Title:  Long-term three dimensional tissue culture system

United States Patent:  6,737,270

Issued: May 18, 2004

Inventors:  Michalopoulos; George (Bethel, PA); Bowen; William C. (White Oak, PA)

Assignee:  University of Pittsburgh of the Commonwealth System of Higher Education (Pittsburgh, PA)

Appl. No.:  455952

Filed:  December 7, 1999

Abstract

A method is provided for long term culture of proliferating hepatocytes that retain hepatic function to produce a hepatic cell culture. Hepatocytes and nonparenchymal cells are co-cultured ex vivo on a matrix coated with a molecule that promotes cell adhesion, proliferation or survival, in the presence of growth factors, resulting in a long-term culture of proliferating hepatocytes that retain hepatic function. The co-culturing method results in the formation of matrix/hepatic cell clusters that may be mixed with a second structured or scaffold matrix that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts. The method can be used to form bio-articial livers through which a subjects blood is perfused. In an embodiment, the hepatocytes and nonparenchymal cells are derived from disaggregated liver tissue and are co-cultured in the presence of epidermal growth factor or heptocyte growth factor and beads coated with extracellular matrix protein, Alternatively, the hepatic cell culture may be implanted into the body of a recipient host having a hepatic disorder. Such hepatic disorders, include, for example, cirrhosis of the liver, induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha₁ antitrypsin deficiency.

SUMMARY OF THE INVENTION

The present invention relates to a novel tissue culture system that provides for long term culture of proliferating hepatocytes that retain their capacity to express hepatic function. The invention generally relates to compositions and methods for generating long term cultures of hepatocytes that can be used to produce three-dimensional hepatic cell culture systems. Such hepatic cell culture systems can be used to form bio-artificial livers that function as perfusion devices. Alternatively, the three-dimensional hepatic cell cultures may be implanted into a subject having a liver disorder.

The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells in the presence of growth factors and a matrix material coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. The co-culturing method results in the formation of matrix/hepatic cell clusters containing a mixture of replicating hepatocytes and nonparenchymal cells. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters in combination with a second structured, or scaffold matrix, that provides a three-dimensional structural support to form structures analogous to liver tissue counterparts.

Compositions of the present invention include populations of matrix/hepatic cell clusters comprising co-cultures of hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival. Further, the invention provides a three-dimensional hepatic cell matrix system comprising a three-dimensional support matrix containing a population of matrix/hepatic cell clusters comprising hepatocytes and nonparenchymal cells bound to a matrix coated with at least one biologically active molecule that promotes cell adhesion, proliferation or survival.

The compositions of the present invention may be used to form bio-artificial livers through which a host's blood is perfused. Alternatively, the three-dimensional hepatic cell matrix system may be transplanted to a recipient host for providing hepatic function in subjects with liver disorders. The three-dimensional matrix system is administered in an effective amount to provide restoration of liver function, thereby alleviating the symptoms associated with liver disorders. The present invention, by enabling methods for generating long-term cultures of hepatocytes, provides a safer alternative to whole liver transplantation in subjects having liver disorders including, but not limited to, cirrhosis of the liver, alcohol induced hepatitis, chronic hepatitis, primary sclerosing cholangitis and alpha,-antitrypsin deficiency.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a novel tissue culture system that provides for long term culture of hepatocytes that retain their capacity to proliferate and express hepatic function. The invention provides compositions and methods for generating long term cultures of hepatocytes that can be used as bio-artificial livers for perfusion purposes. Alternatively, the hepatic cell culture systems may be implanted into a subject having a hepatic disorder to restore or supplement liver function.

The method of the present invention comprises the co-culturing of hepatocytes and nonparenchymal cells, in the presence of growth factors and a matrix material coated with at least one biologically active capable of a molecule promoting cell adhesion, proliferation or survival, thereby, resulting in the formation of matrix/hepatic cell clusters. The method of the present invention may further comprise the mixing of the matrix/hepatic cell clusters with a second matrix material that provides a three-dimensional structural support to form structures analogous to liver tissue found in vivo.

The compositions of the present invention include matrix/ hepatic cell cultures comprising hepatocytes that retain their capacity to proliferate while expressing hepatic function. Further, the invention provides a three-dimensional hepatic cell culture system comprising hepatic cells that retain their capacity to proliferate and express hepatic function growing in a three-dimensional structure.

