Medium for forming bile canaliculi and method for producing bile canaliculi using the same

Abstract

To provide a medium that enables easy formation of bile canaliculi without overlaying an extracellular matrix such as Matrigel(R).SOLUTION: The present invention relates to a medium for culturing hepatocytes to form bile canaliculi, the medium comprising, as an active ingredient, a compound that activates K-Ras protein, and to a method for producing bile canaliculi, the method comprising a step of culturing hepatocytes in the medium.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a medium for preparing bile canaliculi and a method for producing bile canaliculi using the medium. [Background technology]

[0002] The bile canaliculi, which function as a pathway for excreting bile produced in the liver, are also one of the major routes of elimination of drugs. Drugs can inhibit bile excretion, causing it to remain in hepatocytes (cholestasis), leading to drug-induced liver injury, which is one of the major reasons for drug development discontinuation. Therefore, evaluation of bile excretion and risk of cholestasis of candidate compounds are carried out during drug development.

[0003] The above evaluation involves in vitro testing using cells (human primary cultured hepatocytes, iPS-derived hepatocytes, etc.), but it is generally known that bile canaliculi do not form in conventional culture (Non-Patent Document 1). Therefore, conventional bile canaliculi formation methods use the sandwich (SW) culture method, in which hepatocytes are sandwiched between extracellular matrices, and the most common method is Matrigel® SW culture, in which Matrigel® is layered on top of hepatocytes on collagen I (Non-Patent Documents 1 and 2, Patent Documents 1 to 3).

[0004] The present inventors have developed a method for improving the initial adhesion of hepatocytes by culturing them in a medium containing cAMP, a cAMP analog, or a substance that increases the cAMP concentration, thereby activating the cAMP signal (Patent Document 4). Furthermore, they have confirmed that bile canaliculi can be formed by SW culture of hepatocytes using the developed medium (Non-Patent Document 3). Regarding the adhesiveness of hepatocytes, it has been reported that oncostatin M (OsM) activates K-Ras protein to form adherens junctions (Non-Patent Document 4).

[0005] Because SW culture requires an extracellular matrix (typically, Matrigel®) overlay, there are concerns that test drugs may not be able to reach hepatocytes. Furthermore, Matrigel® is derived from mouse sarcoma, resulting in unclear composition and large lot-to-lot variations. It also raises concerns about infection by pathogenic substances and immunogenicity. Furthermore, it gels at room temperature, making it difficult to handle. Therefore, there is a need for a method of forming bile canaliculi without an extracellular matrix overlay (without using Matrigel®).

[0006] Techniques for forming bile canaliculi without using SW culture include a report that bile canaliculi can be formed using a hepatocyte culture device that promotes the accumulation and excretion of hepatic metabolites in bile canalicular-like structures by co-culturing a culture containing multiple hepatocytes (first cell culture) with a cell culture (second cell culture) that can increase the excretion activity of hepatic metabolites in the culture (Patent Document 5), and a report that bile canaliculi can be formed by co-culturing HeLa cells (HeLa / CLDNs) that have been forced to express claudins (CLDNs), which are involved in bile canalicular formation, with HepG2 cells (human liver cancer cell line, HepG2 / CLDNs) (Non-Patent Document 5).

[0007] However, these techniques that do not use SW culture require special culture equipment or co-culture, and therefore are not necessarily sufficient from the viewpoint of practicality. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Japanese Patent Application Laid-Open No. 2013-117411 [Patent Document 2] Special Publication No. 2003-502016 [Patent Document 3] Special Publication No. 2008-503204 [Patent Document 4] International Publication No. 2022 / 138922 [Patent Document 5] International Publication No. 2016 / 158417 [Non-patent literature]

