Seeded hepatocytes and their preparation and uses

Abstract

The present invention provides a product comprising human hepatocytes seeded on a surface, at least some of the seeded hepatocytes being in one or more hepatocyte clusters on feeder cells and attached to the surface. A method for preparing the seeded human hepatocytes is also provided. The preparation method includes applying the human hepatocytes to a surface in the presence of feeder cells, co-culturing the applied hepatocytes with the feeder cells, and forming one or more hepatocyte clusters by the co-cultured hepatocytes on the feeder cells attached to the surface. The seeded hepatocytes can be used for various purposes, including preparing a hepatitis B virus (HBV)-infected hepatocyte culture model and drug testing.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 562,127, filed September 22, 2017, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates generally to seeded human hepatocytes and their preparation and uses. [Background technology]

[0003] Primary human hepatocytes isolated from donors have been used in two-dimensional in vitro culture systems to study drug metabolism or drug-induced liver injury. In two-dimensional in vitro culture systems, primary human hepatocytes are maintained in culture medium and attached to a surface (e.g., a plate) to generate a monolayer of hepatocytes suitable for conducting drug metabolism, toxicity, or viral infection studies. For two-dimensional in vitro culture systems, isolated primary human hepatocytes with at least 85% or greater plateability are desirable for long-term studies, e.g., at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, or 49 days; isolated primary human hepatocytes with less than 60% plateability are generally considered less desirable and less suitable for long-term experiments. A high percentage of cryopreserved primary human hepatocyte batches fall into the latter category of non-adherent or poorly adherent hepatocytes, limiting the availability of many batches with desirable donor specifications for applications such as testing drug metabolism, toxicity, or viral infection, such as non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH). There is a need to improve the adhesion capacity, functional stability, and culture longevity of primary human hepatocytes. Summary of the Invention

[0004] The present invention relates to seeded human hepatocytes and their preparation and uses.

[0005] A product is provided that includes human hepatocytes seeded on a surface. At least 70% of the seeded hepatocytes are in one or more hepatocyte clusters on feeder cells. The feeder cells are endothelial cells and fibroblasts and are attached to the surface. In the product, at least 90% of the seeded hepatocytes in one or more hepatocyte clusters can be in direct hepatocyte-to-hepatocyte contact. The one or more hepatocyte clusters can cover at least 50% of the surface. The surface can have a shortest diameter of at least 3 mm.

[0006] The product may further comprise a culture medium wherein at least 90% of the seeded hepatocytes remain on the surface for at least 7 or 42 days.

[0007] The seeded hepatocytes may produce albumin and / or express cytochrome P450.

[0008] The endothelial cells may be human cells. For example, the endothelial cells may be human umbilical vein endothelial cells (HUVECs). The endothelial cells may not be hepatocytes. The endothelial cells may not be proliferative. The endothelial cells may be primary cells or may be cultured for up to seven passages. The endothelial cells may not be immortal.

[0009] The fibroblasts may be human cells. The fibroblasts may be human skin fibroblasts. The fibroblasts may not be hepatocytes. The fibroblasts may not be proliferative. The cells may be primary cells or may have been cultured for up to 7 passages. The fibroblasts do not have to be immortal.

[0010] In the product, the seeded hepatocytes, endothelial cells and fibroblasts may have a cell ratio of 3:1:1 to 24:1:1.

[0011] The seeded hepatocytes can be obtained from one donor. The seeded hepatocytes can be obtained from two or more donors, each of which may have microsteatosis and / or nonalcoholic steatohepatitis (NASH).

[0012] A method for preparing seeded human hepatocytes is also provided. The method includes applying human hepatocytes to a surface in the presence of feeder cells, the feeder cells being endothelial cells and fibroblasts. After the applying step, the applied hepatocytes are co-cultured with the feeder cells, and the co-cultured hepatocytes on the feeder cells attached to the surface form one or more hepatocyte clusters. At least 85% of the co-cultured hepatocytes are in one or more hepatocyte clusters. The applied hepatocytes may have an adhesion capacity of less than 60% to the surface in the absence of feeder cells. The applied hepatocytes may have an adhesion capacity of at least 85% to the surface in the absence of feeder cells. The one or more seeded hepatocyte clusters may cover at least 50% of the surface. The surface may have a shortest diameter of at least 3 mm.

[0013] Depending on the preparation method, at least 90% of the seeded hepatocytes can remain on the surface for at least 7 or 42 days. Feeder cells can be attached to the surface before the application step or during the co-culture step. The hepatocytes can be frozen before the application step.

[0014] The preparation method may exclude the addition of extracellular matrix proteins.

[0015] For each preparation method, seeded hepatocytes prepared according to the method are provided.

[0016] A method for testing a pharmaceutical agent is provided. The method comprises administering to the seeded hepatocytes an amount of a pharmaceutical agent effective to alter the properties of the seeded hepatocytes. The seeded hepatocytes may be in a product of the invention or prepared according to the method of the invention. The pharmaceutical agent may be selected from the group consisting of a small molecule, an antibody, a live virus, a viral vector, an oligonucleotide, and a cell.

[0017] A method for testing drug metabolism is provided, comprising administering an effective amount of a drug to seeded hepatocytes and determining the amount of drug in the seeded hepatocytes. The seeded hepatocytes may be in the product of the invention or prepared according to the method of the invention.

[0018] A method for testing drug transport is provided, comprising administering an effective amount of a drug to seeded hepatocytes and determining the location of the drug in the seeded hepatocytes. The seeded hepatocytes may be in the product of the invention or prepared according to the method of the invention.

[0019] A method for testing the toxicity of a drug is provided, comprising administering an effective amount of the drug to seeded hepatocytes and detecting surviving seeded hepatocytes. The seeded hepatocytes may be in the product of the invention or prepared according to the method of the invention.

[0020] A method for preparing a hepatitis B virus (HBV)-infected hepatocyte culture model is provided. The method comprises inoculating seeded hepatocytes with hepatitis B virus (HBV) and incubating the infected seeded hepatocytes for at least 14 days. The seeded hepatocytes may be in the product of the invention or may be prepared according to the method of the invention.

[0021] A kit for seeding hepatocytes is provided. The kit includes a first cryopreservation vial containing primary hepatocytes, a second cryopreservation vial containing endothelial cells, a third cryopreservation vial containing fibroblasts, and instructions for preparing human hepatocytes seeded with primary hepatocytes, endothelial cells, and fibroblasts according to the method of the present invention. The second cryopreservation vial and the third cryopreservation vial may be the same. The first cryopreservation vial, the second cryopreservation vial, and the third cryopreservation vial may be the same. [Brief explanation of the drawings]

