Human hepatocyte culture medium and conditioned medium of in vitro cultured human hepatocytes and use thereof

Abstract

Provided are methods of culturing, differentiating or suspending hepatocytes, iPS cell-derived hepatocyte-like cells (iHeps) or chemically-induced liver progenitor (CLiP) cells for recovery and establishment of liver function after isolation and / or storage.SOLUTION: Culturing an amount of hepatocytes, iHeps or CLiP cells in a first phase with a first cell culture medium for a first period of time, removing the first cell culture medium from the cultured culture; And subsequently culturing the hepatocytes, iHeps or CLiP cells in a second phase with a second cell-culture medium for a second period of time, wherein the first cell-culture medium comprises a cell-culture medium supplemented with dimethyl sulfoxide (DMSO) and the second cell-culture medium comprises a cell-culture medium supplemented with DMSO, DMSO2 or TMSO.SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Patent Application Publication No. 63 / 250,529, filed September 30, 2021. Specification and U.S. Patent Application Publication No. 63 / 250,541, filed September 30, 2021 Claim the benefit under 35 U.S.C. § 119(e) of the specification, and incorporate each of the contents thereof in its entirety. The entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a cell culture medium and uses thereof. [Background technology]

[0003] Non-cancerous terminally differentiated human hepatocytes (HH) cultured in vitro have been shown to enhance liver biology and It is the primary in vitro experimental platform for the study of liver diseases and various liver diseases. HH includes, but is not limited to, assessing the drug toxicity of newly developed clinical compounds. It is also a fundamental tool for preclinical evaluation of drug metabolism and pharmacokinetics (DMPK). The global market size for HH for research use is estimated at $50 million and is steadily growing. is estimated to grow.

[0004] However, to date, the usefulness of HH has not yet reached a level that meets the needs of researchers. Cold and warm ischemia, which leads to a rapid decline in cell quality, and collagenase perfusion are also involved. Cell damage resulting from the cell acquisition process, including the isolation procedure and freeze-thaw storage process Given the injury and stress, this lack is primarily due to the in vitro refinement of HH. As a result, there is no established method for culturing or maintaining liver cells. The performance / function of in vitro cultured HH in disease and DMPK studies is desirable. Not likely.

[0005] Furthermore, crucial techniques support long-term cell fate maintenance of mature HH in vitro. The lack of a method for analyzing liver function hinders our understanding of the molecular and cellular biology of the liver. However, there is no definitive technique to support the complete maturation of stem cell- or iPS cell-derived HH. Improved long-term cell fate maintenance of mature HH in vitro and complete maturation of iHep Improving this will lead to the successful establishment of hepatocyte transplantation therapy.

[0006] Currently, liver transplantation (LT) is performed for the treatment of chronic viral hepatitis (HBV and HCV), alopecia, and rheumatoid arthritis. Alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD) and liver Decompensated cirrhosis and liver damage caused by various types of chronic liver disease, including many genetic disorders However, LT is an invasive procedure. The nationwide organ shortage greatly limits its usefulness. The immunosuppression is associated with significant morbidity, including the development of opportunistic infections and malignant neoplasms. You will need to undergo therapy for the rest of your life.

[0007] Hepatocyte transplantation technology is expected to replace LT therapy in the future; however, However, many technical hurdles remain to be overcome. One of the most important technical barriers is The problem is that there is no definitive method that allows for the full maturation of iHep. The degree of maturation of ep is still far inferior to that of primary human hepatocytes (PHH). Indeed, iHep is a rather complex cell population consisting of both immature hepatocytes and cholangiocyte-like cells. The heterogeneity of iHep cell populations is due to the fact that the degree of maturation of iHep varies from one hepatic bipotential progenitor cell to another. The degree of hepatocyte maturation can be determined by the expression abundance of "hepatocyte marker genes" and Immunodeficient mice expressing toxic genes in endogenous mouse hepatocytes [humanized liver chimeras] The mouse strain used to generate HLCM was evaluated by its increased capacity in the liver. Therefore, the currently available iHep can be expanded in the liver of HLCM host mice. Therefore, the final maturation process of hepatic progenitor cells, including iHep, cannot be achieved. There remains a significant unmet need for technologies that enable this. Summary of the Invention

[0008] All publications herein are incorporated by reference as if each individual publication or patent application were incorporated by reference. Each of the preceding claims is incorporated by reference to the same extent as if specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. Any of the information provided is prior art or relevant to the presently claimed invention that any of the publications specifically or implicitly referenced is prior art. This does not constitute an endorsement of the

[0009] Exemplary embodiments are illustrated in the referenced figures. The embodiments and figures disclosed herein are , are intended to be considered illustrative rather than limiting. [Brief explanation of the drawings]