[0009] [Non-Patent Document 1] B. Swift et al., “Sandwich-Cultured Hepatocytes: An In Vitro Model to Evaluate Hepatobiliary Transporter-Based Drug Interactions and Hepatotoxicity”, Drug Metab Rev. 2010, 42(3), p446-471. [Non-patent document 2] LeCluyse et al., “Formation of extensive canalicular networks by rat hepatocytes cultured in collagen-sandwich configuration”, Am J Physiol Cell Physiol, 1994, vol.266, p1764-1774. [Non-patent document 3] Helena, GA et al., “Activation of cAMP (EPAC2) signaling pathway promotes hepatocyte attachment”, Sci Rep 13, 12352 (2023). [Non-patent document 4] Matsui, T et al., “K-Ras mediates cytokine-induced formation of E-cadherin-based adherens junctions during liver development”, EMBO J., 2002, 21, p.1021-1030 [Non-Patent Document 5] Hiroshi Arakawa et al., “Induction of open-form bile canaliculus formation by hepatocytes for evaluation of biliary drug excretion”, Commun. Biol., 2023, 22; 6(1) Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide a culture medium for easily forming bile canaliculi without overlaying an extracellular matrix such as Matrigel (registered trademark). [Means for solving the problem]

[0011] In the course of intensive research to solve the above-mentioned problems, the inventors discovered that culturing hepatocytes in a medium containing as an active ingredient a compound that activates K-Ras protein can promote the formation of bile canaliculi without the need for an extracellular matrix overlay such as Matrigel (registered trademark). As a result of further research, they have completed the present invention.

[0012] The present invention relates to the following inventions. [1] A medium for culturing hepatocytes to form bile canaliculi, the medium comprising, as an active ingredient, a compound that activates K-Ras protein. [2] The medium described in [1] above, wherein the compound that activates the K-Ras protein is oncostatin M (OsM), an interleukin (IL)-6 family cytokine, or a guanine nucleotide exchange factor (GEF) for K-Ras. [3] The medium according to [1] or [2], which does not contain albumin or contains less than 0.1 w / v% albumin. [4] The medium according to any one of [1] to [3] above, which does not contain one or more selected from the group consisting of bovine serum albumin (BSA), 6-bromoindirubin-3'-oxime (BIO), and calcitriol. [5] The medium according to any one of [1] to [4] above, which does not contain any of cAMP, cAMP analogs, and substances that increase intracellular cAMP concentrations. [6] A method for producing bile canaliculi, comprising the step of culturing hepatocytes in the medium according to any one of [1] to [5] above. [7] The method for production described in [6] above, wherein the hepatocytes are primary cultured hepatocytes. [8] The method according to [6] or [7], further comprising changing the culture medium 1 to 8 hours after the start of culture. [9] The method for producing a hepatocyte according to any one of [6] to [8] above, which does not include a step of layering solubilized basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma on hepatocytes.

[10] The method for producing the present invention according to any one of [6] to [8] above, which does not include a step of layering an extracellular matrix on the hepatocytes. [Effects of the Invention]

[0013] The bile canaliculi of hepatocytes cultured using the medium of the present invention have superior functionality to bile canaliculi formed by the Matrigel® sandwich (SW) culture method (conventional method) using a commonly used commercially available medium. In particular, the use of the medium of the present invention results in the formation of more elongated bile canaliculi than by the conventional method.

[0014] The method using the medium of the present invention allows the formation of bile canaliculi, essential for bile excretion and stasis tests, without the need for an extracellular matrix overlay such as Matrigel®, thereby eliminating the risk of drugs being trapped in the extracellular matrix before reaching the hepatocytes. Therefore, using bile canaliculi formed by the method of the present invention allows for more reliable data to be obtained than with cell models constructed using conventional techniques. Furthermore, the absence of an extracellular matrix such as Matrigel® clarifies the medium composition, facilitating mechanistic analysis and contributing to further research in this field. Furthermore, in addition to significantly simplifying the culture process, the absence of Matrigel® also offers the benefit of reduced costs. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows MRP2 immunostained images of hepatocytes cultured up to day 7 (SW culture and conventional culture) in the medium of the present invention and in each of the commercially available media A to C. [Figure 2] FIG. 2 shows phase contrast microscopic images and fluorescence microscopic images of CDFDA accumulation in bile canaliculi on day 7 of culture in the medium of the present invention (normal culture) and commercially available media A and B (SW culture). [Figure 3] FIG. 3 shows phase contrast microscopic images and fluorescence microscopic images of CDFDA accumulation in bile canaliculi on the fourth day of culture in the medium of the present invention (normal culture) and commercially available medium D (normal culture and SW culture). [Figure 4] FIG. 4 shows phase-contrast microscopic images (top row) and the accumulation of CDFDA in bile canaliculi (fluorescence microscopic images; bottom row) on the fourth day of culture in the medium of the present invention and in the medium obtained by removing one or two components from the medium. [Figure 5] Figure 5 shows the accumulation of CDFDA in the bile canaliculi (fluorescence microscopy images) of hepatocytes cultured up to day 4 (normal culture) in the medium of the present invention minus OsM (negative control), the medium of the present invention (positive control), and the negative control medium plus IL-6, LIF, CNTF, or Ras-GRF. DETAILED DESCRIPTION OF THE INVENTION