[0022] [Figure 1]Schematic #1 shows the human hepatocyte co-culture according to one embodiment of the present invention. This schematic provides the preparation of the cell culture and a time-frame analysis. According to Schematic #1, the hepatocyte co-culture is prepared by seeding human hepatocytes onto feeder cells, which are endothelial cells and fibroblasts, pre-seeded on day 0. EC, endothelial cells; Fb, fibroblasts; MG, MATRIGEL. [Figure 2] Schematic #2 for human hepatocyte co-culture according to another embodiment of the present invention. This schematic provides cell culture preparation and time-frame analysis. According to Schematic #2, hepatocyte co-culture is prepared by seeding human hepatocytes with feeder cells, which are endothelial cells and fibroblasts, on day 0. EC, endothelial cells; Fb, fibroblasts; MG, MATRIGEL. [Figure 3] Morphology of human cells used in hepatocyte co-cultures: skin fibroblasts alone (top left), immortalized liver sinusoidal endothelial cells alone (top center), primary hepatocytes alone (top right), a mixture of fibroblasts and endothelial cells (bottom left), and a mixture of primary hepatocytes, endothelial cells, and fibroblasts (bottom right). [Figure 4] Figure 1 shows the expression levels of albumin, CyP450 1A2, CyP450 2B6, and CyP450 3A4 genes in hepatocyte monoculture (monocult), hepatocyte coculture with feeder cells (coculture), or feeder cell culture (feeder) one week after seeding, as measured by qRT-PCR. The expression levels of each of these four genes were significantly higher in the coculture than in the monoculture. *, p<0.05; **, p<0.01. [Figure 5] Shown are the activities of CyP450 1A2, CyP450 2B6, or CyP450 3A4 normalized based on the number of hepatocytes seeded in monoculture or coculture 7 days after seeding, before and after induction. *, p<0.05; **, p<0.01. [Figure 6]Figure 1 shows the secretion of albumin (ALB) and urea measured by ELISA over a 2-week period of hepatocyte culture. The levels of secreted albumin and urea were significantly higher in co-cultured hepatocytes than in monocultures. Mono, monoculture; Co, coculture; *, p<0.05; **, p<0.01. [Figure 7] Microscopic observation of suspension-grade human primary hepatocytes in monoculture (left) or coculture with endothelial cells and fibroblasts (right). Hepatocytes in monoculture began to detach after seeding, whereas the majority of hepatocytes seeded in coculture remained attached with good quality. [Figure 8] Co-cultured suspension-grade hepatocytes show similar or higher expression levels of CyP450 1A2 (top) and CyP450 3A4 (bottom) compared to mono-cultured adherent-grade hepatocytes. Both adherent and suspension-grade hepatocytes showed higher metabolic activity in co-culture compared to mono-culture. Mono-U, mono-culture uninduced; Mono-I, mono-culture induced; Co-U, co-culture uninduced; Co-I, co-culture induced. [Figure 9] Suspension-grade hepatocytes in co-cultures show similar or higher secretion levels of albumin (left) and urea (right) compared to adherent-grade hepatocytes in monoculture. Both adherent and suspension-grade hepatocytes showed higher functional albumin and urea secretion in co-cultures compared to monocultures. Mono-U, monoculture uninduced; Mono-I, monoculture induced; Co-U, coculture uninduced; Co-I, coculture induced. [Figure 10] This figure shows human primary hepatocytes maintained in culture for approximately 7 days under standard sandwich culture conditions (monoculture). Six batches of hepatocytes, including three batches of adherent-grade hepatocytes and three batches of suspension-grade hepatocytes, were observed over a 4-week culture period. Most hepatocytes detached around day 7 of culture. [Figure 11] Shown are hepatocytes seeded onto a mixture of endothelial cells and fibroblasts in co-culture maintained for up to 6 weeks. 10x magnification. [Figure 12]Immunocytochemical staining of hepatocytes 43 days after seeding in co-culture. Staining for albumin (bottom right) and CD31 (bottom left) showed a geographic distribution of hepatocytes, endothelial cells, and fibroblasts. 10x magnification. [Figure 13] Figure 1 shows hepatocytes exhibiting higher levels of CyP450 3A4 activity in co-cultures compared to mono-cultures over 6 weeks of culture. CyP450 3A4 activity levels peaked at week 3, and co-cultures maintained metabolic activity for 6 weeks, whereas mono-cultures discontinued metabolic activity. **, p<0.01. [Figure 14] Functional bile secretion from human hepatocytes in co-culture observed by imaging the efflux of FITC-conjugated CDFDA into the bile canaliculi. 10x magnification. [Figure 15] Hepatocytes from three separate batches and three pooled batches were grown under co-culture conditions maintained for up to six weeks. The hepatocytes were seeded onto the endothelial cell and fibroblast mixture and overlaid with MATRIGEL the day after. 10x magnification. [Figure 16] Hepatocytes pooled from three different batches could be maintained in co-culture for up to 6 weeks. Hepatocytes were cultured in a mixture of endothelial cells and fibroblasts without being overlaid with MATRIGEL. 10x magnification. [Figure 17] Figure 1 shows that the secretion levels of albumin (left) and urea (right) from hepatocytes co-cultured without MATRIGEL tapping were similar to those of hepatocytes co-cultured with MATRIGEL tapping. MG, MATRIGEL tapping; no MG, no MATRIGEL tapping; D4, day 4; D7, day 7. [Figure 18] Figure 1 shows that the expression levels of CyP450 3A4 from hepatocytes co-cultured without MATRIGEL tapping were similar to those from hepatocytes co-cultured with MATRIGEL tapping for up to 6 weeks. MG, MATRIGEL tapping; no MG, no MATRIGEL tapping. [Figure 19]We show that metabolic activity, measured by CyP450 3A4, from three different batches of human primary hepatocytes was similar to the metabolic activity of pooled hepatocytes from three different batches in co-culture for up to 6 weeks. [Figure 20] We demonstrate that endothelial cells from different sources can support hepatocyte culture. Both immortalized hepatic sinusoidal endothelial cells (SECs) and human umbilical vein endothelial cells (HUVECs) were able to support hepatocyte culture. [Figure 21] Similar expression levels of CyP450 1A2 and CyP450 3A4 by hepatocytes co-cultured with immortalized hepatic sinusoidal endothelial cells (SECs) or human umbilical vein endothelial cells (HUVECs) are shown. D4, day 4; D7, day 7. [Figure 22] Similar levels of albumin and urea are shown by hepatocytes co-cultured with immortalized hepatic sinusoidal endothelial cells (SECs) or human umbilical vein endothelial cells (HUVECs). D4, day 4; D7, day 7. [Figure 23] Morphology of hepatocytes in co-cultures with different cell ratios: HH, human hepatocytes; Fb, fibroblasts; EC, endothelial cells. [Figure 24] Figure 1 shows the expression levels of CyP450 1A2 and CyP450 3A4 by hepatocytes co-cultured with different ratios of endothelial cells and fibroblasts. A 12:1:1 cell mixture of hepatocytes, endothelial cells, and fibroblasts shows the highest expression levels of CyP450 1A2 and CyP450 3A4 compared to cell mixtures with other ratios. CTRL, uninduced control; Ind, induced. [Figure 25] Figure 1 shows the expression levels of sodium taurocholate cotransporter (NTCP), a cellular receptor for hepatitis B virus (HBV), in co-cultured hepatocytes at multiple time points. [Figure 26] Figure 1 shows the geographic distribution of hepatocytes and feeder cells in the co-culture. The size of the nuclei was measured using ImageJ software from the overall image area (left) and hepatocyte clusters (right). [Figure 27] FIG. 1 shows a schematic diagram of a co-culture system according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention provides seeded hepatocytes and their preparation and use. The present invention is based on the discovery that co-culturing human hepatocytes with endothelial cells and fibroblasts improves the adhesion ability, functional stability and culture lifespan of human hepatocytes.

[0024] As used herein, the term "seeded hepatocytes" refers to human hepatocytes that have attached directly or indirectly to a surface or a feeder cell layer. As used herein, the term "plateability" or "adherence" refers to the ability of hepatocytes to attach to a surface, such as a plastic or treated surface (e.g., a culture vessel or a multi-well plate), within a predetermined time (e.g., 0.5, 1, 2, 3, 4, 5, 6, or 12 hours) after exposure of the hepatocytes to the surface. The adhesive ability of hepatocytes can be characterized by the percentage of cells that can attach to a surface directly or indirectly, for example, via feeder cells or non-cellular material (i.e., non-cellular material), within a predetermined time.

[0025] The term "feeder cells" as used herein refers to cells other than hepatocytes that help maintain the survival and function of hepatocytes. Examples of feeder cells include fibroblasts and endothelial cells. The non-cellular material may be chemical compounds and / or biological molecules. Examples of non-cellular material include extracellular matrix proteins, hemodynamic flow conditions, paracrine and autocrine factors, adhesive proteins, and polysaccharides.

[0026] Hepatocytes can be considered adherent if at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of the cells are able to attach to a surface, for example, within a given period of time.

[0027] Hepatocytes can be considered non-adherent or poorly adherent if only 70%, 65%, 60%, 55%, 50%, 45% or 40% of the cells are able to attach to a surface, for example, within a given period of time.

