-cell interactions, allowing for a more natural development of cell phenotypes and tissue microenvironments. The matrix may also allow cells to continue to actively grow, proliferate and differentiate to produce a new morphology of organ enhancement structures that can also support the growth, proliferation and differentiation of other cultured cell populations.

According to the invention, the cells grown on the matrix material can grow in layers, forming a cellular structure similar to the biological conditions present in vivo. The matrix can support the proliferation of different types of cells and form a variety of different tissues. Examples include, but are not limited to, kidney, heart, skin, liver, pancreas, adrenal gland and nervous tissue, as well as gastrointestinal and genitourinary tract tissue, and circulatory system.

The inoculated substrates of the present invention can be used for a variety of purposes. For example, the matrix can be transplanted into a subject. According to the present invention, the graft may be used to replace or augment existing tissue. For example, a subject suffering from kidney disease is treated by enhancing the native kidney. The subject may be monitored for remission of a renal condition following transplantation of the matrix graft.

Also within the scope of the present invention are compositions and methods for organ enhancement using a novel morphology of an organ enhancement structure having a cultured cell population for the generation of a tissue layer from the single cell population. In addition, the new forms of organ augmentation constructs may include multilayers derived from at least two different cell populations, e.g., a smooth muscle cell population, and a urothelial cell population. In preferred embodiments, the new morphology of the organ enhancement structure comprises a new morphology of the organ enhancement structure having the original vascular system and at least one other tissue layer derived from parenchymal cells.

Once infused onto the matrix material, the endothelial cells proliferate and develop on the polymer matrix to form an endothelial tissue layer. During in vitro culture, the endothelial cells develop and differentiate to produce primitive vasculature capable of developing into mature vasculature, and are also capable of further development and also support the growth of parenchymal cells perfused into the matrix material. Importantly, because the polymer matrix has a basic structure that allows the culture medium to reach the endothelial tissue layer and parenchymal cells, different cell populations continue to grow, divide, and remain functionally active. The parenchymal cells proliferate and differentiate into a new morphology of organ structure having a morphology similar to that of living structures. The extent of endothelial and parenchymal cell growth prior to use in vivo may vary depending on the type of organ to be enhanced. Organs that can be enhanced include, but are not limited to, the heart, kidneys, liver, pancreas, spleen, bladder, ureters, and urethra.

(i) Polymer matrix

In a preferred embodiment, the matrix material is a polymer matrix. Examples of suitable polymers include, but are not limited to, collagen, poly (alpha esters) such as poly (lactic acid), poly (glycolic acid), polyorthoesters and polyanhydrides and copolymers thereof, cellulose ethers, cellulose esters, fluorinated polyethylene, phenols, poly-4-methylpentene, polyacrylonitrile, polyamides, polyamideimides, polyacrylates, polybenzoxazoles, polycarbonates, polycyanoarylethers, polycarbonates, polyethers, polyetheretherketones, polyetherimides, polyetherketones, polyethersulfones, polyethylenes, polyfluoroolefins, polyimides, polyolefins, polyoxadiazoles, polyphenylenes, sulfides, polypropylene, polystyrene, polysulfides, polysulfones, polytetrafluoroethylene, polythioethers, polytriazoles, polyurethanes, polyvinylidene 1, 1-difluoroethylene, regenerated cellulose, urea-formaldehyde, or copolymers or physical mixtures of said materials.

Polymers, such as polyglycolic acid, are biocompatible structures suitable for the production of organ augmentation structures. The biocompatible polymer may be formed by methods such as solvent casting, pressure forming, wire drawing, netting, leaching, weaving, and coating.

In solvent casting, a solution of one or more polymers in a suitable solvent such as methylene chloride is cast in a branched form of a reduced pressure structure. After evaporation of the solvent, a film was obtained.

In pressure forming, the polymer is compressed into a suitable form at a pressure of up to 30000 pounds per square inch. Drawing includes drawing from molten polymer, and netting includes forming a screen by compressing fibers into a felt-like material.

In leaching, a solution containing both materials is dispersed into a shape that approximates the final form of the organ. A solvent is then used to dissolve one of the components to give a porous structure (see Mikos, US 5,514,378, incorporated herein by reference).

In nucleation, an organ-shaped film is exposed to radioactive fission products, which can create a track of radiation-damaged material. The polycarbonate sheet is then etched with an acid or base to convert the trajectory of the radiation-damaged material into holes. Finally, laser forming may be used, and individual holes fired through many materials to form organ structures with uniform pore size.

The polymer matrix can be fabricated with a controlled pore structure that allows nutrients to pass from the culture medium to the deposited cell population, but prevents the cultured cells from migrating through the pores. Cell attachment and cell viability in vitro can be assessed by scanning electron microscopy, histology, and quantitative assessment with radioisotopes.

The polymer matrix can be molded into any of a variety of desired shapes to meet a variety of overall system, geometric or spatial constraints. The polymer matrix may be formed into different sizes to fit the organs of patients of different body shapes. The polymer matrix may also be shaped to facilitate the particular needs of the patient, for example, to meet the needs of a disabled patient who may have a different abdominal space, may require a reconstructed organ or part of an organ to fit in the space.

In other embodiments, the polymer matrix is used to treat a layered structure in the body, such as the urethra, vas deferens, fallopian tubes, lacrimal duct. In such applications, the polymer matrix may be formed into a hollow tubular shape.

The new forms of the organ enhancement structures of the invention, which function to enhance the organ, may be flat, tubular or have complex geometries. The shape of the organ is determined by its intended use. The artificial organ may be transplanted in order to repair, enhance or replace diseased or damaged portions of the organ. Flat sheets or wafers may be used. The flat sheet may be formed into a desired shape and geometry to fit a target site of an organ, for example, wound into a cylindrical or tubular shape. For example, tubular grafts may be used to replace tubular organs, such as the esophagus, organs, intestines, and fallopian tubes, in transverse sections. The organ has a substantially tubular shape with an outer surface and a luminal surface.

The polymer matrix may be infiltrated with a material, for example, a liquefied copolymer (poly-DL-lactide-co-glycolide 50: 5080 mg/ml dichloromethane), to modify its mechanical properties. This can be achieved by applying one or more layers until the desired mechanical properties are obtained. The size of the polymer matrix is determined according to the degree of organ enhancement desired. For example, the substrate may have dimensions of about 1mm long by 1mm wide by 1mm thick. The shape and size of the matrix depends on the location of the organ to be enhanced.

In one embodiment, the organ to be enhanced is a kidney, and the substrate may be a flat sheet having dimensions of about 1 cm by 1 cm and a thickness of less than about 1 mm. The length of the largest dimension of the substrate is greater than 0.2 mm and less than 100mm, more preferably from about 0.50 mm to about 30 mm. In a preferred embodiment, the mini-matrix is substantially flat in shape and the ratio of its largest dimension to its thickness is greater than 5: 1, more preferably greater than 10: 1.