[0016] The medium of the present invention is a medium for culturing hepatocytes to form bile canaliculi, and contains a compound that activates K-Ras protein as an active ingredient. The hepatocytes are preferably derived from humans, but may also be derived from animals other than humans, such as mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, cows, horses, goats, and monkeys.

[0017] The hepatocytes used may be hepatocytes differentiated or induced from pluripotent stem cells or hepatocytes obtained by further maturing such hepatocytes, but preferably, hepatocytes collected from a living body, such as primary cultured hepatocytes, are used. Here, the meaning of "primary cultured hepatocytes" can be interpreted as the meaning normally used by those skilled in the art, but "primary cultured hepatocytes" can also be defined as hepatocytes that are first seeded and cultured with tissues or cells collected from a living body. Primary cultured hepatocytes are classified into plateable type and suspension type, and either type of primary cultured hepatocytes may be used in the present invention.

[0018] Compounds that activate K-Ras protein include, but are not limited to, oncostatin M (OsM), interleukin (IL)-6 family cytokines, and K-Ras guanine nucleotide exchange factors (GEFs). IL-6 family cytokines include, for example, IL-6, IL-11, IL-27, IL-35, IL-39, leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), cardiotrophin 1 (CT-1), and cardiotrophin-like cytokine factor 1 (CLCF1). K-Ras GEFs include, for example, CDC25, SDC25, Ras-GRF, and SOS. OsM or IL-6 family cytokines are particularly preferred.

[0019] The medium of the present invention contains a compound that activates K-Ras protein in a general basal medium. Examples of basal media include BME medium, BGjB medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM medium, IMDM medium, Medium 199 medium, Eagle's MEM medium, αMEM medium, DMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, William's E medium, and mixtures thereof, but are not particularly limited as long as they can be used to culture animal cells. These media are commercially available.

[0020] The medium of the present invention may contain other additives, such as lipids, amino acids (e.g., non-essential amino acids), vitamins, sugars, hormones (e.g., dexamethasone), growth factors (e.g., HGF), cytokines, insulin, transferrin, antioxidants, serum replacements, 2-mercaptoethanol, pyruvate, dimethyl sulfoxide (DMSO), adenylyl cyclase activators (e.g., forskolin), Epac activators (e.g., Sp-8-BnT-cAMPS (S-220)), buffers (e.g., HEPES), inorganic salts, antibiotics (e.g., penicillin and streptomycin), or antibacterial agents (e.g., amphotericin B).

[0021] In one embodiment, the medium of the present invention contains no albumin or less than 0.1 w / v% albumin. Limiting the amount of albumin in the medium can improve hepatocyte adhesion and maintain or improve hepatocyte function. The albumin concentration in the medium should be less than 0.1 w / v%, but is preferably 0.03 w / v% or less, and more preferably 0.01 w / v% or less. Here, hepatocyte function refers to, for example, the activity of enzymes such as cytochrome P450 (e.g., CYP3A4).

[0022] In one embodiment, the medium of the present invention does not contain one or more selected from the group consisting of bovine serum albumin (BSA), 6-bromoindirubin-3'-oxime (BIO) and calcitriol. Here, bovine serum albumin (BSA) also includes bovine serum albumin (fatty acid-free) (BSA-FAF). These components are not found to contribute to bile canaliculus formation, so they do not need to be essential components in the medium of the present invention.