[0028] As used herein, the term "hepatocyte cluster" refers to a group of two or more hepatocytes in a three-dimensional structure. At least about 70%, 80%, 90%, 95%, or 99% of the hepatocytes in the hepatocyte cluster may be in direct hepatocyte-cell contact and express gap junction proteins (e.g., connexin 32) or tight-gap junction-associated proteins. This can be evidenced by the presence of occludin, ZO-1, claudin-1, and claudin-4.

[0029] In addition to cell-cell interactions and cell binding, the overall shape and three-dimensional structure of hepatocytes in the co-culture system maintain normal structure and function, including cell signaling pathways and normal gene expression programs. The co-culture system maintains the three-dimensional structure and shape of hepatocytes over time, thus enhancing cell-cell interactions and preventing loss of normal hepatic phenotype and cell spreading. In contrast, Over time, hepatocytes maintained as monocultures, especially in subconfluent conditions, lose cell shape and structure, begin to stretch, and become "thinner" (lose height) in the absence of surrounding stromal cells and their supporting ECM factors. The altered cell shape of hepatocytes due to extensive cell stretching begins to show a corresponding reduction and loss of important functions, altered gene expression programs, and altered responses to drug compounds.

[0030] As used herein, the term "functional stability" or "functionally stable" refers to the stability of one or more basic hepatocyte functions over a given time period. Basic hepatocyte functions include albumin and urea synthesis rates, cytochrome P450 enzyme activity rates, and inducible cytochrome P450 levels. Functional stability of hepatocytes can be initially characterized by the percentage of basic hepatocyte function within an initial period after isolation from a donor, e.g., within the first 4-6 days, and maintained in culture for a given period, e.g., for the following 1, 2, 3, 4, 5, or 6 days or for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks (e.g., up to 28 or 42 days). Hepatocytes are considered functionally stable if at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the basic hepatocyte function of the cells is retained after the given period, without deviations of more than 5, 10, 15, 20, or 25%.

[0031] As used herein, the term "culture longevity" refers to the lifespan of hepatocytes that remain viable and functional. Cells attached to a surface are also referred to as seeded cells. At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the seeded cells can remain viable attached to the surface for a predetermined period of time, e.g., 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks. Viable hepatocytes are functional, as evidenced, for example, by albumin and urea production and cytochrome P450 enzyme activity.

[0032] As used herein, the term "hepatocytes" refers to primary hepatocytes isolated or obtained from one or more donors and that have not been cultured for a predetermined number of passages (e.g., 0, 1, 2, 3, 4, or 5 passages). As used herein, the term "pooled hepatocytes" refers to primary hepatocytes isolated from two or more donors and then mixed. Hepatocytes can be selected from donors based on specific genotyping information.

[0033] The term "donor" as used herein refers to a living mammal having a liver. The mammal may be a human, cow, pig, dog, cat, non-human primate, rodent such as a rat or mouse, horse, goat, sheep, or deer. The donor may also be a human with a liver disease or condition, such as microsteatosis, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), or hepatitis (e.g., A, B, C, D, and E). Hepatocytes from livers with NASH disease may express markers such as cytokeratin 18.

[0034] The term "endothelial cells" as used herein refers to any endothelial cell. The endothelial cells may be primary human cells, such as human umbilical vein endothelial cells (HUVECs). The endothelial cells may not be hepatocytes. The endothelial cells may be isolated from the umbilical cord, liver, lung, kidney, brain, pancreas, lymph nodes, heart, intestine, or other arteries, blood vessels, or capillaries. The endothelial cells may not be proliferative. The endothelial cells may be primary cells or may be cultured for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 passages. The endothelial cells may not be immortal.

[0035] The term "fibroblast" as used herein refers to any fibroblast cell. The fibroblast may be a primary human cell, e.g., a human skin fibroblast. The fibroblast may be isolated from skin, lung, bladder, cornea, or other tissue types. The fibroblast may not be a liver cell. The fibroblasts may not be proliferative. The fibroblasts may be primary cells or may be cultured for up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 passages. The fibroblasts may not be immortal.

[0036] Feeder cells can be prepared by inactivating and freezing dermal fibroblasts and HUVECs cultured at or before the seventh passage of culture. Feeder cell inactivation can be completed with mitomycin C or gamma irradiation, followed by verification by either PCNA or BrdU or cell counting. Inactivated feeder cells can be frozen to prepare one plate of co-culture per vial. Each lot of feeder cells can be tested to ensure quality criteria, including but not limited to, cell number, viability, cell doubling time, sterility, and purity. The fibroblasts and endothelial cells used herein may not interfere at all with the hepatocytes used herein, or may interfere only minimally. The term "interference" as used herein refers to the normal characteristics of hepatocytes, such as their biological activity. For example, only about 0%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 7%, 10%, 20%, 30%, or 40% of the fibroblasts and endothelial cells may possess the albumin secretion, metabolic clearance, induction, transporter activity, or other biological activity of the hepatocytes used in coculture. The fibroblasts and endothelial cells may have cell viability of about 70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater. The ratio of fibroblasts to endothelial cells, such as HUVECs, may range from about 1:10 to about 10:1, e.g., 1:1, 1:2, 2:1, or any other desired ratio, as evidenced by, for example, fibroblast markers (e.g., TE-7) and endothelial cell markers (e.g., CD31). Feeder cells may be free of mycoplasma and endotoxin.

[0037] As used herein, the term "co-culture" refers to a culture containing two or more cell types in culture medium. As used herein, the term "hepatocyte co-culture" refers to a culture containing hepatocytes, endothelial cells, and fibroblast feeder cells in culture medium. The cell ratio of hepatocytes to feeder cells can range from about 1:40 to about 40:1, and the cell ratio of fibroblasts to endothelial cells on feeder cells can range from about 1:10 to about 10:1. The cell ratio of hepatocytes to endothelial cells to fibroblasts can be 3:1:1, 6:1:1, 12:1:1, or 24:1:1. The cell ratio of hepatocytes to feeder cells and the cell ratio of fibroblasts to endothelial cells on feeder cells can be adjusted to improve the adhesion ability or lifespan of hepatocytes in co-culture.

[0038] Compositions are provided that include cells in a culture medium. The cells may consist of hepatocytes (e.g., human hepatocytes) and feeder cells, and may include endothelial cells and fibroblasts. The hepatocytes may be adherent. The hepatocytes may be functionally stable. The hepatocytes may have a cell life span.

[0039] Cells are typically cultured in media developed to promote desired cell characteristics (e.g., proliferation). Hepatocytes are typically cultured in hepatocyte seeding medium within a desired cell concentration range. Fibroblasts are typically cultured in fibroblast medium within a desired cell concentration range. Endothelial cells are typically cultured in endothelial cell medium within a desired cell concentration range. When hepatocytes are co-cultured with endothelial cells and fibroblasts, the hepatocyte seeding medium, fibroblast medium, and endothelial cell medium contribute to the co-culture seeding medium used to seed and / or co-culture the cells.

[0040] The composition of the co-culture seeding medium can be adjusted to achieve a desired cell ratio of hepatocytes to endothelial cells and fibroblasts. The co-culture seeding medium can include hepatocyte seeding medium, fibroblast medium, and endothelial cell medium in a volume ratio of about (5-30):(1-5):(1-5), e.g., about 10:1:1 to about 20:1:1. In one embodiment, the co-culture seeding medium can have a ratio of hepatocyte medium, endothelial cell medium, and fibroblast medium of 12:1:1, which is calculated by dividing 12 parts of hepatocyte medium by fibroblast medium. It represents one part hepatocyte seeding medium, one part endothelial cell medium and one part fibroblast medium.

[0041] Hepatocyte seeding medium may be DMEM (Dulbecco's Modified Eagle's Medium, Gibco 21063-029) supplemented with 10% heat-inactivated FBS (Gibco 10082-147), 1x NEAA (non-essential amino acid, Sigma M7145), 1 mM sodium pyruvate (Gibco 11360-070), 5 μg / mL insulin, and 5 μM dexamethasone.Hepatocyte seeding medium may be WEM (William's E Media, Gibco A1217601) supplemented with 10 mM HEPES (Fisher BP299100), 1x Glutamax (Gibco 35050061), 1x ITS-A (Gibco 51300-044), and 10 μM dexamethasone.