In another embodiment, the polymer matrix may be wound into a tubular shape after seeding the cells to provide a greater volume of the organ structure. The polymer matrix may be in the size and shape of a wafer, rolled wafer, square and rectangular, and the like. The shape of the polymer matrix is determined according to the area to be enhanced, the organ to be enhanced. The size and shape of the polymer matrix is selected so that the ratio of the largest dimension of the resulting new morphology of the organoenhancing structure to its thickness is greater than 5: 1, more preferably greater than 10: 1.

In one embodiment, an organ comprising multiple layers, such as a bladder, may be reinforced with the enhanced organ structure. This object is achieved by preparing a tissue layer from one side of said polymer matrix by applying a suspension of a first homogeneous cell population, such as kidney cells, to one side of said polymer matrix. Culturing the first homogenous cell suspension in a culture medium until the cells develop and proliferate to produce a monolayer, and the cells of the monolayer are attached to the polymer matrix. Once the monolayer is formed, the first homogenous cell suspension is deposited on the first monolayer, and the cells are cultured until they develop and proliferate to produce a second cell monolayer on the first monolayer, thereby forming a bilayer. Repeating the above process until a plurality of layers including the plurality of layers of the first uniform cell population is generated. Culturing the multilayers so as to produce tissue layers having morphological and functional characteristics that enable it to differentiate into organ-enhancing structures.

In another embodiment, multiple layers of uniform cell populations are generated using both sides of the polymer matrix. This is achieved by coating one side of the polymer matrix with a suspension of a homogeneous cell population, such as kidney cells, and culturing the cells until they develop into a monolayer. The above process is repeated on the opposite side of the polymer matrix. The above process is repeated on both sides of the polymer until multilayers comprising multilayers of uniform cell populations are produced on both sides of the matrix. The polymer matrix comprising the multilayers on both sides is cultured in order to produce tissue layers with morphological and functional characteristics enabling it to differentiate into organ-enhancing structures. In another embodiment, multiple layers of different cell populations can be prepared using both sides of the polymer matrix. This is achieved by applying a suspension of a first homogeneous cell population, such as endothelial cells, to one side of a polymer matrix and culturing the cells until they develop into a monolayer. The process is repeated with a different homogenous cell population, such as kidney cells, on the opposite side of the polymer matrix.

In one embodiment, the organ to be enhanced is a kidney. The organ enhancement structure is prepared by seeding kidney cells, or isolated distal tubule cells, proximal tubule cells, or glomerular cell populations in or on a matrix material. The kidney may be surgically opened along its longitudinal axis and the new configuration of organ augmentation structure is placed at least one target site within the kidney. In another embodiment, multiple new forms of organ augmentation structures may be prepared and added at multiple target sites within the kidney. The amount of new morphology of organ enhancement structure to be added depends on the extent to which the kidney is damaged. For example, if one-half of the upper kidney is damaged, about 1 to about 10 wafers of the enhancer construct can be placed equidistantly along the upper kidney. The number of reinforcing constructs to be implanted also depends on the size of the wafers used to produce them. If the wafers have large dimensions, such as 1 cm x 1mm x 1 cm, a smaller number of wafers are required to strengthen the upper part of the kidney. In addition, if the wafer is small, for example, 1mm x 1mm, many such wafers are required to strengthen the upper part of the kidney.

(ii) Hydrogels

In one embodiment, the matrix material is a hydrogel of a crosslinked polymer network, which is generally insoluble or poorly soluble in water, but which is capable of swelling to an equilibrium size in the presence of excess moisture. For example, the cells may be placed into a hydrogel and the hydrogel injected into a desired location within an organ. In one embodiment, the cells can be injected with collagen alone. In another embodiment, the cells can be injected with collagen and other hydrogels. The composition of the hydrogel may include, but is not limited to, for example, poly (esters), poly (hydroxy acids), poly (lactones), poly (amides), poly (ester-amides), poly (amino acids), poly (anhydrides), poly (orthoesters), poly (carbonates), poly (phosphazenes), poly (thioesters), polysaccharides, and mixtures thereof. Additionally, the composition may also include, for example, poly (hydroxy) acids, including poly (alpha-hydroxy) acids and poly (beta-hydroxy) acids. The poly (hydroxy) acids include, for example, polylactic acid, polyglycolic acid, polycaproic acid, polybutanoic acid, polypentanoic acid, and copolymers and mixtures thereof. Various types of hydrogels have been synthesized and characterized due to their unique properties and their potential use in the field of controlled drug delivery. Most of this work has focused on lightly crosslinked, homogeneous homopolymers and copolymers.

Bulk polymerization, i.e., polymerization of monomers to produce a homogeneous hydrogel in the absence of added solvent, can produce a glassy, transparent polymer matrix that is very rigid. When soaked in water, the glassy matrix swells, becoming soft and flexible. Porous hydrogels, typically prepared by solution polymerization techniques, are capable of polymerizing monomers in a suitable solvent. The nature of the synthetic hydrogel (whether it is a dense gel or a loose polymer network) depends on the type of monomer, the amount of diluent in the monomer mixture, and the amount of crosslinking agent. The pore size can also be increased to the micrometer range as the amount of diluent (typically water) in the monomer mixture is increased. Hydrogels with effective pore sizes of 10-100 nanometers, and 100 nanometers-10 microns, are referred to as "microporous" and "macroporous" hydrogels, respectively. The microporous and macroporous structure of hydrogels can be distinguished from non-hydrogel porous materials such as porous polyurethane powders. In the field of plastic foams, micropores and macropores are defined as having pores smaller than 50 microns and 100-300 microns, respectively. One of the reasons for this difference is that hydrogels with pores larger than 10 microns are uncommon, while porous plastics with pores of 100-300 microns are very common.

Microporous and macroporous hydrogels, commonly referred to as polymeric "sponges". When monomers such as hydroxyethyl methacrylate (HEMA) are polymerized in water at a starting monomer concentration of 45 (w/w)% or more, a hydrogel is produced having a porosity greater than that of the homogeneous hydrogel. Matrix materials of the present invention include conventional foam or sponge materials, as well as so-called "hydrogel sponges". For further description of hydrogels, see U.S. Pat. No. 5,451,613 (Smith et al).

(iii) Acellular parts of biological structures

In another embodiment, portions of a native decellularized organ can be used to prepare a new form of an organ enhancement structure. Biological structures, or portions of organs, can be decellularized by removing intact cellular and tissue components from the organ. The decellularization process comprises a series of sequential extractions. A key feature of this extraction process is that a rough extraction of complex underlying structures that may interfere with or destroy the biological structure can be avoided. The first step involves the elimination of cell debris and lysis of the cell membrane. Following this step, lysis of the cytoplasmic and nuclear components of the cell nucleus follows.

The biological structure, e.g., a portion of an organ, is preferably decellularized by removing cell membranes and cell debris surrounding the portion of the organ by mild mechanical disruption. The gentle mechanical disruption method must be sufficient to disrupt the cell membrane. However, the decellularization process should avoid damaging or interfering with the complex infrastructure of the biological structure. Gentle mechanical disruption methods include scraping the surface of the organ portion, agitating the organ portion, or agitating the organ in a suitable volume of fluid, such as distilled water. In a preferred embodiment, the gentle mechanical disruption method comprises agitating the organ portion in a suitable volume of distilled water until the cell membranes are disrupted and cell debris is removed from the organ.