[0023] In one embodiment, the medium of the present invention does not contain cAMP, cAMP analogs, or substances that increase intracellular cAMP concentrations. These components are not found to contribute to bile canalicular formation, and therefore do not need to be essential components of the medium of the present invention. Examples of cAMP analogs include cAMPS-Sp, 6-Bnz-cAMP, 8-Bromo-cAMP, Dibutyryl-cAMP, 8-CPT-2Me-cAMP, and 8-pCPT-2-O-Me-cAMP-AM.

[0024] Substances that increase intracellular cAMP levels include phosphodiesterase inhibitors and adenylyl cyclase activators. Phosphodiesterase inhibitors include IBMX (3-isobutyl-1-methylxanthine), theophylline, papaverine, caffeine, rolipram, sildenafil, milrinone, cilostazol, vinpocetine, theobromine, and resveratrol. Adenylyl cyclase activators include forskolin.

[0025] The method for producing bile canaliculi of the present invention comprises the step of culturing hepatocytes in the above-described medium of the present invention. In one embodiment of the method for producing bile canaliculi of the present invention, the hepatocytes are primary cultured hepatocytes. In the culture method of the present invention, it is preferable to change the medium 1 to 8 hours after the start of culture. Such a change of the medium can improve the adhesiveness of hepatocytes and maintain or improve the function of hepatocytes. The timing of the change of the medium may be any time between 1 and 8 hours after the start of culture, but is preferably between 3 and 5 hours, and more preferably between 3.5 and 4.5 hours after the start of culture.

[0026] The culture method of the present invention is usually carried out in a CO2 incubator at a culture temperature suitable for culturing hepatocytes (usually 30 to 40°C, preferably about 37°C). The specific culture period is usually 0.5 to 30 days, preferably 1 to 14 days, and more preferably 3 to 7 days.

[0027] In one embodiment, the method for producing bile canaliculi of the present invention does not include the step of layering solubilized basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma on hepatocytes, such as Matrigel® (Corning). In one embodiment, the method for producing bile canaliculi of the present invention does not include a step of layering extracellular matrix on hepatocytes, such as solubilized basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma or collagen.

[0028] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. [Example]

[0029] 1. Materials and Methods [Preparation of the medium of the present invention] The medium of the present invention was prepared according to the medium composition shown in Table 1, using William's E Cica-modified 2 (BioConcept) as the basal medium. [Table 1]

[0030] [Regular hepatocyte culture] Frozen stocks of human primary hepatocytes (PHH) were thawed using a commercially available thawing medium, and cell numbers were measured using trypan blue. PHH were plated at 1.0 × 10 cells / well onto a collagen I-coated 96-well plate. 5 The cells were seeded using the medium of the present invention, commercially available medium A, commercially available medium B, or commercially available medium C at a seeding density of 100 cells / well and a medium volume of 75 μL / well, and cultured at 37°C under 5% CO2 conditions. Four hours after seeding, and one day, two days, and four days later, the medium was completely replaced with each medium to give a medium volume of 75 μL / well, and the cells were cultured until day 7 of culture.

[0031] [Culture of hepatocytes derived from human liver chimeric mice] The seeding procedure for human liver chimeric mouse-derived hepatocytes (HepaSH®) was performed according to the manufacturer's recommended protocol. 1.0 × 10 cells were seeded onto a collagen I-coated 96-well plate. 5 Cells were seeded in the medium of the present invention or the medium recommended by the commercial manufacturer at a seeding density of 100 cells / well, with a medium volume of 75 μL / well, and cultured under normal conditions at 37°C and 5% CO2. Four hours, one day, two days, three days, and four days after seeding, the entire medium was replaced with the medium of the present invention or commercially available medium D to a volume of 75 μL / well, and the cells were cultured until day 4. For SW culture conditions, one day after seeding, commercially available medium D containing Matrigel® at a final concentration of 0.25 mg / mL was used, and 4 hours, two days, three days, and four days after seeding, a medium replacement was performed with commercially available medium D without Matrigel® at a volume of 75 μL / well.

[0032] [Preparation of Matrigel®-containing medium] Matrigel (registered trademark) was added to the medium of the present invention, commercial medium A, commercial medium B, and commercial medium C at a final concentration of 0.25 mg / mL.