[0042] Endothelial cell medium can be purchased from LifeLine, Inc. The endothelial cell medium may be VasulLife Basal Medium, 5 ng / mL rh-FGF-β, 50 μg / mL ascorbic acid, 1 μg / mL hydrocortisone, 2% FBS, 10 mM L-glutamine, 15 ng / mL rh IGF-1, 5 ng / mL rh EGF, 5 ng / mL rh VEGF, and 0.75 U / mL heparin sulfate.

[0043] Fibroblast medium may be DMEM high glucose (Gibco 11995-065) supplemented with 10% FBS.

[0044] In one embodiment, at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100, e.g., at least 85%, of the hepatocytes are adherent in the presence of endothelial cells and fibroblasts. In another embodiment, less than 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10%, e.g., less than 60%, of the hepatocytes may be adherent in the absence of endothelial cells and fibroblasts.

[0045] The hepatocytes (e.g., human hepatocytes) in the composition can be seeded, and at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, e.g., 90%, of the seeded hepatocytes can remain viable and functional while attached to the surface for a predetermined period of time, e.g., at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, or 49 days, e.g., at least 7 or 42 days.

[0046] A product is provided that includes human hepatocytes seeded on a surface. At least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100, e.g., at least 70%, of the seeded hepatocytes are in one or more hepatocyte clusters on feeder cells. The feeder cells are endothelial cells or fibroblasts and are attached to the surface. At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100, e.g., at least 90%, of the seeded hepatocytes in one or more hepatocyte clusters may be in direct hepatocyte-cell contact. The one or more hepatocyte clusters may cover at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100, e.g., at least 50%, of the surface. The surface may have a shortest diameter of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 100 mm.

[0047] The product may further comprise a culture medium, which may be a mixture of hepatocyte seeding medium, endothelial cell medium, and fibroblast medium. At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100, e.g., at least 90%, of the seeded hepatocytes remain on the surface for at least 3, 4, 5, 6, 7, 14, 21, 28, or 42 days.

[0048] The seeded hepatocytes can exhibit one or more basic hepatocyte functions. For example, seeded hepatocytes can produce albumin and / or express cytochrome P450.

[0049] The endothelial cells may be human cells. For example, the endothelial cells are human umbilical vein endothelial cells (HUVECs). The endothelial cells may not be hepatocytes. The endothelial cells may not be proliferative. The endothelial cells may be primary cells or may be cultured for up to 3, 4, 5, 6, 7, 8, 9, 10, e.g., 7 passages. The endothelial cells may not be immortal.

[0050] The fibroblasts may be human cells. The fibroblasts may be human skin fibroblasts. The fibroblasts may not be hepatocytes. The fibroblasts may not be proliferative. The fibroblasts may be primary cells or may be cultured for up to 3, 4, 5, 6, 7, 8, 9, 10, e.g., 7 passages. The fibroblasts may not be immortal.

[0051] In the product, the seeded hepatocytes, endothelial cells and fibroblasts may have any cell ratio that improves the adhesion ability of hepatocytes, for example, 3:1:1, 6:1:1, 12:1:1 to 24:1:1.

[0052] The seeded hepatocytes can be obtained from one, two, or more donors, each of which may have a liver disease or disorder, such as microsteatosis and / or nonalcoholic steatohepatitis (NASH).

[0053] A method for preparing seeded human hepatocytes is provided. The method includes applying human hepatocytes to a surface in the presence of feeder cells, the feeder cells being endothelial cells and fibroblasts. After the applying step, co-culturing the applied hepatocytes with the feeder cells and forming one or more hepatocyte clusters by the co-cultured hepatocytes on the feeder cells attached to the surface. At least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, for example at least 70%, of the co-cultured hepatocytes are in one or more hepatocyte clusters.

[0054] The applied hepatocytes may have an adhesive capacity of less than 70%, 65%, 60%, 55%, 50%, 45%, or 40%, for example less than 60%, to a surface in the absence of feeder cells. The applied hepatocytes may have an adhesive capacity of at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, for example at least 85%, to a surface in the absence of feeder cells.

[0055] The one or more hepatocyte cell clusters may cover at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100, e.g., at least 50%, of the surface. The surface may have a shortest diameter of at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 100 mm. At least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100, e.g., at least 90%, of the seeded hepatocytes of the one or more hepatocyte cell clusters may be in direct hepatocyte-to-hepatocyte contact.

[0056] At least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%, e.g., at least 90%, of the seeded hepatocytes remain adherent, i.e., remain attached to the surface, in the presence of endothelial cells and fibroblasts for a predetermined period of time, e.g., at least 1, 2, 3, 4, 5, 6, 7, 14, 21, 28, 35, 42, or 49 days, e.g., at least 7 or 42 days. The hepatocytes may be co-cultured with the endothelial cells and fibroblasts for as little as 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day, e.g., as little as 3 days.

[0057] According to the preparation method of the present invention, endothelial cells and / or fibroblasts are attached to the surface of hepatocytes. The endothelial cells and / or fibroblasts can be attached to the surface before, during, or after the co-culture step.

[0058] The preparation method may exclude additional extracellular matrix proteins, which may be selected from the group consisting of collagen matrices, such as BioCoat, or rat tail collagen, HuGentra Matrix, MATRIGEL, and HuBiogel.

[0059] The preparation method further comprises freezing the seeded hepatocytes.

[0060] For each preparation method, the seeded hepatocytes prepared are provided.

[0061] A method for testing a pharmaceutical agent is provided. The method comprises administering to the seeded hepatocytes an amount of the pharmaceutical agent effective to alter the properties of the seeded hepatocytes. The seeded hepatocytes may be in the product of the invention or prepared according to the method of the invention. The pharmaceutical agent may be selected from the group consisting of a small molecule, an antibody, a live virus (hepatitis B or hepatitis C), a viral vector, an oligonucleotide, and a cell.

[0062] A method for testing drug metabolism is provided. The method includes administering an effective amount of a drug to seeded hepatocytes. The seeded hepatocytes may be in the product of the invention or may be prepared according to the method of the invention. As used herein, the term "drug metabolism" refers to the conversion or clearance of a drug. The method further includes determining the amount of the drug in the seeded hepatocytes.

[0063] Methods for testing drug transport are provided. The methods include administering an effective amount of a drug to seeded hepatocytes. The seeded hepatocytes may be in the article of manufacture of the invention or may be prepared according to the methods of the invention. The methods further include determining the cellular uptake and distribution of the drug in the seeded hepatocytes.

[0064] A method for testing the toxicity of a drug is provided. The method comprises administering an effective amount of the drug to seeded hepatocytes. The seeded hepatocytes may be in a product of the invention or prepared according to the method of the invention. The method further comprises detecting a toxic event. Detection of a toxic event can be evidenced by a decreased proportion of remaining viable seeded hepatocytes.

[0065] A method for preparing a hepatitis B virus (HBV)-infected hepatocyte culture model is provided. The method includes inoculating seeded hepatocytes with hepatitis B virus (HBV) and incubating the infected seeded hepatocytes for at least 7, 14, or 21 days, e.g., 14 days. An HBV-infected hepatocyte culture model is prepared. The seeded hepatocytes may be in the product of the present invention or prepared according to the method of the present invention. The method further includes determining the transcription or expression level of a liver-specific bile acid transporter, e.g., sodium-taurocholate cotransporter (NTCP), in hepatocytes, selecting a batch of hepatocytes with the desired transcription or expression level of NTCP, seeding the selected batch of hepatocytes according to the method of the present invention, and then inoculating the seeded hepatocytes with hepatitis B virus (HBV). Covering with a protein matrix may not be required in the present invention to increase viral inoculation efficiency. A suspension-grade hepatocyte batch with the desired NTCP level may be used for HBV inoculation and may be suitable for long-term culture studies.