After removal of the cell membrane, the nuclear and cytoplasmic components of the biological structure are removed. This can be achieved by lysing the cellular and nuclear components without destroying the infrastructure. For solubilizing the nuclear components, a nonionic detergent or surfactant may be used. Examples of nonionic detergents or surfactants include, but are not limited to, the Triton family, available from Rohm and Haas corporation of Philadelphia, Pa., which includes Triton X-100, Triton N-101, Triton X-114, Triton X-405, Triton X-705, and Triton DF-16, and are available from a number of vendors. Tween series, such as monolaurate (Tween 20), monopalmitate (Tween 40), monooleate (Tween 80), and polyoxyethylene-23-dodecyl ether (Brij.35), polyoxyethylene ether W-1(Polyox), etc., sodium cholate, deoxycholate, CHAPS, saponin, n-decyl β -D-glucopyranoside, n-octyl β -D-heptyl P-D-glucopyranoside, n-octyl α -D-glucopyranoside, and Nonidet P-40.

It will be appreciated by those skilled in the art that descriptions and vendors for compounds belonging to the above classes are commercially available and can be found in: "Chemical Classification, Emulsifiers and Detergents", McCutcheon's, Emulsifiers and Detergents, 1986, North American and International regulations, McCutcheon Division, MC Publishing Co., Glen Rock, N.J., U.S.A. and Judulth Neugebauer, A Guide to the Properties and uses of Detergents in Biology and Biochemistry, Calbiochem.R., Hoech cell Corp., 1987. In a preferred embodiment, the non-ionic surfactant is of the Triton. series, preferably Triton X-100.

The concentration of the non-ionic detergent may vary depending on the type of biological structure to be decellularized. For example, for delicate tissues such as blood vessels, the concentration of the detergent should be reduced. The preferred concentration range for the nonionic detergent may be from about 0.001 to about 2.0% (w/v). More preferably from about 0.05 to about 1.0% (w/v). More preferably from about 0.1% (w/v) to about 0.8% (w/v). A preferred concentration range is from about 0.001 to about 0.2% (w/v), with from about 0.05 to about 0.1% (w/v) being particularly preferred.

The cytoskeletal components, including dense cytoplasmic filament networks, intercellular complexes, and apical fibrocyte structures, can be solubilized with an alkaline solution such as ammonium hydroxide. Other alkaline solutions consisting of ammonium salts or their derivatives may also be used to dissolve the cytoskeletal components. Examples of such other suitable ammonium solutions include ammonium sulfate, ammonium acetate and ammonium hydroxide. In a preferred embodiment, ammonium hydroxide is used.

The concentration of the alkaline solution, such as ammonium hydroxide, may vary depending on the type of biological structure to be decellularized. For example, for delicate tissues such as blood vessels, the concentration of the detergent should be reduced. A preferred concentration range may be from about 0.001 to about 2.0% (w/v), more preferably from about 0.005 to about 0.1% (w/v). Even more preferably from about 0.01% (w/v) to about 0.08% (w/v).

The decellularized, lyophilized structure can be stored at a suitable temperature until needed for use. Prior to use, the decellularized structure can be equilibrated in a suitable isotonic buffer or cell culture medium. Suitable buffers include, but are not limited to, Phosphate Buffered Saline (PBS), saline, MOPS, HEPES, and Hank's balanced salt solutions, and the like. Suitable cell culture media include, but are not limited to, RPMI 1640, Fisher's, Iscove's, McCoy's, and Dulbecco's media, among others.

Cell adhesion

In certain embodiments, the attachment of the cells to the matrix material is enhanced by coating the matrix material with compounds such as basement membrane components, agar, agarose, gelatin, gum arabic, collagen I, II, III, IV, and V, fibronectin, laminin, glycosaminoglycans, mixtures thereof, and other hydrophilic and peptide attachment materials known to those skilled in the art of cell culture. The preferred material for coating the matrix material is collagen.

For example, in other embodiments, the matrix material may be treated with factors or drugs prior to transplantation, prior to or after coating the matrix material with cultured cells, in order to promote the formation of new tissue after transplantation. Factors including drugs may be incorporated into the matrix material or provided in combination with the matrix. The factors are generally selected based on the tissue or organ to be reconstructed or enhanced to ensure the formation of appropriate new tissue (e.g., additives to promote bone healing) in the transplanted organ or tissue (see, e.g., Kirker-Head, (1995) vet. surg.24: 408-19). For example, when matrix materials are used to enhance vascular tissue, Vascular Endothelial Growth Factor (VEGF) may be used to promote the formation of new vascular tissue (see, e.g., U.S. Pat. No. 5,654,273, issued to Gallo et al). Other useful additives include antimicrobial agents such as antibiotics.

Establishment of endothelial tissue layer

In one aspect, the invention relates to the use of a cultured population of endothelial cells or a portion of an decellularized organ perfused on or within a polymeric matrix material such that the endothelial cells grow and develop, thereby creating a primitive vasculature. The endothelial cells may be derived from organs such as skin, liver, and pancreas, and the cells may be obtained by biopsy (if appropriate) or by autopsy. Endothelial cells may also be obtained from any suitable cadaveric organ. The endothelial cells may be expanded by culturing them to a desired cell density in vitro prior to perfusion into the matrix material.

Endothelial cells can be conveniently isolated by lysing a suitable organ, or portion of an organ or tissue, to be used as a source of the cells. This can be accomplished by using techniques known to those skilled in the art. The tissue or organ may be mechanically isolated and/or treated with digestive enzymes and/or chelators that weaken the association between adjacent cells, allowing the tissue to be dispersed as a suspension of single cells without significant destruction of the cells. Enzymatic dissociation can be accomplished by disrupting the tissue and treating the disrupted tissue with any one or a combination of a plurality of digestive enzymes. Such enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase, and/or hyaluronidase, DNase, pronase, and dispase. Alternatively, mechanical disruption may be employed, and this may be accomplished by a variety of methods including, but not limited to, scraping the surface of the organ, using a grinder, stirrer, screen, homogenizer, pressure chamber, or sonication. For an overview of tissue spreading techniques, see Freshney (1987), animal cell culture, A Manual of Basic Technique, 2d Ed., A.R. Liss, Inc., New York, Ch.9, pp.107-126.

Once the tissue is degraded into a suspension of single cells, the suspension can be separated into subpopulations from which endothelial cells can be obtained. This objective can also be achieved using standard techniques for cell separation including, but not limited to, cloning and screening of specific cell types, selective destruction of unwanted cells (negative selection), separation based on differential cell aggregation forces in mixed cell populations, freeze-thaw methods, differential adhesion properties of cells in mixed cell populations, filtration, conventional and zonal centrifugation, centrifugal elutriation (convection centrifugation), unit gravity centrifugation, convection distribution, electrophoresis, and fluorescence activated cell sorting. (see, e.g., Freshney, (1987), animal cell culture, A Manual of Basic Techniques, 2d Ed., A.R. Liss, Inc., New York, Ch.11 and 12, pp.137-168).