The hepatic cell system can be used for generating bio-artificial livers that function as perfusion devices for restoration of liver function. The three-dimensional matrix hepatic cell system can be administered to an individual for providing hepatic function in subjects with liver disorders. The matrix/hepatic cell system is administered in an effective amount necessary for restoration of liver function, thereby alleviating the symptoms associated with liver disorders.

5.1. Mixed Cultures of Hepatocytes and Nonparenchymal Cells

The present invention relates to methods for generating long term cultures of proliferating hepatocytes that retain their hepatic function. The method generally comprises co-culturing or propagating hepatocytes and nonparenchymal cells on a matrix coated with a biologically active molecule that promotes cell adhesion, in vitro. The cells are cultured under conditions effective and for a time sufficient to allow formation of a culture of proliferating hepatocytes that retain hepatic function. The cells are grown in the presence of growth factors that maintain hepatic cell differentiation and the capacity to proliferate.

Hepatocytes and nonparenchymal cells may be obtained from a variety of different donor sources. In a preferred embodiment, autologous cells are obtained from the subject who is to utilize the bio-artificial liver or receive the transplanted hepatic cells to avoid immunological rejection of foreign tissue. In yet another preferred embodiment of the invention, allogenic liver tissue for use in purifying cells may be obtained from donors who are genetically related to the recipient and share the same transplantation antigens on the surface of their hepatic cells. Alternatively, if a sibling is unavailable, tissue may be derived from antigenically matched (identified through a national registry) donors.

In an embodiment of the invention, hepatic cells and nonparenchymal cells are isolated from a disaggregated liver tissue biopsy. This may be readily accomplished using techniques known to those skilled in the art. For example, the liver tissue can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells, making it possible to disperse the tissue suspension of individual cells. Enzymatic dissociation can be carried out by mincing the liver tissue and treating the minced tissue with any of a number of digestive enzymes. Such enzymes include, but are not limited to, trypsin, chymotrypsin, collagenase, elastase and/or hylauronidase. A review of tissue disaggregation techniques is provided in, e.g., Freshney, Culture of Animal Cells, A Manual of Basic Technique, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 9, pp.107-126. In addition to primary cell cultures, established hepatic cell lines may also be utilized in the methods and compositions of the invention.

The present methods and compositions can also employ hepatic cells genetically engineered to enable them to produce a wide range of functionally active biologically active proteins, including but not limited to growth factors, cytokines, hormones, inhibitors of cytokines, peptide growth and differentiation factors. Additionally, the cells may be genetically engineered to increase their proliferative capacity, i.e, the cells may be immortalized. Methods which are well known to those skilled in the art can be used to construct expression vectors containing a nucleic acid encoding the protein coding region of interest operatively linked to appropriate transcriptional/translational control signals. See, for example, the techniques described in Sambrook, et al., 1992, Molecular Cloning, A Laboratory Manuel, Cold Spring Harbor Laboratory, N.Y., and Ausebel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, N.Y., incorporated herein by reference.

Once isolated, the hepatic and nonparenchymal cells can be grown in any culture medium known to those skilled in the art to support the growth and proliferation of such cells. For example, the mixed cultures of cells can be grown in chemically defined hepatocyte growth medium (HGM) supplemented with specific growth factors and regulatory factors. Such factors can be added to the culture media to enhance, alter or modulate proliferation and/or differentiation of the cultured hepatocytes and nonparenchymal cells. In a preferred embodiment of the invention, the culture media may be supplemented with growth factors such as hepatocyte growth factor (HGF) and/or epidermal growth factor (EGF), or functional homologs thereof, to impart phenotypic stability in terms of differentiated hepatocyte gene expression and the ability to proliferate.

In addition, the co-cultures of cells are propagated in the presence of a natural or synthetic matrix that provides support for hepatic cell growth during in vitro culturing. The type of matrix that may be used in the practice of the invention is virtually limitlessness. The matrix will have all the features commonly associated with being "biocompatible", in that it is in a form that does not produce an adverse, or allergic reaction when administered to the recipient host. In a preferred embodiment of the invention, the matrix is in the form of a bead to which the cultured cells may adhere. The beads may be composed of variety of different substances including, but not limited to, synthetic materials or naturally derived materials. The type of matrix material to be used will depend on the desired use of the hepatocyte cultures. For example, when the matrices are to be transplanted into a subject it is preferred that a biodegradable matrix material be used. For purposes of forming bio-artificial livers, the matrix may be composed of any suitable material to which the hepatocytes and nonparenchymal cells will adhere and proliferate.