[0066] In accordance with the present invention, a kit for seeding hepatocytes is provided. The kit includes a first cryopreservation vial containing primary hepatocytes, a second cryopreservation vial containing endothelial cells, and a third cryopreservation vial containing fibroblasts. The kit includes a cryopreservation vial and instructions for preparing human hepatocytes seeded with primary hepatocytes, endothelial cells, and fibroblasts according to the method of the present invention. The second cryopreservation vial and the third cryopreservation vial may be the same. The first cryopreservation vial, the second cryopreservation vial, and the third cryopreservation vial may be the same. The kit may include hepatocyte cryopreservation vials from a single donor or hepatocyte cryopreservation vials from multiple donors. The kit includes test results or a Certificate of Analysis (COA) for the hepatocyte co-culture system. The kit includes an instruction protocol for the preparation method. The kit includes media for thawing, seeding, and culturing hepatocytes, fibroblasts, and endothelial cells.

[0067] A method for preparing a hepatocyte co-culture system is provided. The method includes isolating hepatocytes from a donor's liver, adding fibroblasts and endothelial cells to the isolated hepatocytes, and freezing the hepatocytes mixed with the fibroblasts and endothelial cells. The method further includes growing the cells in a culture medium, wherein the cells consist of hepatocytes, fibroblasts, and endothelial cells. The donor may be an animal, preferably a human.

[0068] A ready-to-use hepatocyte co-culture system is provided. The co-culture hepatocyte system includes a tissue culture plate having culture wells with or without an ECM substrate coating, a fibroblast and endothelial cell layer attached to the surface of the culture well, and a hepatocyte layer attached to the fibroblast and endothelial cell or cell layer, forming hepatocyte clusters or hepatocyte islands. The hepatocyte clusters, also known as hepatocyte islands, self-organize after being seeded on or together with the fibroblasts and endothelial cells. Hepatocytes within the hepatocyte clusters or hepatocyte islands maintain direct hepatocyte-cell contact, and the hepatocytes produce hepatocyte-cell adhesion molecules, such as cadherins and connexins. More than 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the hepatocytes in the co-culture system are distributed in hepatocyte clusters and maintain direct hepatocyte-cell contact. More than 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the surface of the culture well is covered by hepatocyte clusters. The hepatocytes used herein can be derived from a single donor or multiple donors. The hepatocytes and endothelial cells of the co-culture system can form vessel-like structures during culture (Figure 27). [Example]

[0069] Example 1 Co-culture of human hepatocytes Human primary hepatocytes were co-cultured with human dermal fibroblasts and endothelial cells and characterized. In this example, suspension-grade hepatocytes had less than 70% adhesive capacity, while adherent-grade hepatocytes had at least 70% adhesive capacity.

[0070] 1. Method 1.1 Isolation and cryopreservation of human primary hepatocytes Human primary hepatocytes were isolated from human donors and then stored in liquid nitrogen.

[0071] 1.2 Source of human dermal fibroblasts and endothelial cells Human dermal fibroblasts were isolated from adult skin and characterized by TE-7 expression. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and characterized by CD31 and endothelial (VE)-cadherin expression. Immortalized human liver sinusoidal endothelial cells (SECs) were purchased from abm Inc. Both dermal fibroblasts and endothelial cells were frozen in freezing medium at passage #0 or #1 containing 10% DMSO (Sigma).

[0072] 1.3 Preparation of inert feeder cell layer for co-culture Adult dermal fibroblasts were grown to confluence in DMEM (Dulbecco's Modified Eagle's Medium, Gibco) high glucose supplemented with 10% fetal bovine serum. Both HUVECs and SECs were grown to confluence in endothelial cell growth medium purchased from Lonza. Cells were grown to a density of 10,000–15,000 cells / cm. Upon confluence, cells were passaged by enzymatic digestion with TrypLE (Gibco) or trypsin-EDTA (Gibco). Either enzyme was used to detach the cells from the tissue culture surface, forming a suspension of cells that could be counted. Cells in suspension were then seeded at a lower density by increasing the volume of the cell suspension and transferring the cell suspension to tissue culture plastic with a larger surface area than the one on which they were previously seeded. Cells were seeded at a density of 10,000–15,000 cells / cm. 2 Cells were seeded at a density of 1000 x g and passaged approximately every 5-7 days upon reaching confluency. Cells were passaged up to 5 times and then rendered mitotically inactive and frozen.

[0073] Because mitomycin C or gamma irradiation inactivates cells by causing double-strand breaks, mitomycin C was used to inactivate confluent fibroblasts and / or endothelial cells. Inactivated cells became mitotically inactive and did not proliferate. Briefly, cells were rinsed three times with Dulbecco's phosphate-buffered saline (DPBS, Gibco, Paisley, UK), incubated with 10 μg / ml mitomycin C (Sigma) for 3 hours at 37°C, and then rinsed three times with DPBS. After inactivation, fibroblasts and endothelial cells were immediately lysed with TrypLE for cell count and then resuspended in cryopreservation medium at a concentration of 1–2 million cells / ml. Cells were then aliquoted into cryovials and stored overnight at -80°C. The next day, cells were transferred to liquid nitrogen for long-term storage.

[0074] 1.4 Suspension and adherent grade human hepatocytes are used as inert feeder cells, endothelial cells, and Seeding the mixture of cells and fibroblasts Feeder cells consisting of human dermal fibroblasts and endothelial cells were thawed and plated at 25,000 cells / cm in either collagen-coated (BioCoat, Corning) or regular tissue culture plastic 24- or 96-well plates without matrix coating. 2 The density of the test feeder cells was 12,500 cells / cm. 2 ~100,000 cells / cm 2 It was.

[0075] Thirty minutes after seeding the feeder cells, primary human hepatocytes reached 150,000 cells / cm 2 Traditionally, hepatocytes are seeded on collagen-coated tissue culture plates at 250,000 cells / cm (schematic #1). 2Hepatocytes were seeded. The cell density of the hepatocytes was adjusted in the coculture system to allow for some hepatocyte interaction, which can explain the hepatocyte attachment rate. Alternatively, feeder cells and hepatocytes can be seeded together by mixing the hepatocytes and feeder cells into a uniform suspension and then seeding the feeder cells and hepatocytes together on a collagen-coated plate (schematic diagram #2). Four to six hours after seeding the hepatocytes, the medium was changed to remove unattached cells and cell debris. The incubation time or shaking conditions before changing the medium can be varied depending on the type of culture plate (e.g., 6-well, 12-well, 24-well, 48-well, 96-well, or 384-well plate).

[0076] 16-20 hours after hepatocyte seeding, excess cell matrix such as MATRIGEL was added to the medium to induce hepatocyte maturation. The culture platform was successful without the addition of excess cell matrix.

[0077] 1.5 Long-term culture of hepatocytes under co-culture conditions Traditional adherent hepatocyte monoculture models allow for up to 7 days of culture, allowing the analysis of high-grade hepatocytes, which begin to spread due to membrane disruption and lose their functionality. Adherent-grade and suspension-grade hepatocytes were seeded onto feeder cells in a co-culture model to study their morphology and function for up to 6 weeks. The medium was changed daily throughout the culture period. The hepatocyte medium consisted of HEPES (Fisher), Glutamine, and PEG-400. It contained aMAX (Gibco), ITS+ (insulin, transferrin, selenium complex, BSA and linoleic acid, Gibco), dexamethasone (Sigma), and sodium pyruvate (Gibco) in William's E medium.

[0078] Over a 6-week culture period, the co-cultured hepatocytes were fed daily with 12:1:1 HHCM medium, a mixture of hepatocyte medium, endothelial cell medium, and fibroblast medium in a volume ratio of 12:1:1, in tests of their typical cuboidal morphology, bile canalicular formation, CyP450 enzyme activity, and albumin and urea secretion.

[0079] 1.6 Eliminating donor-to-donor variability by pooling hepatocytes from multiple donors Hepatocytes vary in their adherence rate, morphology, protein expression, and enzyme functionality, largely due to the donor. Hepatocytes from multiple donors, regardless of suspension or adherent grade, were pooled to determine whether pooling reduced or eliminated some of these variations. Hepatocytes from 3 to 10 or more different donors were individually thawed, counted, and mixed together in equal amounts. Pooled hepatocytes and hepatocytes from each donor were cultured at 150,000 cells / cm. 2 The hepatocytes were pooled together before and after the hepatocytes were thawed.