The growth of cells in a matrix material, such as a polymer matrix, can be achieved by adding or coating proteins (e.g., collagen, elastic fibers, reticular fibers) glycoprotein glycosaminoglycans (e.g., heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, keratan sulfate, etc.), cell matrices, and/or other materials onto the matrix material.

After perfusion of the endothelial cells, the matrix material should be incubated in a suitable nutrient medium. Many commercial media, such as RPMI 1640, Fisher's, Iscove's, McCoy's, Dulbecco's, etc., may be suitable for use. The medium should also be replaced periodically to remove used medium, reduce released cells, and add new medium. It is important to allow the endothelial cells to grow to such a stage: wherein, prior to perfusing the endothelial tissue layer with the parenchymal cells, an endothelial tissue layer including the native vasculature has been formed.

Perfusing parenchymal cells onto the endothelial cell layer of the matrix material

Once the endothelial tissue layer has reached a suitable degree of growth and has developed to the point of producing the original vasculature, additional cultured cell populations, such as parenchymal cells, may be added to the endothelial tissue layer. Parenchymal cells perfused onto the endothelial tissue may be cultured so as to adhere the cells to the endothelial tissue layer. The parenchymal cells may be cultured in vitro in a culture medium, allowed to grow and develop until the cells develop morphology and structure similar to native tissue. Growth of parenchymal cells on the endothelial tissue layer results in differentiation of parenchymal cells into appropriate new forms of organ-enhancing structures.

In addition, after perfusion of the parenchymal cells, the matrix may be transplanted into the body without prior in vitro culture of the parenchymal cells. The selection of parenchymal cells for perfusion depends on the organ to be enhanced. For example, enhancement of the kidney involves perfusion of cultured endothelial cells into or onto the matrix material, culturing the cells until they develop into an endothelial tissue layer that includes the original vasculature. The endothelial tissue can then be perfused with cultured kidney cells and cultured in vitro until the kidney cells begin to differentiate to form nephron structures.

The parenchymal cells may be obtained from a cell suspension prepared by isolating the desired tissue using standard techniques as described above. The cells are then cultured in vitro to the desired density. After the desired density is reached, the cultured cells can be used to perfuse a matrix material with the endothelial tissue layer. The cells will proliferate, mature and differentiate on the endothelial tissue layer. The choice of parenchymal cells depends on the organ to be augmented, e.g., in augmenting the kidney, the matrix material, e.g., polymer matrix and endothelial tissue layer, are perfused with cultured kidney cells. In augmenting the liver, the polymer matrix and endothelial tissue layers are perfused with cultured hepatocytes. In augmenting the pancreas, the polymer matrix and endothelial tissue layers are perfused with cultured pancreatic endocrine cells. In augmenting the pancreas, the polymer matrix and endothelial tissue layers are perfused with cultured pancreatic endocrine cells. When augmenting the heart, the polymer matrix and the endothelial tissue layer are perfused with cultured heart cells. For a review of the methods that can be used to obtain parenchymal cells from various tissues, see Freshney (1987) animal cell culture, AManual of Basic technology, 2d Ed., A.R. Liss, Inc., New York, Ch.20, pp.257-288. The cells are cultured until they differentiate to produce a new morphology of organ enhancing structures similar to the morphology of native in vivo tissues.

Growth factors and regulatory factors may be added to the culture medium in order to enhance, alter or modulate proliferation and cell maturation and differentiation of the culture. The growth and activity of cells in culture can be affected by a variety of growth factors, such as insulin, growth hormones, growth regulators, colony stimulating factors, erythropoietin, epidermal growth factor, hepatic hematopoietic factor (erythropoietin), and hepatocyte growth factor. Other factors that regulate proliferation and/or differentiation include prostaglandins, interleukins, and naturally occurring statins.

Formation of three-dimensional biomatrix

In one aspect, the invention relates to the formation of a three-dimensional biological matrix, or organ enhancement structure/construct. In one embodiment, the organ augmentation structure is prepared for use in the treatment of a particular disease or disorder that disrupts the function of the organ. For example, the organ enhancement structure can be a specific enhancement structure for enhancing, and thereby alleviating, glomerulopathies associated with abnormal glomerular function (e.g., glomerulopathies such as primary glomerulopathies associated with impaired glomerular filtration (e.g., acute nephritis syndrome, Rapidly Progressing Glomerulonephritis (RPGN), glomerulosclerosis, nephritic syndrome, asymptomatic urinary sediment abnormalities (hepaturia, proteinuria), and chronic glomerulonephritis), or secondary glomerulopathies associated with systemic disease (e.g., diabetic nephropathy and immune-mediated multiple system disease). So as to enhance and thereby alleviate renal tubular diseases such as proximal tubular dysfunction and renal tubular acidosis. This makes the methods and compositions of the present invention useful for treating specific conditions and diseases associated with specific sites of the nephron. Proximal tubular dysfunction may exhibit nonselective resorption defects that may lead to hypokalemia, amino acid urine, diabetes, phosphate urine, uric acid urine, or bicarbonate urine. Renal Tubular Acidosis (RTA) is caused by filtered HCO₃The reabsorption defect of (b), the excretion of H, or both. Tubular acidosis is characterized by hyperchloremia and normal glomerular function. RTAs can be divided into a distal RTA (RTA-1), a proximal RTA (RTA-2) and a hyperkalemic RTA (RTA-4). The last case occurs in a variety of waysIn a hyperkalemia state, and the defect is such that the tubules are unable to secrete sufficient NH₄Characteristically, it is a direct consequence of increased cellular potassium retention.

In another embodiment, the organ enhancement structure may be a universal enhancement structure prepared from a mixture of cells isolated from the organ to be enhanced. For example, a new morphology of an organ-enhancing structure seeded with renal cells, including a mixture of distal tubule cells, proximal tubule cells, loop of Henle cells, and glomerular cells.

In another embodiment, the enhancing construct may be used to enhance cardiac function. In this example, a new form of the organogenic structure may be prepared by seeding the matrix material with a population of heart cells.

In another embodiment, the enhancing construct may be used to enhance bladder function. In this example, the enhancing construct may be made by seeding the matrix material with an isolated urothelial cell population, or a cell population comprising a mixture of smooth muscle and urothelial cells.

The biological matrix comprises a matrix material that has been perfused with at least one cultured cell population and incubated until it forms a monolayer, and the incubation of the cultured cells is continued until it forms a multilayer consisting of multiple cell monolayers and ultimately forms a tissue layer, e.g., a layer of kidney tissue, or a layer of endothelial cells with the original vasculature.

Continued active proliferation of the tissue layer ultimately results in the tissue layer resembling the equivalent parenchymal tissue of an organ within the body. This may be due in part to the process by which the multilayer is produced. Produced by first culturing a first homogenous population of cells on the matrix material once until each layer of cells is actively proliferating. Incubating the plurality of layers until the cells develop and reproduce a structure and morphology similar to an equivalent parenchymal tissue of an organ in vivo.