Further, to improve hepatic cell adhesion, proliferation or survival, the matrix is coated on its external surface with factors known in the art to promote cell adhesion, growth or survival. Such factors include cell adhesion molecules, extra-cellular matrix molecules and/or growth factors for hepatocytes and/or nonparenchymal cells. Matrices may also be designed to allow for sustained release of growth factors over prolonged periods of time. Thus, appropriate matrices will ideally provide factors known to promote hepatic cell adhesion, growth or survival, and also act as a support on which the cultured cells differentiate and proliferate. In a preferred embodiment of the invention, the hepatic cell cultures are propagated in media containing matrices coated with collagen type I protein for promotion of cell adhesion and proliferation of bound hepatocytes.

The method of the present invention involves the co-culturing of hepatic and nonparenchymal cells in the presence of the selected matrix material. Although the cells may be propagated under static conditions, it is preferred that the cells arc propagated under mixing or stirring conditions wherein a cell suspension is combined with matrix, and mixed or stirred, to enhance the number and frequency of cell contacts with the matrix to maximize cell adhesion to the matrix, but not disrupt adherence to cells. Such conditions may be generated in variety of different ways including, for example, the use of roller bottles to provide continuous stirring or mixing of the culture. Preferably, the stirring is continued throughout the culturing of the hepatic and nonparenchymal cells.

The conditions of long-term matrix-cell culturing will preferably be maximized to enhance hepatocyte proliferation while maintaining hepatic function. Although certain variations in cell number, seeding techniques, culture media, incubation temperatures and incubation times, may be utilized, such variations would be routine to those skilled in the art and are encompassed by the present invention.

5.2. Preparation of Three-dimensional Culture Systems

The present invention further relates to the use of the matrix/hepatic/nonparenchymal cell clusters, produced as described in Section 5.1, for generation of three-dimensional hepatic cell culture systems to form structures analogous to liver tissue counterparts. The method of the invention comprises growing hepatic and nonparenchymal cells on a three-dimensional matrix in vitro under conditions effective and for a period of time sufficient to allow proliferation of the cells to form a three-dimensional structure.

The three-dimensional matrices to be used are structural matrices that provide a scaffold for the cells, to guide the process of tissue formation. Cells cultured on a three-dimensional matrix will grow in multiple layers to develop organotypic structures occurring in three dimensions such as ducts, plates, and spaces between plates that resemble sinusoidal areas, thereby forming new liver tissue. Thus, in preferred aspects, the present invention provides a three-dimensional, multi-layer cell and tissue culture system. The resulting liver tissue culture system survives for prolonged periods of time and performs liver-specific functions for use as a perfusion device or following transplantation into the recipient host.

A wide variety of structural matrices may be used in the context of the present invention for preparation of a three-dimensional hepatic cell culture system. In preferred embodiments, the matrices are bio-compatible matrices that provide a scaffold for the cells to guide the development of tissue. Preferred matrices are generally those that define a space for subsequent tissue development. Such matrices include hydrogels, biomatrix gels, or porous materials such as fiber based or sponge like matrices. The culture system described herein provides for the proliferation of cells to form structures analogous to liver tissue counterparts in vivo.

In certain embodiments, synthetic matrices, such as synthetic polymer matrices, may be used. Such matrices include, but are not limited to, nylon, dacron, polystyrene and homopolymers or heterpolymers such as polylactic acid (PLA) polymers, polyglycolic acid (PGA) polymers and polylactic acid-polyglycolic acid (PLGA) copolymer matrices. In other embodiments, matrices for use in the invention may be naturally-derived matrices extracted from or resembling extracellular matrix materials such as a collagen matrix, such as type I collagen. Other naturally derived matrix materials include laminin-rich gels, alginate, agarose and other polysaccharides, gelatin and hyaluronic acid derivatives. Certain matrix materials may not support efficient cellular attachment and, in such instances, it may be advantageous to coat the matrix with molecules that promote cell adhesion, such as extracellular matrix proteins or, specifically, collagen type I.

To generate the three-dimensional hepatic cell cultures, matrix/hepatic/nonparenchymal cell clusters generated as described above in Section 5.1 are isolated from cell culture suspensions. For example, the cell clusters may be isolated by low speed gravity sedimentation. The matrix/hepatic/nonparenchymal cell clusters are then exposed to a second structural matrix material in the presence of an appropriate culture media, thereby providing an environment for three-dimensional hepatic cell growth. Many commercially available culture media, supplemented in some instances with growth factors and the like, may be suitable for use. In addition, the culture media may be replenished periodically to provide a fresh supply of nutrients. The three-dimensional hepatic cell culture system is cultured for a sufficiently long period of time to allow the hepatic cells to replicate to form a three-dimensional cell or tissue structure.