[0080] 1.7 Characterization of hepatocytes in co-culture conditions Suspension or adherent grade hepatocytes at 25,000 cells / cm 2 The cells were seeded onto inactive feeder cells and co-cultured for 6 weeks. Their typical cuboidal and multinucleated hepatocyte morphology was visualized by efflux of 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFDA, Invitrogen) in the bile canaliculi over the 6-week culture period.

[0081] 1.8 CyP450 activity in co-cultured hepatocytes The CyP450 1A2, CyP450 2B6, and CyP450 3A4 activities of the co-cultured hepatocytes were analyzed using Promega's P450-Glo™ assay according to the manufacturer's instructions. Samples were induced for 48 hours with 100 μM omeprezole for CyP450 1A2, 100 nM CITCO for CyP450 2B6, and 25 μM rifampicin for CyP450 3A4.

[0082] When multi-week studies were performed, samples were allowed to be collected for 5 days, after which the medium was switched to induction medium for another 48 hours before analysis. Activity levels in induced and uninduced samples were analyzed for 6 weeks.

[0083] 1.9 Protein and Gene Expression Spent medium samples were collected and assessed for albumin and urea levels from co-cultured hepatocytes over a 6-week culture period. Gene expression levels for albumin, urea, CyP450 1A2, CyP450 2B6, and CyP450 3A4 were assessed weekly over a 6-week culture period. Co-cultured hepatocytes were fixed after 1, 2, and 6 weeks of culture and stained for albumin, CD31, CD90, CyP450 1A2, CyP450 2B6, and CyP450 3A4.

[0084] 1.10 Bile canaliculi formation The efflux of 5-(and-6)-carboxy-2',7'-dichloro-fluorescein diacetate (CDFDA) was visualized under a fluorescent microscope inside the co-cultured hepatocytes. The co-cultured hepatocytes were rinsed twice with DPBS (-Ca / -Mg) and then incubated in 3:1:1 HHCM at 37°C with 5% CO for 10 min, followed by incubation in 3:1:1 HHCM containing 5 μM CDFDA and 5% CO at 37°C for 2 min. The cells were then rinsed twice with DPBS (-Ca / -Mg) and imaged in complete medium without phenol red.

[0085] 2. Schematic of human hepatocyte co-culture in a mixture of endothelial cells and fibroblasts: analysis by cell culture and time frame 2.1 Schematic diagram #1 In scheme #1, human primary hepatocytes were seeded onto surfaces pre-seeded with a mixture of endothelial cells and fibroblasts as feeder cells (Figure 1).

[0086] Thirty to sixty minutes after seeding the feeder cells, hepatocytes were added to the feeder cells to establish hepatocyte co-cultures with hepatocytes, endothelial cells, and fibroblasts at a cell ratio of 3:1:1 in a seeding medium mixture of hepatocyte seeding medium, endothelial cell medium, and fibroblast medium (also known as 3:1:1 HHPM). The co-cultured cells were placed at 37°C in 5% CO2 and shaken four times every 15 minutes in a normal and then an abnormally wide (NEW) pattern for the first hour of hepatocyte co-culture.

[0087] Four to six hours after hepatocyte seeding, the seeding medium mixture was changed to a medium mixture of endothelial cell medium, fibroblast medium, and hepatocyte medium in a volume ratio of 3:1:1.

[0088] Twenty to 24 hours after hepatocyte seeding, the medium mixture was replaced with a fresh mixture of spent medium, hepatocyte seeding medium, endothelial cell medium, and fibroblast medium in a volume ratio of 3:1:1, also known as 3:1:1 HHCM. If an extracellular matrix, such as MATRIGEL, was diluted into the medium mixture and added to the cells, the medium mixture was subsequently replaced with fresh 3:1:1 HHCM daily.

[0089] Cytochrome P450 (CyP450) levels were determined on either day 4 or day 7 with Promega's P450Glo™ assay.

[0090] Bile canaliculi were visualized by CDFDA efflux on day 5 of the 1-week assay, which could be repeated multiple times over extended culture.

[0091] Protein expression was detected by immunocytochemical (ICC) staining and ELISA. Albumin and urea were detected and quantified using ELISA. ELISA was performed on 300 μl of spent medium collected after 24 hours of culture. Uninduced and induced samples were fixed for ICC at day 7 and various time points throughout long-term culture. Cell samples were also snap-frozen in Trizol for subsequent RNA extraction and qPCR analysis.

[0092] 2.2 Schematic diagram #2 In Scheme #2, human primary hepatocytes, endothelial cells, and fibroblasts were seeded together (Figure 2). Scheme #2 is identical to Scheme #1, except that non-expanding feeder cells were mixed with the human hepatocytes before freezing, then simultaneously revived, centrifuged, and resuspended at 100,000 cells / cm, depending on the intended application and grade of hepatocytes. 2 ~775,000 cells / cm 2 The cells were seeded onto collagen-coated tissue culture plastic plates at a density of 1000 x g.

[0093] 3. Co-cultured vs. mono-cultured hepatocytes Human primary hepatocytes were seeded onto a mixture of feeder cells, endothelial cells, and fibroblasts to establish a co-culture, as shown in Schematic #1 or 2, or seeded onto the surface in the absence of feeder cells to establish a monoculture. There were no significant differences in hepatocyte characteristics (e.g., morphology or gene expression). Under schematic diagram #1 or #2, co-cultured hepatocytes showed better hepatocyte characteristics (e.g., morphology or gene expression) than mono-cultured hepatocytes.

[0094] Co-cultured hepatocytes showed better culture lifespan and better hepatocyte morphology than mono-cultured hepatocytes (Figure 3).

[0095] Co-cultured hepatocytes showed significantly higher gene expression of CyP450 1A2, CyP450 2B6, and CyP450 3A4 than mono-cultured hepatocytes on day 7 (FIG. 4).

[0096] Co-cultured hepatocytes exhibited higher CyP450 1A2 and CyP450 2B6 activities after 48 hours of induction than mono-cultured hepatocytes on day 7. Furthermore, on day 7, co-cultured hepatocytes exhibited similar induced CyP450 3A4 activity as mono-cultured hepatocytes (Figure 5).

[0097] Co-cultured hepatocytes secreted significantly higher levels of albumin and urea during 2 weeks of culture, as measured by ELISA, compared with mono-cultured hepatocytes (FIG. 6).

[0098] 4. Improved Performance of Co-cultured Suspension-grade Human Primary Hepatocytes vs. Mono-cultured Adherent-grade Hepatocytes Hepatocytes characterized as suspension-grade cells are unable to maintain a confluent monolayer for multiple days. Adjusting the donor attachment rate by adding more cells does not improve hepatocyte attachment and confluence. Even when more hepatocytes are seeded and the attachment rate of a particular donor is adjusted, additional debris likely prevents cells from attaching. The inability of suspension-grade hepatocytes to attach prevents them from exhibiting typical hepatocyte morphology or function. The addition of a feeder cell layer consisting of fibroblasts and endothelial cells to hepatocyte cocultures improved the attachment, morphology, and functionality of suspension-grade hepatocytes (Figures 7-9).

[0099] The majority of co-cultured suspension-grade human primary hepatocytes remained adherent, whereas the majority of mono-cultured suspension-grade human primary hepatocytes did not attach or detached from the surface by day 4 (Figure 7).

