FIELD OF THE INVENTION

The present disclosure relates to compositions and methods for culturing a population of hepatocytes in vitro, comprising co-culturing the population of hepatocytes with at least one non-parenchymal cell population and incubating the co-culture in culture medium, wherein the co-culture is incubated in culture medium comprising serum for at least one time period of about 1 hour to about 6 weeks and is incubated in culture medium that does not comprise serum (serum-free culture medium) for at least one time period of about 1 hour to about 6 weeks.

BACKGROUND OF THE INVENTION

Hepatocytes, the main cell type of the liver, rapidly lose their phenotypic functions under conventional culture formats that rely exclusively on extracellular matrices (ECM). Co-culture of hepatocytes with stromal cells from both within and outside the liver in either 2-dimensional or 3-dimensional configurations can prolong hepatic phenotype to ˜4 weeks in vitro. The lifetime of a hepatocyte in vivo is around 150 days; however, no current culture platform can keep hepatocytes highly functional for that length of time. The culture medium plays an important role in hepatocyte survival in any culture configuration; however, most models do not maintain hepatic functions past 3-4 weeks of co-culture in current media formulations. There is a need in the art for improving the culture media formulation and timed administration of culture media to further prolong the lifetime of hepatocytes in vitro. Such a medium can result in hepatocyte cultures that have applications in testing the effects of chronically administered drugs and diseases that affect the liver, such as hepatitis, type 2 diabetes, malaria, liver fibrosis, liver cancer and fatty liver disease.

SUMMARY OF THE INVENTION

In an aspect, the disclosure provides a method of culturing a population of hepatocytes in vitro, comprising co-culturing the population of hepatocytes with at least one non-parenchymal cell population and incubating the co-culture in culture medium, wherein the cell populations are disposed in a micropattern on a culture substrate and the micropattern comprises a predetermined two-dimensional pattern of multiple microdots, the micropattern defined by a microdot diameter and a center-to-center spacing between each of any two neighboring microdots, and the microdots comprise the hepatocytes and the space between the microdots comprises the non-parenchymal cell population, and wherein the co-culture is incubated in culture medium comprising serum for at least one time period of about 1 hour to about 6 weeks and is incubated in culture medium that does not comprise serum (serum-free culture medium) for at least one time period of about 1 hour to about 6 weeks. Each microdot can have a diameter of about 500 μm and the center-to-center spacing between each of any two neighboring microdots is about 1200 μm. The co-culture can be incubated in culture medium comprising serum for at least one time period of at least 1 week before the composition is incubated in serum-free culture medium. The co-culture can be incubated in culture medium comprising serum for at least one time period of at least 2 weeks before the composition is incubated in serum-free culture medium. The co-culture can be incubated in serum-free culture medium for at least one time period of about 1 hour to about 3 days. The co-culture can be incubated serum-free culture medium for at least one time period of about 2 days. Specifically, the co-culture can be (a) incubated in serum-free culture medium for a time period of about 1 hour to about 3 days and (b) incubated in culture medium comprising serum for a time period of about 5 to about 8 days, wherein (a) and (b) are alternated for up to 6 weeks.

The co-culture can exhibit at least one indicator of hepatocyte function at least 4 weeks in culture. The co-culture can exhibit at least one indicator of hepatocyte function at least 6 weeks in culture. The at least one indicator of hepatocyte function can be selected from albumin production, urea production, ATP production, glutathione production, enzyme activity, lipid accumulation, liver gene expression, liver protein expression, or CYP2C9 activity, CYP3A4 activity, CYP1A1 activity, CYP1A2 activity, CYP2B6 activity, CYP2A6 activity, CYP2D6 activity, CYP2C8 activity, CYP2C19 activity, or CYP2E1 activity.

The co-culture can retain hepatocyte morphology at least 4 weeks in culture. The co-culture can retain hepatocyte morphology at least 6 weeks in culture.

The serum-free culture medium can comprise glucose. The serum-free culture medium can comprise glucagon.

The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can compromise human hepatocytes. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can compromise primary human hepatocytes.

The co-culture of a population of hepatocytes with at least one non-parenchymal cell population wherein at least one of the non-parenchymal cell populations can comprise non-human stromal cells. The non-human stromal cells can be selected from the group consisting of fibroblasts, fibroblast-derived cells, macrophages, endothelial cells, stellate cells, pericytes, inflammatory cells, cholangiocytes and other types of stromal cells, and combinations thereof. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise murine embryonic fibroblasts. Specifically, the co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise 3T3-J2 murine embryonic fibroblasts. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise growth arrested stromal cells. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise stromal cells that are not growth arrested.

