Long-term three dimensional tissue culture system

Details Long-term three dimensional tissue culture system Long-term three dimensional tissue culture system

1999-12-07

2004-05-18

Naff, David M. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of co-culturing cells

C424S093700, C435S177000, C435S180000, C435S395000, C435S402000, C435S405000

Reexamination Certificate

active

06737270

ABSTRACT:

1. INTRODUCTION
The present invention relates to a novel tissue culture system that provides for the long term culture of proliferating hepatocytes that retain hepatic function. Disclosed are methods and compositions for ex vivo culturing of hepatocytes and nonparenchymal cells 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 hepatic cell culture system can be used to form bio-artificial livers through which a subjects blood is perfused. Alternatively, the novel hepatic cell culture system 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 alpha, antitrypsin deficiency.
The present invention is based on the discovery that mixed cultures of proliferating hepatocytes and nonparenchymal cells, grown on a collagen-coated matrix in medium containing hepatocyte growth factor (HGF) and epidermal growth factor (EGF), maintain their capacity to proliferate while retaining hepatic functions.
2. BACKGROUND OF INVENTION
One of the major functions of the liver is to break down harmful substances absorbed from the intestine or manufactured elsewhere in the body, followed by their excretion as harmless by-products into the bile or blood. Abnormalities of liver function caused by insult to and/or death or malfunction of the cells in the liver can lead to a variety of different hepatic disorders including cirrhosis of the liver or hepatitis. Treatment of such disorders may include whole liver transplants, although this treatment is limited by organ availability, surgical complications, and immunologically-mediated graft rejection normally associated with liver transplantation.
While hepatocyte transplantation has been considered as an alternative to whole-organ transplantation, major technical barriers such as the inability to transfer donor hepatocytes into the liver of a recipient, in numbers to provide a beneficial result, have limited the usefulness of this approach. One of the major difficulties in constructing artificial liver tissue is that, to function effectively, the artificial liver tissue requires functionally active, differentiated hepatocytes present at high densities. Future success with artificial liver tissue will depend on the development of systems in which hepatocytes attached to matrices and packed at high density can retain long term their full functional capacity.
To generate artificial liver tissue, it will be necessary to provide in vitro cultures of hepatocytes. Unfortunately, one of the problems associated with the culturing of hepatocytes is that gene expression deteriorates rapidly as the hepatocytes proliferate. Likewise, long-term cultures of hepatocytes having stable gene expression can only be maintained in the absence of cell proliferation. Thus, one of the long-standing goals of culturing hepatocytes is the establishment of proliferating cultures with long-term gene expression.
A number of culture techniques have been developed that permit primary hepatocyte cultures to grow and/or express complex patterns of hepatocyte differentiation (Mitaka, et al., 1995,
Biochem Biophys Res Commun
214: 310-317; Cable, 1997,
Hepatology
26: 1444-1445; Block, et al., 1996,
J. Cell Biol.
132: 1133-1149). Conditions have also been established that allow mature hepatocytes to enter into clonal expansion in cell culture (Block, et al., 1996,
J. Cell Biol.
132: 1133-1149). For example, hepatocytes cultured in chemically defined hepatocyte growth medium (HGM) enter into DNA synthesis in response to polypeptide mitogens, notably epidermal growth factor (EGF), transforming growth factor-&agr; (TGF-&agr;), and hepatocyte growth factor (HGF). These mitogens induce multiple rounds of DNA synthesis and expansion of the cell population. The proliferating cells, however, lose most markers of hepatocyte differentiation while they retain expression of hepatocyte associated transcription factors HNF1, HNF4, and HNF3. In addition, proliferation of adult hepatocytes has been observed in serum-free medium supplemented with nicotinarnide and epidermal growth factor (EGF) (Mitaka, T., et al., 1991,
Hepatology
12: 21-30; Mitaka, T., et al., 1992,
Hepatology
10:440-447; Mitaka, T., et al., 1993,
J. Cell Physiol,
147: 461-468; Mitaka, T., et al.,
Cancer Res,
1993, 53: 3145-3148; Block, G. D., et al., 1996,
J. Cell Biol.
132:1133-1149; Tateno, C., et al., 1996,
Am J. Pathol
148: 383-392).
Previous studies have indicated that a fundamental parameter that best determines hepatocyte gene expression in culture is the surrounding matrix. Hepatocytes embedded in complex matrices, such as Matrigel or type I collagen gels, maintain stable phenotypic expression, however, at the expense of cell proliferation. Recently, Mitaka, T. et al. (1999,
Hepatology
29: 111-125) showed that small hepatocytes could differentiate to mature hepatocytes that interact with hepatic nonparenchymal cells and extracellular matrix. The mature hepatocytes reconstructed three-dimensional structures, expressed proteins known to be expressed in highly differentiated hepatocytes and the cells survived for more than 3 months, while maintaining hepatic differentiated functions. In addition, Landry et al. (1985,
J. Cell Biol.
101:914-923) demonstrated the reconstruction of a three-dimensional cyto-architecture consisting of differentiated hepatocytes, bile duct-like cells and deposited extracellular matrix by the use of spheroidal aggregate culture of hepatic cells isolated from newborn rats. Three-dimensional cell culture systems have also been disordered in which hepatocytes are grown on a pre-established stromal tissue (U.S. Pat. No. 5,624,840). Attempts have also been made to grow a three-dimensional hepatic organoid using a co-culture of hepatocytes and fibroblasts (Senoo, et al., 1989,
Cell Biol. Internat. Reports
13:197-206; Takezawa, et al., 1992,
J Cell Sci
101:495-501).
A number of devices which perform the function of the liver and involve blood perfusion have been described (Hagger et al., 1983,
ASAIO J.
6:26-35; U.S. Pat. No. 5,043,260; U.S. Pat. No, 5,270,192: Demetriou et al., 1986,
Ann. Surg
9:259-271). However, a number of problems are associated with the use of such devices for treatment of patients suffering from hepatic failure or dysfunction. Perhaps, the most significant problem is the inability to culture hepatocytes that retain hepatic function for prolonged periods of time, although, attempts have been made to circumvent this problem through the use of transformed hepatocytes that are capable of proliferating indefinitely (U.S. Pat. No. 4,853,324).
Development of a stable support system that would maintain hepatic functions and be useful in stabilizing patients in partial or complete hepatic failure has been a long-term scientific goal in the field of hepatology. Similar devices have revolutionized the treatment of patients with kidney failure and have allowed long-term stabilization of a large population of patients. Currently the use of such devices in treatment of liver failure is quite limited and existing devices are based on rapidly assembled hepatocyte support systems which partially sustain the patient over a very limited period of time, i.e, 24 to 48 hours with declining function over more prolonged term use.
3. 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 generat

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