[0100] Co-cultured hepatocytes also demonstrated improved cell function over mono-cultured hepatocytes. Co-cultured suspension-grade hepatocytes demonstrated improved CyP450 1A2 and CyP450 3A4 activity compared with mono-cultured suspension-grade hepatocytes on days 4 and 7, and maintained higher levels throughout day 7. CyP450 1A2 activity in co-cultured suspension-grade hepatocytes was similar to that of mono-cultured adherent-grade hepatocytes on days 4 and 7. CyP450 3A4 activity in co-cultured suspension-grade hepatocytes was higher than that of adherent-grade hepatocytes on days 4 and 7 (Figure 8). Co-cultured suspension-grade and adherent-grade hepatocytes demonstrated and maintained higher albumin and urea expression than mono-cultured suspension-grade and adherent-grade hepatocytes, as detected by ELISA. Co-cultured suspension-grade hepatocytes showed higher albumin and urea expression than mono-cultured adherent-grade hepatocytes on days 4 and 7 (Figure 9).

[0101] 5. Lifespan, Morphology, Marker Expression, Metabolic Activity, and Functional Bile Secretion of Co-cultured Hepatocytes The majority of monocultured suspension-grade human primary hepatocytes did not adhere to the surface, and those that did not began to detach from the surface after up to one week in culture. Hepatocytes initially attached to the surface with high efficiency but began to detach from the surface and lose their cuboidal morphology by day 7 of culture. Adherent hepatocytes did not remain attached for more than 11 days in culture (Figure 10).

[0102] Co-cultured suspension-grade hepatocytes were maintained for over 6 weeks. Cubic hepatocyte morphology was clearly maintained for hepatocytes that remained attached to small islets on a non-proliferating feeder layer of fibroblasts and endothelial cells for 6 weeks (Figure 11). Longer time points were not tested. Interactions between hepatocytes and feeder cells were visualized by albumin and CD31 staining. Hepatocytes integrated into the supportive feeder layer as a complex network of cells. Hepatocyte function was maintained in this network, as visualized by albumin and CyP450 3A4 staining (Figure 12). CyP450 3A4 activity of co-cultured suspension-grade hepatocytes was maintained for 6 weeks. The high activity of co-cultured suspension-grade hepatocytes contrasted with the low CyP450 3A4 activity of the same hepatocytes cultured as sandwich monocultures (Figure 13). Co-cultured suspension-grade hepatocytes expressed and maintained a functional network of bile canaliculi during 6 weeks of culture (FIG. 14).

[0103] 6. Lifespan of co-cultured hepatocytes: Induction of hepatocyte maturation in co-culture with or without MATRIGEL Co-cultured hepatocytes maintained their morphology, function, or lifespan without the need for MATRIGEL or any other extracellular matrix. The morphology of suspension-grade hepatocytes co-cultured with and without MATRIGEL was very similar over 1 to 6 weeks (Figures 15 and 16). Hepatocytes with and without MATRIGEL formed networks with small cellular islands and their feeder layers. The interaction between feeder cells and hepatocytes was visualized by ICC after 6 weeks of culture, and bile canaliculi were observed for 6 weeks (data not shown). No differences in morphology were observed between hepatocytes cultured with or without MATRIGEL. The presence of MATRIGEL did not affect the functionality of co-cultured hepatocytes. Albumin and urea concentrations and CyP450 3A4 activity remained constant over 6 weeks of co-cultured hepatocytes with and without MATRIGEL (Figures 17 and 18).

[0104] Monocultured and cocultured hepatocytes from different donors showed significant variations in morphology, protein expression, and enzyme activity. Hepatocytes from multiple donors were pooled together to normalize these variations. The attachment rate, confluency, and lipid accumulation of pooled hepatocytes from three donors in the coculture system appeared normalized (Figures 15 and 16). CyP450 3A4 enzyme activity of pooled hepatocytes appeared average or slightly higher than average when compared with hepatocytes from individual donors (Figure 19). The normalizing effect of pooling hepatocytes from three or more donors is useful for in vitro testing applications.

[0105] The feeder layer may consist of multiple types of endothelial cells and fibroblasts. The morphology and function of human hepatocytes co-cultured on feeder cell layers containing sinusoidal endothelial cells or HUVECs were compared. No significant differences were observed in morphology (Figure 20), CyP450 activity (Figure 21), or albumin and urea secretion (Figure 22).

[0106] 7. Optimal Ratio of Co-cultured Hepatocyte Cell Mixture 12,500 cells / cm 2 ~100,000 cells / cm 2 Feed layer densities of 12,500 cells / cm were tested in the co-culture system. The morphology of individual hepatocytes at different feeder cell densities appeared similar, but lower feeder cell densities allowed more hepatocyte-cell interactions (Figure 23). 2 (24:1:1) or 25,000 cells / cm 2 Hepatocytes co-cultured at a feeder cell density of (12:1:1) were cultured at 50,0 00 cells / cm 2 (6:1:1) or 100,000 cells / cm 2 Hepatocytes at a higher density (3:1:1) showed higher CyP450 1A2 and CyP450 3A4 activities (FIG. 24).

[0107] 8. Multiple Types of Culture Plates for Hepatocyte Co-culture Hepatocytes co-cultured on endothelial cells and fibroblasts in either 24- or 96-well plates exhibited similar characteristics, including morphology (FIG. 25, left and center panels), levels of albumin secretion (FIG. 26), and biomarker expression (FIG. 27). Hepatocytes co-cultured on non-collagen-coated standard tissue culture plastic exhibited similar morphology (FIG. 25, right panel) and biomarker expression patterns (FIG. 27) compared to hepatocytes co-cultured on collagen-coated BioCoat plates.

[0108] Although only suspension-grade hepatocytes were co-cultured, it is expected that adherent-grade hepatocytes will maintain similar high-grade morphology and function for over 6 weeks. It is expected that higher-grade cells may even develop more complex networks with each other and with feeder cells more rapidly.

[0109] Example 2 HBV infection model (NTCP expression) NTCP (sodium / taurocholate cotransporter) expression is required for HBV uptake by hepatocytes. Co-cultured hepatocytes were found to express NTCP on days 7, 11, 14, 19, and 21, as detected by immunocytochemistry. Cells were fixed, permeabilized, and incubated with primary antibody SLC10A1 (Abcam 131084) at a concentration of 1:100 overnight at 4°C. Secondary antibody Alexa Fluor 488 goat anti-rabbit (Invitrogen A11008) was incubated at a concentration of 1:1000 for 1 hour at 4°C to detect the primary antibody. Cells were counterstained with Hoechst. NTCP expression was quantified by whole-well fluorescence scanning at 483 nm and 530 nm on a BMG CLARIOstar (Figure 25). Thus, NTCP expression was maintained by co-cultured hepatocytes for 2 weeks.

[0110] Example 3 Optimal ratios for cells and medium Feeder cell seeding densities of 12,500, 25,000, 50,000, and 100,000 cells per square centimeter were tested for hepatocyte attachment and cluster formation. Hepatocyte seeding densities of 150,000, 250,000, and 375,000 cells per square centimeter were also tested. The ideal cell ratios for media ratios of hepatocyte medium, endothelial cell medium, and fibroblast medium were also tested. Feeder cell ratios of 12,500 to 25,000 cells per square centimeter and hepatocyte seeding densities of 150,000 cells per square centimeter demonstrated the highest attachment rates, cluster formation, and hepatocyte function. Media ratios matching the cell ratios were also found to best support hepatocyte morphology and function.

[0111] Example 4 Hepatocyte Clusters A layer of feeder cells, consisting of fibroblasts and endothelial cells, attached to the tissue culture plastic surface within minutes after seeding. The feeder cells were seeded at a low enough density to allow the feeder cells to spread, migrate, and cover the tissue culture plastic surface. Hepatocytes then attached to the top of the feeder cells and migrated toward each other, forming clustered hepatocytes. This hepatocyte migration occurred within the first hour or up to 48 hours, depending on the characteristics of the hepatocytes. Once hepatocyte clusters formed, adjacent hepatocytes formed bile canaliculi and tight junctions. Bile canaliculi were visualized by CDFDA efflux starting on day 4. Junctional proteins occludin 1 and connexin-32 were visualized on day 7 using immunocytochemistry. At least 90% of the hepatocytes in the clusters formed tight junctions. Gap junctions are shown, indicating direct hepatocyte-cell contact.