Prior to use of three-dimensional hepatic cell cultures, the cultures may be contacted with a number of different growth factors that can regulate tissue regeneration by affecting cell proliferation, and gene expression. Such growth factors include those capable of stimulating the proliferation and/or differentiation of hepatic progenitor cells. For example, epidermal growth factor (EGF), transforming growth factor .alpha. (TGF-.alpha.) or hepatocyte growth factor (HGF) may be utilized. The hepatic cells may be stimulated in vitro prior to transplantation into the recipient subject, or alternatively, by injecting the recipient with growth factors following transplantation.

5.3. Use of the Hepatic Cell Cultures

The hepatic cell cultures of the invention can be used as bio-artificial livers for use by subjects having liver disorders that result in hepatic failure or insufficiency. The use of such bio-artificial livers involves the perfusion of the subject's blood through the bio-artificial liver. In the blood perfusion protocol, the subject's blood is withdrawn and passes into contact with the hepatocyte cell cultures. During such passage, molecules dissolved in the patient's blood, such as bilirubin, are taken up and metabolized by the hepatocyte cultures. In addition, the cultured hepatocytes provide factors normally supplied by liver tissue.

To form the bio-artificial liver the three-dimensional hepatocyte cell cultures of the invention are grown within a containment vessel containing an input and output outlet for passage of the subjects blood through the containment vessel. The bio-artificial liver further includes a blood input line which is operatively coupled to a conventional peristaltic pump. A blood output line is also included. Input and output lines are connected to appropriate arterial-venous fistulas which are implanted into, for example, the forearm of a subject. In addition, the containment vessel may contain input and output outlets for circulation of appropriate growth medium to the hepatocytes for continuous cell culture within the containment vessel.

In an embodiment of the invention, semipermeable membranes may be included in the bio-artificial livers to prevent direct contact of the subject's blood with the three-dimensional hepatocyte cultures. In such instances, the molecules dissolved in the subject's blood will diffuse through the semipermeable membrane and are taken up and metabolized by the hepatocycte cultures.

The use of the cultured hepatocyte systems of the invention to form bio-artificial livers provides a method which may be utilized to provide liver function to subjects suffering from hepatic failure or insufficiency.

The three-dimensional hepatic cell cultures can also be administered or transplanted to the recipient in an effective amount to achieve restoration of liver function, thereby alleviating the symptoms associated with liver disorders. When the hepatic cell cultures are to be administered to a recipient, it is desirable to form the hepatocyte cultures with hepatocytes and nonparenchymal cells derived from the recipient so as to avoid tissue rejection.

The number of cells needed to achieve the purposes of the present invention will vary depending on the degree of liver damage and the size, age and weight of the host. For example, the cells are administered in an amount effective to restore liver function. Determination of effective amounts is well within the capability of those skilled in the art. The effective dose may be determined by using a variety of different assays designed to detect restoration of liver function. The progress of the transplant recipient can be determined using assays that include blood tests known as liver function tests. Such liver function tests include assays for alkaline phosphatase, alanine transaminase, aspartate transaminase and bilirubin. In addition, recipients can be examined for presence or disappearance of features normally associated with liver disease such as, for example, jaundice, anemia, leukopenia, thrombocytopenia, increased heart rate, and high levels of insulin. Further, imaging tests such as ultrasound, computer assisted tomography (CAT) and magnetic resonance (MR) may be used to assay for liver function.

The three-dimensional hepatic cell system can be administered by conventional techniques such as injection of cells into the recipient host liver, injection into the portal vein, or surgical transplantation of cells into the recipient host liver. In some instances it may be necessary to administer the hepatic cell composition more than once to restore liver function. In addition, growth factors, such as G-CSF, or hormones, may be administered to the recipient prior to and following transplantation for the purpose of priming the recipients liver and blood to accept the transplanted cells and/or to generate an environment supportive of hepatic cell proliferation.

Claim 1 of 6 Claims

We claim:

1. A population of hepatocytes and nonparenchymal cells, derived using a method comprising:

co-culturing hepatocytes and nonparenchymal cells, derived from disaggregated liver tissue, in the presence of

(a) one or more growth factors that support the growth of hepatocytes comprising epidermal growth factor or hepatocyte growth factor and

(b) beads coated with extracellular matrix protein that promotes cell adhesion under conditions sufficient to allow for the proliferation of said hepatocytes while retaining hepatic faction of said hepatocytes.

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