Thus, the multilayers formed by the methods of the invention can produce the proteins, growth factors and regulatory factors required to support long-term proliferation of the uniform cell population. After the first multilayer is formed, a surface for producing a second multilayer is provided therefrom. The second plurality includes a second homogeneous population of cells different from the first homogeneous population of cells. The second plurality is formed by culturing the second uniform population of cells one layer at a time until each layer of cells is actively proliferating to produce a plurality of layers of cells, and eventually forming a tissue layer.

By further in vitro incubation or in vivo incubation, the tissue layers are capable of differentiating into organ enhancement structures. Growth of cells in the tissue layer may be further enhanced by the addition of factors such as nutrients, growth factors, cytokines, extracellular matrix components, differentiation inducers, secretion products, immunomodulators, bioactive compounds capable of enhancing or allowing cell network growth or neurofibrillary proteins, glycoproteins, and glycosaminoglycans.

In one embodiment, the matrix material used to prepare the biomatrix is a polymer matrix. The tissue layers may be formed on one side of the polymer matrix, or on both sides of the polymer matrix, until tissue layers are produced having morphology and histology that allow differentiation into organ-enhancing structures. In another embodiment, the polymer matrix is a hydrogel into which the cultured cell population has been mixed. Incubating the cells in the hydrogel until they form a tissue layer that can differentiate into an organ enhancing structure.

Transplantation of three-dimensional biomatrix

The three-dimensional biomatrix or organ proliferation structure is transplanted into an organ in need of enhancement by standard surgical methods. The surgical method may vary depending on the organ to be enhanced. For kidney transplantation, it may be desirable to transplant a series of three-dimensional biomatrix into an incision made along a avascular plane of the kidney, or into a region of the organ with minimal blood vessels. In other applications, the constructs of the invention may be introduced by less invasive means, for example, a catheter, needle, trocar or cannula type instrument.

Use of

The methods and compositions of the present invention are useful for enhancing organ function in a variety of organs.

(i) Kidney (A)

In one embodiment, the present invention relates to methods and compositions for enhancing kidney function. Since virtually all kidney diseases can lead to kidney failure, in most cases the main focus of treatment is to preserve kidney function. Subjects typically have more than required kidney function, and most kidney diseases do not cause significant problems or symptoms until 90% of kidney function is lost. Thus, the methods and compositions of the present invention can be used to enhance kidney function in a kidney where at least about 2% function, preferably about 5% function, more preferably about 10% function, more preferably about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90% function. The methods and compositions of the present invention may also be used to alleviate symptoms of acute and chronic renal failure. Kidney diseases that may be enhanced include, but are not limited to (e.g., glomerulopathies, such as primary glomerulopathies associated with impaired glomerular filtration (e.g., acute nephritic syndrome, Rapidly Progressing Glomerulonephritis (RPGN), glomerulosclerosis, nephritic syndrome, asymptomatic urinary sediment abnormalities (hepatoria, proteinuria), and chronic glomerulonephritis), or secondary glomerulopathy associated with systemic diseases (e.g., diabetic nephropathy and immune-mediated multiple system diseases). this makes the methods and compositions of the invention useful for treating specific conditions and diseases associated with specific sites of the nephron.proximal tubular dysfunction may exhibit nonselective defect in reabsorption, which may lead to hypokalemia, aminouria, diabetes, phosphouria, uricuria, or bicarbonate uremia.₃The reabsorption defect of (b), the excretion of H, or both. Kidney smallTubular acidosis is characterized by hyperchloremia and normal glomerular function. RTAs can be divided into a distal RTA (RTA-1), a proximal RTA (RTA-2) and a hyperkalemic RTA (RTA-4). The last condition occurs in a variety of hyperkalemic states, and the defect is such that the tubule is unable to secrete sufficient NH₄Characteristically, it is a direct consequence of increased cellular potassium retention.

(ii) Heart disease

In another embodiment, the methods and compositions of the invention can be used to enhance cardiac function in a subject having a cardiac disease or disorder. In the united states, heart failure is one of the leading causes of morbidity and mortality. Heart failure can be caused by any condition that can impair the ability of the heart to pump blood. Most commonly, heart failure is caused by a decrease in myocardial contractility, which is caused by a decrease in coronary blood flow. Heart failure may be caused by a number of other factors, including damage to heart valves, vitamin deficiencies, and primary cardiomyopathy (Guyton (1982) Human Physiology and mechanics of Disease, third edition, w.b. saunders co., philiadelphia, Pa., p.205). Heart failure is often manifested as a correlation with myocardial infarction (Manual of Medical Therapeutics (1989) Twenty-Sixth Edition, Little, Brown & Co., Boston (W.C. Dunagan and M.L.Ridner, eds.), pp.106-09).

Heart failure in humans begins with a decrease in myocardial contractility, which leads to a decrease in cardiac output. The methods and compositions of the present invention may be used to enhance cardiac function. For example, by creating a new form of an organoaugmentation construct, it is possible to augment a portion of the heart that has been damaged or infarcted or ischemic.

Heart diseases include, but are not limited to, angina pectoris, myocardial infarction, and chronic ischemic heart disease.

(iii) Disorders of the urogenital tract

In another embodiment, the methods and compositions of the present invention may be used to enhance urogenital organ function in a subject having a disease or condition of the urogenital organ. Examples of urogenital diseases include, but are not limited to, diseases associated with the bladder, urethra and ureter.

(iv) Spleen disorders

The spleen is a small organ near the stomach, which is part of the lymphatic system. The spleen helps protect the body from infection and filters blood. Patients who have had their spleens removed are more susceptible to certain types of infection. Thus, the methods and compositions of the invention can be used to ameliorate and control a spleen disorder, for example, by using recombinantly modified cells that express an agent that controls the disease or disorder. Examples of spleen disorders include, but are not limited to, idiopathic purpura, Felty's syndrome, Hodgkin's disease, and immune-mediated spleen destruction.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All U.S. patents and other documents referred to herein are expressly incorporated by reference for all purposes.

Examples

Example 1: isolation of renal cells

The small kidneys, such as from a week-old C7 black mouse, were decapsulated, dissected, and ground and suspended IN Dulbecco's modified Eagles's medium (DMEM; Sigma, St. Louis, Mo) containing 15mM Hepes, pH7.4, and 0.5. mu.g/ml insulin, 1.0mg/ml collagenase, and 0.5mg/ml dispase, neutral protease from Bacillus polymyxa (Boehringer Mannheim, Indianapolis, IN).

Large kidneys such as porcine kidney were perfused arterially with calcium-free Eagles minimal essential medium for 10 minutes at 37 ℃ within 3 hours of extraction. Then, the mixture was supplemented with 1.5mM MgCl₂And 1.5mM CaCl₂0.5mg/ml collagenase (IV) in the same bufferType, Sigma, st. The kidneys were then decapsulated, dissected, crushed and suspended IN Dulbecco's modified Eagles's medium (DMEM; Sigma, St. Louis, Mo.) containing 15mM hepes, pH7.4, and 0.5. mu.g/ml insulin, 1.0mg/ml collagenase and 0.5mg/ml dispase, neutral protease from Bacillus polymyxa (Boehringer Mannheim, Indianapolis, IN).