[0112] Example 5 Cluster Measurements Adherent quality of hepatocytes was assessed by analyzing the nuclear features of the total area and areas not occupied by hepatocyte clusters relative to the total cell area. Suspension quality of hepatocytes was assessed by analyzing the area occupied by clusters relative to the total cell culture area. Feeder cells and hepatocytes were differentiated by their respective nuclear sizes. Fibroblasts and endothelial cells have larger average nuclear sizes than hepatocytes. The average nuclear size of feeder cells was established by measuring the nuclear size of feeder cells alone in co-cultures. A threshold of the mean feeder cell size + one standard deviation was used to distinguish hepatocytes from feeder cells. The threshold used was a hepatocyte nuclear area of ​​less than 345 pixels^2. All measurements were taken in ImageJ (Figure 26).

[0113] All documents, books, manuals, articles, patents, filed patent applications, guides, abstracts, and / or other references mentioned herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A product comprising a tissue culture surface or cell culture surface, feeder cells and human hepatocytes, wherein the product comprises human hepatocytes seeded on feeder cells attached to the tissue culture surface or cell culture surface, at least 70% of the seeded hepatocytes being one or more hepatocyte clusters on the feeder cells, and the feeder cells being endothelial cells and fibroblasts.

2. 10. The article of manufacture of claim 1, wherein at least 90% of the seeded hepatocytes in the one or more hepatocyte clusters are in direct hepatocyte-to-hepatocyte contact.

3. 10. The article of manufacture of claim 1, wherein the one or more hepatocyte clusters cover at least 50% of the tissue culture or cell culture surface.

4. 10. The article of manufacture of claim 1, wherein the tissue culture or cell culture surface has a shortest diameter of at least 3 mm.

5. 10. The article of manufacture of claim 1, further comprising a culture medium, wherein at least 90% of the plated hepatocytes remain on the feeder cells attached to the tissue culture surface or cell culture surface for at least 7 days.

6. 10. The article of manufacture of claim 1, further comprising a culture medium, wherein at least 90% of the seeded hepatocytes remain on the feeder cells attached to the tissue culture surface or cell culture surface for at least 42 days.

7. 10. The article of manufacture of claim 1, wherein the seeded hepatocytes produce albumin.

8. 10. The article of manufacture of claim 1, wherein the seeded hepatocytes express cytochrome P450.

9. The article of manufacture of claim 1 , wherein the endothelial cells are human cells.

10. 10. The article of manufacture of claim 1, wherein the endothelial cells are human umbilical vein endothelial cells (HUVECs). 。

11. The article of manufacture of claim 1 , wherein the endothelial cells are not hepatocytes.

12. The article of manufacture of claim 1 , wherein the endothelial cells do not proliferate.

13. 10. The article of manufacture of claim 1, wherein the endothelial cells are primary cells or have been cultured for up to 7 passages.

14. The article of manufacture of claim 1 , wherein the endothelial cells are not immortal.

15. 2. The article of manufacture of claim 1, wherein the fibroblast cells are human cells.

16. 2. The article of manufacture of claim 1, wherein the fibroblasts are human dermal fibroblasts.

17. The article of manufacture of claim 1 , wherein the fibroblasts are not hepatocytes.

18. The article of manufacture of claim 1 , wherein the fibroblasts do not proliferate.

19. 10. The article of manufacture of claim 1, wherein the fibroblasts are primary cells or have been cultured for up to 7 passages.

20. The article of manufacture of claim 1 , wherein the fibroblasts are not immortal.

21. 2. The article of manufacture of claim 1, wherein the seeded hepatocytes, endothelial cells, and fibroblasts have a cell ratio of 3:1:1 to 24:1:

1.

22. 10. The article of manufacture of claim 1, wherein the seeded hepatocytes are obtained from a single donor.

23. 23. The article of manufacture of claim 22, wherein the donor is suffering from microsteatosis.

24. 23. The article of manufacture of claim 22, wherein the donor has nonalcoholic steatohepatitis (NASH).

25. 10. The article of manufacture of claim 1, wherein the seeded hepatocytes are obtained from two or more donors.

26. (a) applying human hepatocytes to a tissue culture surface or a cell culture surface in the presence of feeder cells, wherein the feeder cells are endothelial cells and fibroblasts; (b) after step (a), co-culturing the applied hepatocytes with the feeder cells; and (c) forming one or more hepatocyte clusters on the feeder cells by the co-cultured hepatocytes, wherein the feeder cells are attached to the tissue culture surface or cell culture surface, and at least 85% of the co-cultured hepatocytes are in one or more hepatocyte clusters, thereby preparing seeded human hepatocytes.

27. 27. The method of claim 26, wherein in step (a), the applied hepatocytes have an adhesive capacity of less than 60% on the tissue culture surface or cell culture surface in the absence of feeder cells.

28. In step (a), the applied hepatocytes are cultured in the tissue in the absence of feeder cells.

27. The method of claim 26, wherein the culture surface or cell culture surface has an adhesion capacity of at least 85%.

29. 27. The method of claim 26, wherein the one or more seeded hepatocyte cell clusters cover at least 50% of the tissue culture or cell culture surface.

30. 27. The method of claim 26, wherein the tissue culture or cell culture surface has a shortest diameter of at least 3 mm.

31. 27. The method of claim 26, wherein at least 90% of the plated hepatocytes remain on the feeder cells attached to the tissue culture surface or cell culture surface for at least 7 days.

32. 27. The method of claim 26, wherein at least 90% of the seeded hepatocytes remain on the feeder cells attached to the tissue culture surface or cell culture surface for at least 42 days.

33. 27. The method of claim 26, wherein the feeder cells are attached to the tissue culture surface or cell culture surface prior to step (a).

34. 27. The method of claim 26, wherein the feeder cells are attached to the tissue culture surface or cell culture surface in step (b).

35. 27. The method of claim 26, wherein the method excludes the addition of extracellular matrix proteins.

36. 27. The method of claim 26, wherein the hepatocytes are frozen prior to step (a).

37. 26. A method for testing a pharmaceutical agent comprising administering to seeded hepatocytes of the product of any one of claims 1 to 25 an amount of the pharmaceutical agent effective to alter the properties of the seeded hepatocytes.

38. 38. The method of claim 37, wherein the pharmaceutical agent is selected from the group consisting of a small molecule, an antibody, a live virus, a viral vector, an oligonucleotide, and a cell.

39. 26. A method of testing drug metabolism comprising administering an effective amount of a drug to the seeded hepatocytes of the product of any one of claims 1 to 25 and determining the amount of the drug in the seeded hepatocytes.

40. 26. A method of testing drug transport comprising administering an effective amount of a drug to the seeded hepatocytes of the article of manufacture of any one of claims 1 to 25 and determining the location of the drug in the seeded hepatocytes.

41. 26. A method for testing the toxicity of a drug, comprising administering an effective amount of the drug to the seeded hepatocytes of the product of any one of claims 1 to 25, and detecting surviving seeded hepatocytes.

42. 1. A method for preparing a hepatitis B virus (HBV)-infected hepatocyte culture model, comprising: (a) inoculating the seeded hepatocytes of the product of any one of claims 1 to 25 with Hepatitis B virus (HBV); (b) incubating the infected seeded hepatocytes for at least 14 days, thereby preparing the HBV-infected hepatocyte culture model.

43. A kit for seeding hepatocytes, comprising: (a) a first cryopreservation vial containing primary hepatocytes; (b) a second cryopreserved vial containing endothelial cells having 0% to 10% interference with the biological activity of said hepatocytes; (c) a third cryopreservation vial containing fibroblasts having 0% to 10% interference with the biological activity of the hepatocytes; and (d) instructions for preparing seeded human hepatocytes using the primary hepatocytes, the endothelial cells, and the fibroblasts according to the method of any one of claims 26 to 36; The kit.

44. 44. The kit of claim 43, wherein the second cryopreservation vial and the third cryopreservation vial are the same.

45. 44. The kit of claim 43, wherein the first cryopreservation vial, the second cryopreservation vial, and the third cryopreservation vial are the same.

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