[0033] [Matrigel® Sandwich (SW) Culture of Hepatocytes] Frozen stocks of PHH were thawed using a commercially available thawing medium, and the cell number was measured using trypan blue. 1.0 × 10 PHH were placed in a collagen I-coated 96-well plate. 5 The cells were seeded using the medium of the present invention, commercial medium A, commercial medium B, or commercial medium C at a seeding density of 100 cells / well and a medium volume of 75 μL / well. Four hours after seeding, the medium was replaced with each medium, and one day later, the medium was replaced with Matrigel®-containing medium (75 μL / well) for each condition. Subsequently, after two and four days of culture, the medium was replaced with the medium of the present invention, commercial medium A, commercial medium B, or commercial medium C without Matrigel® at a medium volume of 75 μL / well.

[0034] [Confirmation of bile acid excretion transporter expression by immunohistochemistry] On day 7 of culture, the medium was removed, 4% paraformaldehyde (PFA) was added at 100 μL / well, and the cells were incubated at room temperature for 15 minutes before washing three times with PBS(-) (cell fixation). 1% Triton-X 100 was added at 100 μL / well, and the cells were incubated at room temperature for 10 minutes before washing once with PBS(-). Blocking buffer was added at 200 μL / well, and the cells were incubated at room temperature for 1 hour. The blocking buffer was then removed, and anti-MRP2 antibody was added at 100 μL / well. The following day, the primary antibody solution was removed, and the cells were washed three times with PBS(-). Secondary antibody was added at 100 μL / well and incubated at room temperature for 2 hours in the dark. After incubation, the cells were washed three times with PBS(-) and observed under a fluorescent microscope.

[0035] [Excretion of fluorescent substrates into bile canaliculi by bile acid excretory transporters] On day 7 of culture, the medium was removed, and 100 μL / well of 4 μmol / L CDFDA solution was added. The cells were then incubated for 10 minutes in a CO2 incubator (5% CO2, 37°C). After washing three times with HBSS(+), 75 μL / well of the medium for each condition was added. The cells were then incubated for 10 minutes in a CO2 incubator (5% CO2, 37°C) and observed under a fluorescent microscope.

[0036] [Verification of the effect of each component of the medium of the present invention on bile canaliculi formation] Eight types of medium were prepared using the medium of the present invention and the medium of the present invention minus six major components (HGF, OsM, DEX, Frk, DMSO, and S-220) (excluding two components). Standard culture was performed using each medium. On day 4 of culture, the medium was removed, and 100 μL / well of 4 μmol / L CDFDA solution was added. The cells were then incubated for 10 minutes in a CO2 incubator (5% CO2, 37°C). After washing three times with HBSS(+), 75 μL / well of the medium was added. The cells were then incubated for 10 minutes in a CO2 incubator (5% CO2, 37°C) and observed under a fluorescence microscope.

[0037] [Effectiveness of K-Ras activators on bile canaliculi formation] Two types of medium were used as controls: the medium of the present invention minus OsM (negative control), and the medium of the present invention (positive control). Four types of medium were prepared by adding K-Ras activators other than OsM (IL-6, LIF, CNTF, Ras-GRF) to the negative control (six types in total). Standard culture was performed using each medium, and on day 4, the medium was removed. 100 μL / well of 4 μmol / L CDFDA solution was added. The cells were incubated for 10 minutes in a CO2 incubator (5% CO2, 37°C) and then observed under a fluorescent microscope.

[0038] 2.Results [Confirmation of bile acid excretory transporter expression by immunohistochemistry] Under normal culture conditions in the medium of the present invention, significant expression of MRP2 was observed. On the other hand, under normal culture conditions in commercially available media A, B, and C, almost no MRP2 expression was observed. Furthermore, under normal culture conditions in the medium of the present invention, MRP2 expression was observed to be greater than under SW culture conditions using commercially available media A, B, and C (conventional method) (Figure 1). When SW culture was performed using the medium of the present invention, MRP2 expression was observed to be equal to or greater than that observed when SW culture was performed using commercially available media A, B, and C. Compared to normal culture using the medium of the present invention, higher MRP2 expression was observed in normal culture.