The suspension of renal cells from the large or small kidney was gently stirred in a water bath at 37 ℃ for 30 minutes. The cells and fragments were recovered by centrifugation at 50g for 5 minutes. The pellet was resuspended in DMEM containing 10% fetal bovine serum (Biowhittaker, Walkersville, Maryland) to terminate proteolysis, and the turbid solution was passed through a sterile 80 mesh nylon sieve to remove large fragments. The cells were recovered by centrifugation and washed 2 times with Dulbecco's modified Eagles's medium without calcium.

Example 2: in vitro culture of renal cells

I. Isolation of rat tail collagen

The tendons were stripped from the rat tail and stored in a 0.12M acetic acid solution prepared with deionized water in a 50ml tube. After 16 hours, at 4 ℃ overnight.

The dialysis bag is pre-treated to ensure uniform pore size and removal of heavy metals. Briefly, the dialysis bag was soaked in a solution of 2% sodium bicarbonate and 0.05% EDTA and boiled for 10 minutes. Sodium bicarbonate and 0.05% EDTA were removed by multiple distilled water rinses.

A0.12M solution of acetic acid including rat tendons was placed in the treated dialysis bag and dialyzed for a period of 2-3 days, and the acetic acid was removed. The dialysis solution was changed every 3-4 hours.

(ii) Coating the tissue culture plates:

with a solution containing about 30. mu.g/ml collagen (Vitrogen or rat tail collagen), about 10. mu.g/ml human fibronectinSolutions of white (Sigma, St. Louis, Mo.) and approximately 10. mu.g/ml bovine serum albumin (Sigma, St. Louis, Mo.) were incubated at 75cm²The culture flask of (4) was coated in a total volume of about 2ml of supplemented medium and incubated at 37 ℃ for 3 hours.

(iii) Cell culture

Digesting singly suspended kidney cells at about 1X 10⁶Cells/ml were plated onto a modified collagen matrix and incubated at a concentration of about 10% fetal bovine serum, about 5. mu.g/ml bovine insulin, about 10. mu.g/ml transferrin, about 10. mu.g/ml sodium selenate, about 0.5. mu.M hydrocortisone, about 10ng/ml prostaglandin E₂Approximately 100 units/ml penicillin G, approximately 100. mu.g/ml streptomycin (Sigma, St. Louis, Mo.) in DMEM at 5% CO₂In an incubator, grown at about 37 ℃.

By dissolving in Phosphate Buffered Saline (PBS) without calcium ion (about 1.51mM KH)₂PO₄About 155.17mM NaCl, about 2.8mM Na₂HPO₄·7H₂O), approximately 0.05% trypsin, approximately 0.53mM EDTA (Gibco BRL, GrandIsland, NY) and subcultured to confluent monolayers. Starting from the first generation, cells can be cultured for any time by suspension in medium containing about 10% DMSO, so as to be frozen, and stored in liquid medium.

Example 3: isolation and culture of endothelial cells

Endothelial cells were isolated from dissected veins. Intravenous heparin/papaverine solution (3 mg papaverine hydrochloride diluted in 25ml Hanks Balanced Salt Solution (HBSS) containing 100 units heparin (final concentration 4 μ g/ml)) was used to improve endothelial cell preservation. The proximal filamentous ring is placed around the venous vessel and secured with one node. A small phlebotomy is performed at a site near the segment and the tip of the venous cannula is inserted and secured in place with a second segment. A second minor phlebotomy was performed beyond the proximal segment and the vein was gently flushed with medium 199/heparin solution medium 199(M-199) supplemented with 20% fetal bovine serum, ECGF (100mg/ml), L-glutamine, heparin (Sigma, 17.5u/ml) and an anti-biotin-antifungal agent to remove blood and clots. The dissected vein was rinsed with approximately 1ml of collagenase solution (0.2% Worthington type I collagenase dissolved in 98ml M-199, 1ml FBS, 1ml PSF, 15-30 minutes at 37 ℃, and sterile filtered). The vein was also gently dilated with the collagenase solution and the dilated vein was placed into a 50ml tube containing Hank's Balanced Salt Solution (HBSS). The tube containing collagenase-dilated venous vessels was incubated at 37 ℃ for 12 minutes in order to digest the venous lining. After digestion, the contents of the vein containing the endothelial cells were removed and placed in a sterile 15ml tube. The endothelial cell suspension was centrifuged at 125 Xg for 10 min. Endothelial cells were resuspended in 2ml of dulbecco.'s modified Eagle medium (DMEM/10% FBS) containing 10% FBS and penicillin/streptomycin and plated into 24-well plates coated with 1% difcogelatin. The endothelial cells were incubated overnight at 37 ℃.

After overnight incubation, the cells were rinsed with HBSS and placed in 1ml of fresh DMEM/10% FBS. The medium was changed 3 times per week. When the culture reached confluency (after 3-7 days), the confluent monolayer was sub-cultured by treatment with 0.05% trypsin, 0.53mM EDTA for 3-5 minutes until the cells were dispersed. The dispersed cells were plated on plates coated with 0.1% difcogelatin at a split ratio of 1: 4 to 1: 6. Expanding the endothelial cells until a sufficient number of cells are obtained. Cells were trypsinized, collected, washed, and counted for plating.

Example 4: isolation and culture of urothelial and smooth muscle cells

The harvested cells were cultured according to the previously disclosed methods: atala et al, (1993) j.urol.150: 608, Cilento et al (1994) J.Urol.152: 655, Fauza et al, (1998) J.Ped.Surg, 33, 7-12, all of which are expressly incorporated herein by reference.

a) Culturing urothelial cell populations

Bladder samples were obtained and prepared for culture. The serosal face is marked with sutures to ensure that it is not confused which face represents the urothelial cell face.

The samples were processed in a laminar flow cell culture cabinet using a sterile instrument. Preparing a medium supplemented with keratinocyte-SFM (GIBCO BRL (Cat. No.17005), bovine pituitary extract (Cat. No.13028, 25mg/500ml medium), and recombinant epithelial growth factor (Cat. No.13029, 2.5 μ g/500ml medium). at 4 deg.C, 10ml of medium was placed in each of two 10cm cell culture dishes, and 3.5ml was placed in the third dish.blood was removed from the sample by placing the sample in the first dish and gently agitating it back and forth.the above procedure was repeated in the second dish and finally the sample was transferred to the third dish.the urothelial cell surface was gently scraped with a No. 10 dissecting blade without cutting through the sample. And inoculated into 6 wells of a 24-well cell culture plate, and approximately 0.5-1ml of the medium was inoculated into each well, so that the total volume of each well was 1-1.5 ml. 5% CO at 37 ℃₂Incubating the cells.

The next day (first day after harvest), the medium was aspirated from the 6 wells, and new medium was added. The cells were centrifuged at 1000rpm for 4 minutes and the supernatant was removed. The cells were resuspended in 3-4.5ml of fresh medium and heated to 37 ℃ on a 24-well plate.