In another aspect, the disclosure provides a composition comprising a population of hepatocytes in vitro, comprising co-culturing the population of hepatocytes with at least one non-parenchymal cell population and incubating the co-culture in culture medium, wherein the cell populations are disposed in a micropattern on a culture substrate and the micropattern comprises a predetermined two-dimensional pattern of multiple microdots, the micropattern defined by a microdot diameter and a center-to-center spacing between each of any two neighboring microdots, and the microdots comprise the hepatocytes and the space between the microdots comprises the non-parenchymal cell population, and wherein the co-culture is incubated in culture medium comprising serum for at least one time period of about 1 hour to about 6 weeks and is incubated in culture medium that does not comprise serum (serum-free culture medium) for at least one time period of about 1 hour to about 6 weeks. Each microdot can have a diameter of about 500 μm and the center-to-center spacing between each of any two neighboring microdots is about 1200 μm. The co-culture can be incubated in culture medium comprising serum for at least one time period of at least 1 week before the composition is incubated in serum-free culture medium. The co-culture can be incubated in culture medium comprising serum for at least one time period of at least 2 weeks before the composition is incubated in serum-free culture medium. The co-culture can be incubated in serum-free culture medium for at least one time period of about 1 hour to about 3 days. The co-culture can be incubated serum-free culture medium for at least one time period of about 2 days. Specifically, the co-culture can be (a) incubated in serum-free culture medium for a time period of about 1 hour to about 3 days and (b) incubated in culture medium comprising serum for a time period of about 5 to about 8 days, wherein (a) and (b) are alternated for up to 6 weeks.

The co-culture can exhibit at least one indicator of hepatocyte function at least 4 weeks in culture. The co-culture can exhibit at least one indicator of hepatocyte function at least 6 weeks in culture. The least one indicator of hepatocyte function can be selected from albumin production, urea production, ATP production, glutathione production, enzyme activity, lipid accumulation, liver gene expression, liver protein expression, or CYP2C9 activity, CYP3A4 activity, CYP1A1 activity, CYP1A2 activity, CYP2B6 activity, CYP2A6 activity, CYP2D6 activity, CYP2C8 activity, CYP2C19 activity, or CYP2E1 activity.

The co-culture can retain hepatocyte morphology at least 4 weeks in culture. The co-culture can retain hepatocyte morphology at least 6 weeks in culture.

The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can compromise human hepatocytes. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can compromise primary human hepatocytes.

The co-culture of a population of hepatocytes with at least one non-parenchymal cell population wherein at least one of the non-parenchymal cell populations can comprise non-human stromal cells. The non-human stromal cells can be selected from fibroblasts, fibroblast-derived cells, macrophages, endothelial cells, stellate cells, pericytes, inflammatory cells, cholangiocytes and other types of stromal cells, and combinations thereof. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise murine embryonic fibroblasts. Specifically, the co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise 3T3-J2 murine embryonic fibroblasts. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise growth arrested stromal cells. The co-culture of a population of hepatocytes with at least one non-parenchymal cell population can further compromise stromal cells that are not growth arrested.

In still another aspect, the disclosure provides a method of identifying a candidate test compound for use in treating a disorder of the liver, the method comprising: contacting a composition of the disclosure with the test compound; maintaining the composition for a time and under conditions sufficient to allow an effect of the test compound on the hepatocytes; measuring at least one indicator of hepatic function in the hepatocytes to obtain a test measurement, or applying hepatocyte imaging technology (HIAT) to the hepatocytes to obtain a test image; and comparing the test measurement to a control measurement from the hepatocytes before contact with the test compound, or the test image to a control image of the hepatocytes before contact with the test compound, wherein a difference between the test and control is indicative of whether the test compound is a candidate for use in treating a disorder of the liver. The at least one indicator of hepatic function can be selected from the group consisting of albumin production, urea production, ATP production, glutathione production, enzyme activity, lipid accumulation, liver gene expression, and liver protein expression in the hepatocytes. The at least one inducible liver enzyme can be selected from CYP2C9, CYP3A4, a combination of CYP1A1, CYP1A2, CYP2B6, CPY2A6, and CYP2D6, and any combination thereof.