[0039] [Excretion of fluorescent substrates into bile canaliculi by bile acid excretory transporters] On the seventh day of culture, CDFDA accumulation in the bile canaliculi was significantly greater under normal culture conditions using the medium of the present invention than under conditions (conventional methods) in which commercially available medium A or commercially available medium B was used to culture in Matrigel (registered trademark) SW (Figure 2).

[0040] [Verification of the effectiveness of this medium for hepatocytes derived from human liver chimeric mice] In fluorescence microscopy images taken on day 4 of culture, CDFDA accumulated in dot-like patterns in the bile canaliculi under normal culture conditions with commercial medium D, while CDFDA accumulated in tubular patterns in some areas of the bile canaliculi under SW culture conditions with commercial medium D. On the other hand, in the medium of the present invention, tubular accumulation of CDFDA in the bile canaliculi was observed throughout the entire field of view (Figure 3).

[0041] [Verification of the effect of each component of the medium of the present invention on bile canaliculi formation] Phase-contrast microscopy images on day 4 of culture showed that in the OsM-free medium condition, almost no intercellular boundaries were observed, whereas in the other conditions, intercellular boundaries were observed. Furthermore, fluorescence microscopy images on day 4 of culture showed that in the OsM-free medium condition, almost no CDFDA accumulation in the bile canaliculi was observed, whereas in the other conditions, CDFDA accumulation in the bile canaliculi was observed (Figure 4).

[0042] [Effectiveness of K-Ras activators on bile canaliculi formation] Fluorescence microscopy images on day 4 of culture showed that CDFDA accumulated in the bile canaliculi in a dot-like pattern in the negative control, whereas CDFDA accumulated in a tubular pattern in the bile canaliculi under the four culture conditions containing K-Ras activators. In particular, CDFDA accumulation was observed to be comparable to that observed in OsM (positive control) under the IL-6-containing medium (Fig. 5).

[0043] 3. Discussion The above results in OsM-free media suggest that OsM contributes to the promotion of bile canaliculi formation. Furthermore, the above results in the validation of the effectiveness of K-Ras activators suggest that OsM strengthens cell-cell adhesion by activating K-Ras, a protein involved in the formation of adherens junctions between cells. This may promote bile canaliculi formation without sandwich culture. Furthermore, seeding primary hepatocytes (PHH) in albumin-free media allows PHH to rapidly adhere to the cultureware, reducing damage from thawing and seeding and preserving their relatively high functionality. This may also contribute to the promotion of bile canaliculi formation. [Industrial Applicability]

[0044] The present invention enables the simple and efficient formation of bile canaliculi from hepatocytes without using the sandwich culture method, which involves layering extracellular matrix. The bile canaliculi produced by the present invention are relatively homogeneous and can be used in a wide range of fields, including testing and research, such as screening candidate drugs.

Claims

1. A medium for culturing hepatocytes to form bile canaliculi, the medium comprising, as an active ingredient, a compound that activates K-Ras protein.

2. The medium of claim 1, wherein the compound that activates K-Ras protein is oncostatin M (OsM), an interleukin (IL)-6 family cytokine, or a guanine nucleotide exchange factor (GEF) for K-Ras.

3. The medium of claim 1, which is albumin-free or contains less than 0.1 w / v% albumin.

4. 2. The medium of claim 1, which does not contain one or more selected from the group consisting of bovine serum albumin (BSA), 6-bromoindirubin-3'-oxime (BIO), and calcitriol.

5. The medium according to claim 1, which does not contain any of cAMP, cAMP analogs, and substances that increase intracellular cAMP concentrations.

6. A method for producing bile canaliculi, comprising a step of culturing hepatocytes in the medium according to any one of claims 1 to 5.

7. The production method according to claim 6, wherein the hepatocytes are primary cultured hepatocytes.

8. The production method according to claim 6, which comprises changing the medium 1 to 8 hours after the start of culture.

9. 7. The method of claim 6, which does not include a step of layering solubilized basement membrane extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma onto hepatocytes.

10. The method of claim 6, which does not include a step of layering an extracellular matrix on the hepatocytes.

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