The medium was removed and PBS/EDTA (37 ℃, pH7.2, 0.53mM EDTA (0.53ml of 0.5M EDTA, pH8.0, in 500ml PBS respectively)) was added to each well of the 24-well plate or 10ml was added to each 10cm petri dish. The cells were then passaged in 2 10cm culture dishes. Then, when the cells reached 80-90% confluency, the cells were passaged without allowing the cells to reach 100% confluency.

The cells were observed under a phase contrast microscope. When the cell-cell junctions were separated for most cells (approximately 5-15 minutes), PBS/EDTA was removed, and 300. mu.l of either was added to each well of a 24-well plate or 7ml of trypsin/EDTA (37 ℃, GIBCO BRL, Cat. No.25300-054) was added to each 10cm dish. The plate/dish was periodically agitated. When 80-90% of the cells detached from the plate and began to float (approximately 3-10 minutes), the effect of EDTA was stopped by adding 30. mu.l of soybean trypsin inhibitor (GIBCO BRL, Cat. No.17075-029, 294mg inhibitor in 20ml PBS) to each well of a 24-well plate or to each 10cm petri dish. 0.5ml of medium was added to each well of a 24-well plate, or 3ml of medium was added to each 10cm dish. PBS/EDTA and trypsin/EDTA incubations were performed at room temperature, however, it would be more effective if the plates were incubated at 37 ℃.

The cells were harvested by centrifugation at 1000rpm for 4 minutes and the supernatant removed. The cells were resuspended in 5ml of medium and the number of cells was determined by hemocytometer. Cell viability was determined by standard trypan blue staining test. For a 100mm culture plate, the optimal seeding density is about 1X 10⁶Cells/plate. The required amount of cells was dispensed into the culture dish and a volume of medium was added to bring the total volume to approximately 10 ml/plate.

b) Bladder smooth muscle cells were cultured.

After removal of the urothelial cell layer from the bladder sample as described above, the remaining muscle was dissected into 2-3 mm muscle fragments. Each muscle segment was evenly distributed on 100mm cell culture dishes. The muscle fragment was dried and allowed to adhere to a petri dish (approximately)10 minutes). 20ml of Dulbecco's modified Eagle's medium containing 10% FCS was added to the dried muscle fragments. 5% CO at 37 deg.C₂The muscle fragments were incubated for a period of 5 days without interference. The medium was changed on day 6 and any non-adhering fragments were removed. The remaining fragments were incubated for a total of 10 days, and then all muscle fragments were removed. Cells from the muscle fragments that had adhered to the dish were cultured until islets of cells appeared. The cells were trypsinized, counted, and plated into T75 culture flasks.

The cells were fed every 3 days according to cell density, and passaged when the cells reached 80-90% confluence.

Example 5: isolation and culture of cardiac cells

This example discloses a method of culturing cardiac cells. Cardiac cells, such as from atrial tissue, can be obtained from mammals. Atrial tissue may be obtained from tissue such as the right atrial appendage obtained from a patient undergoing cardiovascular surgery requiring cardiopulmonary bypass surgery. The atrial appendage can be removed and placed in an ice-saline slush for rinsing. The hard pericardial covering may be removed with a surgical blade in order to reduce the amount of connective tissue contained in the cell harvest. The remaining atrial muscle may be minced into small (0.5-1.0 cubic millimeters) pieces and placed in cold Hank's Balanced Salt Solution (HBSS) without calcium or magnesium (Whittaker, walker, Mass.). Minced atrial tissue can be digested at a concentration of 1.43mg/ml in 0.14% collagenase solution (Worthington, Freehold, n.j.). The pieces can be placed in 35ml of this solution and allowed to digest on a shaker at 125RPM for 1 hour at 37 ℃. Supernatants may be removed from the atrial tissue and centrifuged at 3500RPM for 10 minutes at 37 ℃. The minced tissue can be placed into another 35ml of collagenase solution while the supernatant is centrifuged and digestion continued for an additional 1 hour. The supernatant collagenase solution was removed and set aside for a third digestion. The cell pellet can be resuspended in 2ml of Eagle's Minimal Essential Medium (EMEM) with Earle's salts (Whittaker) containing 30% newborn calf serum (Whittaker) and 0.1% antibiotic solution-10,000 units/cc penicillin G, 10,000. mu.g/cc streptomycin, and 25. mu.g/cc amphotericin B (Gibco, Grand Island, N.Y.). This process may continue until digestion is again achieved.

The digests can be pooled and the cell concentration measured by a hemocytometer and adjusted to 1X 10 by EMEM⁵Cells/ml. The cells can be plated onto 35 mm gelatin coated petri dishes (Corning, n.y.) and incubated at 37 ℃ in 5% CO₂Is incubated in the atmosphere of (2). The medium was changed every three days for the first two weeks of growth, and then every 5-7 days. When the cultures were dispersed and nearly confluent, they were treated with trypsin and transferred to EMEM in a 60 mm gelatin coated petri dish (Corning). When the cells are close to confluency, they can be treated with trypsin and transferred to MCDB 107(Sigma, Saint Louis, Mo.) in T-75 flasks (Corning).

A portion of the cells grown in MCDB 107 can be plated onto four-chamber gelatin-coated slide culture plates (Lab Tek, Naperville, III). Control cells may be human umbilical cord endothelial cells and human skin fibroblast cultures (Beaumont Research Institute, Royal Oak, Mich.) which may be grown in M119 containing 20% fetal bovine serum, 1% L-glutamine, 0.1% 5mg/ml insulin, 5mg/ml transferrin, 5. mu.g/ml selenic acid (Collaborative Research.), 0.6ml heparin (0.015% dissolved in M199), 0.1% antibiotic-antifungal solution (Gibco Laboratories: 10,000 units/ml penicillin sodium G, 100,000mcg/ml streptomycin sulfate and 25mcg/ml amphotericin B), and 300. mu.g/ml endothelial Growth Supplement (GS) purchased from Biotechnology Research Institute, Rockville, Md.. When the control culture and harvested cells were spread and near confluency, they could be rinsed with HBSS and fixed with 10% formalin for 10 minutes. The chamber can be removed and the cells left on the plate stained with an immunoperoxidase dye capable of staining for smooth muscle alpha actin (Lipshaw, Detroit, Mich.), striated muscle specific myosin (Sigma, st. louis, Mo.), myoglobin (Dako, Carpinteria, Calif.), factor VIII (Lipshaw, Detroit, Mich.), and atrial natriuretic factor peptides (Research and Diagnostic Antibodies, Berkeley, Calif.). The plates were then examined with an optical microscope.

A portion of the cells grown in MCDB 107 can be plated onto a 96-well gelatin-coated plate (Corning). When the cells were spread and nearly confluent, they were rinsed with HBSS and fixed with 2.5% glutaraldehyde, 0.2M cacodyate buffer, ph7.4(Polysciences, Inc., Warrington, Pa.), post-fixed with 1% osmium tetroxide (Polysciences, Inc.), embedded in Epon LX-112 resin (Ladd's Research, burrington, Va.), stained with 0.03% lead citrate (eastman kodak, Rochester, n.y.) and saturated urea acetate (Pelco., Tustin, Calif.) prepared with 50% ethanol, and then examined under a transmission electron microscope.

Example 6: preparation of decellularized or partial organs

The following methods disclose methods for removing the intact cellular components of an organ or tissue without disrupting the complex three-dimensional infrastructure of the organ or tissue. The kidneys were surgically removed from C7 black mice using standard techniques for tissue removal. The kidneys were placed in flasks containing a suitable volume of distilled water to cover the isolated kidneys. The isolated kidneys were spun in the distilled water at a suitable speed for 24-48 hours at 4 ℃ using a magnetic stir plate and a magnetic stirrer. This process removes the cellular debris and cell membranes surrounding the isolated kidney.

After the first removal step described above, the distilled water was replaced with 0.05% ammonium hydroxide solution containing 0.5% Triton X-100. The kidneys were allowed to rotate in the solution at 4 ℃ for 72 hours using a magnetic stir plate and a magnetic stirrer. This alkaline solution is capable of solubilizing the separated nuclear and cytoplasmic components of the kidney. The nuclear components of the kidney were removed using the detergent Triton X-100, and the separated cell membrane and cytoplasmic proteins of the kidney were lysed using ammonium hydroxide solution.

The isolated kidney was then washed with distilled water at 4 ℃ for 24-48 hours using a magnetic stir plate and a magnetic stirrer. After this washing step, the removal of cellular components from the isolated kidney was confirmed by tissue analysis of a small piece of kidney. The isolated kidney was again treated with ammonium hydroxide solution containing Triton X-100, if necessary, until all cellular contents of the isolated kidney were removed. After removal of the dissolved components, a collagenous three-dimensional framework in the shape of an isolated kidney was prepared.

The decellularized kidney was equilibrated with 1 fold Phosphate Buffered Saline (PBS) including rotating the decellularized kidney at 4 ℃ overnight using a magnetic stir plate and a magnetic stirrer. After equilibration, the decellularized kidneys were freeze-dried under vacuum overnight. The freeze-dried kidneys were sterilized with ethylene oxide gas for 72 hours. The decellularized kidney is used immediately after sterilization, or stored at 4 ℃ or room temperature until use. Before seeding the cultured cells, the preserved organs were placed in tissue culture medium and equilibrated at 4 ℃ overnight.

Example 7: preparation of kidney-enhancing organ structures

This example discloses the preparation of wafers for transplantation to one or more sites of an organ such as a kidney. The size and shape of the kidney tissue matrix (wafer) for placement of the kidney parenchyma is determined. For example, a substrate about 1mm thick, i.e., about 2cm long and wide. Suspending single kidney cell in about 10 × 10⁶A concentration of cm3 of cells was seeded onto the kidney tissue matrix. The cells were allowed to attach to the matrix at 37 ℃ for approximately 2 hours. The matrix is then inverted to the opposite side and a single suspension of kidney cells is seeded onto the kidney tissue matrix. At 37 deg.C, using about 2 hrWhile allowing the cells to attach to the matrix wall. After the incubation was complete, medium was slowly added to the flask so as to cover the entire kidney matrix. Care should be taken not to disturb the cells within the matrix. In the supply of CO₂The substrate was incubated in an incubator at 37 ℃. The medium is changed daily or more frequently depending on the level of lactic acid produced. On day 4 after the start of seeding, the cell-matrix system was placed into a rotating bioreactor system and incubated for an additional 3 days in order to obtain a uniform cell distribution and growth.

Example 8: transplantation of kidney-enhancing organ structures

A kidney enhancing organ wafer is placed into one or more regions of an organ, such as a kidney. The surface of the recipient kidney is exposed. The renal vessels were temporarily clamped with vascular clamps to reduce bleeding. An incision is made in the renal capsule to access the renal parenchyma. Carefully pushing the balloon away from the substance. Renal parenchymal tissue similar in size and shape to the renal tissue matrix is removed without disruption of the collection system during removal. The cell-seeded kidney tissue matrix is placed into the kidney parenchyma, and the kidney capsule is sutured to the transplanted kidney tissue matrix. The vascular clamp is removed to restore circulation. Hemostasis was achieved by pressing gently on the graft. After hemostasis, the wound was closed. After transplantation, the cells were examined for growth and development in kidney-enhancing organ structures. Photographs of the renal biomatrix one week after transplantation showed that the cells were viable and positive at 100-fold magnification with the lipophilic red fluorescent tracer carbocyanine (not shown). Four weeks after transplantation, the formation of tubules and glomerular-like structures was seen (H & E × 200, photos not published). The development of the tubular structures continued until 8 weeks after transplantation (pictures not shown).

Claims (15)

1. An artificial organ augmenting construct for augmenting a function of a native organ, comprising:

a three-dimensional biological matrix formed by perfusing a matrix material with at least one cultured cell population such that the cells attach to the matrix material and produce a tissue layer capable of enhancing organ function at one or more target sites of the native organ, wherein the matrix has a maximum dimension of less than 50 mm and a ratio of its maximum dimension to its thickness of greater than 5: 1.

2. The construct of claim 1, wherein the construct is selected to enhance an organ selected from the group consisting of: heart, kidney, liver, pancreas, spleen, bladder, ureter and urethra.

3. The construct of claim 1, wherein the matrix material is decellularized tissue.

4. The construct of claim 1, wherein the matrix material is a hydrogel.

5. The construct of claim 4, wherein the hydrogel has a volume of 0.01 to 30 cubic millimeters.

6. The construct of claim 4, wherein the hydrogel has a volume of 0.1 to 20 cubic millimeters.

7. The construct of claim 1, wherein the matrix material is a polymer.

8. The construct of claim 1, wherein the matrix has a maximum dimension of less than 25 mm.

9. The construct of claim 1, wherein the matrix has a maximum dimension of less than 10 mm.

10. The construct of claim 1, wherein the matrix has a maximum dimension of less than 2-5 mm.

11. The construct of claim 1, wherein the matrix has a maximum dimension of less than 2-3 mm.

12. The construct of claim 1, wherein the matrix is a substantially flat structure having a ratio of its largest dimension to its thickness of greater than 5: 1.

13. The construct of any one of claims 1 to 12, wherein the construct is a kidney function enhancing construct comprising:

a three-dimensional biological matrix formed by infusing a matrix material with a population of kidney cells such that the kidney cells attach to the matrix and create a tissue layer that differentiates into a nephron structure or a portion thereof, thereby enhancing kidney function.

14. The construct of any one of claims 1 to 12, wherein the construct is a cardiac function enhancing construct comprising:

a three-dimensional biological matrix formed by perfusing a matrix material with a population of heart cells such that the heart cells attach to the matrix and create a tissue layer that differentiates into a cardiac structure or a portion thereof, thereby enhancing cardiac function.

15. The construct of any one of claims 1-12, wherein the matrix is initially perfused with a population of endothelial cells such that the endothelial cells attach to the matrix to create a layer of endothelial tissue comprising the vasculature, and then seeded with a second population of cells such that the second population of cells attach to the layer of endothelial tissue comprising the vasculature and differentiate to enhance organ function.