[0010] [Figure 1]Comparison of hepatocyte culture media: Freshly isolated HHs were cultured for 7 days with HH recovery medium (dHCGM: control) or commercial hepatocyte culture medium. The HHs were then subjected to: (left) immunofluorescence microscopy to detect MRP2 (arrow) (bar indicates 25 µm), (top right) immunoblot analysis to detect the indicated hepatocyte marker genes, or (bottom right) quantification of human albumin in the culture supernatant. Graph bars represent mean ± SD; *** indicates p<0.001 by one-way ANOVA. [Figure 2] Figure 1 shows a comparison of ADH activity in cultured HH and normal human tissues, as well as DMSO inhibition of ADH activity: (A) Cell / tissue homogenates of HH or human liver were subjected to measurement of NADH production rate, a proxy for ADH activity, by absorbance detection at 340 nm. Hepatoma cells (Huh7 and HepG2) were used as negative controls. (B) Relative ADH activity of HH was assessed in the absence of organic compounds (NT) and in the presence of DMSO or DMSO2 using the same method as described in (A). Graph bars represent mean ± SD; *, **, and *** indicate p<0.05, p<0.01, and p<0.001 by one-way analysis of variance; ns (not significant); nd (not detected). [Figure 3] Figure 1 shows DMSO inhibition of DMPK enzymes. Freshly isolated HHs were cultured with DMSO-containing medium (recovery medium) for 7 days. The cells were then used to assess CYP3A4 (left) and CYP2A9 (center) activity in the presence of organic compounds (DMSO or DMSO2) using the P450-Glo™ Assay (Promega). CYP2E1 activity (right) was assessed using the Vivid™ CYP2E1 Blue Screening Kit (Thermo Fisher). Graph bars represent mean ± SD; *, **, and *** indicate p<0.05, p<0.01, and p<0.001 by one-way analysis of variance, respectively; ns (not significant); nd (not detected). [Figure 4]Figure 1. DMSO inhibition of ethanol metabolism on acetate production: Freshly isolated HHs were cultured with HH recovery medium for 7 days. Then, HHs were cultured for 24 hours with either DMSO or DMSO2-containing maintenance / application medium in the absence (0 mM) or presence (0 mM) of 10 mM ethanol. A seal was applied throughout the ethanol treatment course. At the end of the ethanol treatment, cell culture medium was collected, and acetate concentrations were subsequently measured using an Acetate Colorimetric Assay Kit (Biovision). Graph bars represent mean ± SD; *** indicates p<0.001 by one-way ANOVA. nd (not detected). Acetate production rates were normalized by human liver cell number to assess the EtOH metabolic capacity of HHs under each given condition compared with healthy human livers (right). [Figure 5] This figure shows that DMSO withdrawal promotes hepatocyte cell fate loss: Freshly isolated HHs were cultured with HH recovery medium for 7 days, followed by an additional 4 days of incubation with medium containing the indicated concentrations of DMSO. HHs were then subjected to (A) immunofluorescence analysis to detect MRP2 (arrow), (B) immunoblotting, and (C) qPCR analysis to detect the indicated hepatocyte marker molecules. Bars indicate 25 µm. [Figure 6] Figure 1 shows the role of DMSO in cell fate recovery in HH cells. Freshly isolated HH cells were cultured for 7 days with medium containing the indicated concentrations of DMSO. At the indicated time points, the cells were then subjected to (A) immunofluorescence analysis to detect MRP2 (arrow), (B) immunoblotting, and (C) qPCR analysis to detect the indicated hepatocyte marker molecules. [Figure 7] Figure 1 shows the importance of organic compounds in maintaining the expression of hepatocyte marker genes. Freshly isolated HHs were cultured with HH recovery medium for 7 days, followed by maintenance / application medium containing the indicated organic compounds for an additional 7 days. Whole cell lysates were then subjected to immunoblot analysis to detect the indicated molecules. NT; non-treated. [Figure 8]Figure 1 shows the importance of organic compounds in the morphological maintenance of human hepatocytes. Freshly isolated HHs were cultured with HH recovery medium for 7 days, followed by incubation with maintenance / application medium containing the indicated organic compounds for an additional 7 days. HHs were then subjected to immunofluorescence analysis to detect MRP2 (arrow), F-actin, and DAPI. The bar indicates 25 µm. [Figure 9] Figure 1 shows the effect of DMSO on the inducibility of CYP genes. Freshly isolated HHs were cultured with HH recovery medium (dHCGM) for 7 days, followed by maintenance / application medium containing DMSO or DMSO2 for an additional 7 days in the presence of the indicated concentrations of CYP3A4 inducers (rifampicin and carbamazepine) (left) and CYP2E1 inducers (ethanol and isoniazid) (right). Total RNA and cell lysates were then subjected to immunoblot analysis (left) and qPCR analysis (right) to determine the inducibility of the indicated molecules. Graph bars represent the mean ± SD; *, **, and *** indicate p<0.05, p<0.01, and p<0.001 by one-way ANOVA. [Figure 10] Figure 1 shows TMSO inhibition of alcohol-metabolizing enzymes: HH cell homogenates were subjected to measurement of NADH production rate, a proxy for ADH activity, by detecting absorbance at 340 nm in the presence of organic compounds (DMSO, DMSO2, or TMSO). CYP2E1 activity (right) was assessed using the Vivid™ CYP2E1 Blue Screening Kit (Thermo Fisher). Graph bars represent mean ± SD; *** indicates p<0.001 by one-way analysis of variance. ns (not significant); nd (not detected). [Figure 11] Figure 1 shows the application of a sealing device to ethanol treatment of HH: A tight silicone sealing device was applied to the HH culture dish to minimize evaporative loss of ethanol from the culture medium. The image on the left is representative of a standard cell culture dish setup, and the image on the right demonstrates the same cell culture dish with application of the sealing device (top: without lid, and bottom: with lid). [Figure 12] Figure 1 shows an overview of CMHH preparation. Human hepatocytes (e.g., PHH and HLCM-HH) in suspension medium (e.g., DMEM10) are plated onto type I collagen-coated cell culture dishes with a seeding cell number optimized to achieve an adherent cell density of >0.5 x 10 cells / cm. 24 hours after seeding, the plating medium (e.g., DMEM10) is replaced with hepatocyte culture medium (dHCGM containing DMSO), which is replaced every 2–4 days for up to 7 days to complete the recovery phase. Then, during the maintenance / application phase, cells undergo repeated medium changes and cell culture supernatant (CMHH) collection every 48 hours, as described above. The recovered CMHH is then frozen or frozen at -20°C or -80°C until use. [Figure 13] Figure 1 shows the biological effects of CMHH on the cellular structure of HH. Freshly isolated HLCM-HH (lot: JFC) were plated at 1.05 × 10 cells / cm onto type I collagen-coated wells. The plating medium (DMEM10) was replaced with fresh dHCGM (FM) (left) or CMHH (right) 30–60 min after cell seeding, followed by medium changes every 24 h until day 7 (either FM or CMHH). Cells were then subjected to light microscopy analysis. Arrows indicate bile canaliculi formed between hepatocytes, which exhibit a polygonal-cuboidal appearance, characteristic of terminally differentiated human hepatocytes. [Figure 14] Figure 1 shows functional evaluation of the bile secretion mechanism. Freshly isolated HLCM-HH (lot: JFC) were cultured with FM or CMHH as described in Figure 13. On day 7 after seeding, the cells were washed with Hank's balanced salt solution (HBS) and incubated with HBS containing 1.25 nM carboxy-DCFDA [5(6)-carboxy-2',7'-dichlorofluorescein (CDFDA)], an MRP2-specific fluorescent substrate, at 37°C for 10 min. Subsequently, the cells were washed with HBSS (three times) and incubated with HBSS at 37°C for 10 min. The cells were then subjected to fluorescence microscopy to detect CDFDA-filled bile canaliculi (arrows). [Figure 15-1] Figure 15A shows the biological effects of CMHH on HH cell fate. Freshly isolated HLCM-HH (lot: JFC) were cultured with fresh dHCGM (FM) or CMHH (CM) as described in Figure 13. Seven days after seeding, total RNA was extracted using Quick RNA microprep (ZymoResearch) and subsequently synthesized using a cDNA superMix Kit (Quanta Bio). cDNA was then subjected to real-time qPCR for relative quantification of the indicated genes. Bars represent the mean ± SD of expression levels relative to freshly isolated HLCM-HH; *, **, and *** indicate p<0.01, p<0.001, and p<0.0001, respectively, by unpaired t-test. [Figure 15-2] Figure 15B shows the biological effects of DMSO and DMSO2-containing CMHH on HH cell fate. Freshly isolated HLCM-HH were cultured with fresh dHCGM or CM containing either DMSO or DMSO2 for 7 days. Total RNA was then extracted using Quick RNA microprep (ZymoResearch) and subsequently synthesized using the cDNA superMix Kit (Quanta Bio). cDNA was then subjected to real-time qPCR for relative quantification of the indicated genes. Bars represent the mean ± SD of the expression levels relative to freshly isolated HLCM-HH; statistical analysis was performed using Tukey's multiple comparison test, *P<0.0001. [Figure 16]Figure 1 shows the effect of CMHH on HH susceptibility to HBV infection. Freshly isolated HLCM-HH (lot: JFC) were cultured with dHCGM (FM) or CMHH (CM) as described in Figure 13. HLCM-HH incubated with either FM or CM for 7 days were then inoculated with hepatitis B virus (HBV) (genotype A) at 5 Geq / cell using a method established in a previous study (PMID: 25791527). HBV-infected cells were cultured with FM or CM for an additional 14 days with medium changes every 2 days and subsequently analyzed by immunofluorescence microscopy to detect HBV surface antigen (HBsAg) using anti-HBsAg antibody and alexa488-labeled anti-mouse antibody. Arrows indicate active HBV infection in CM-treated HLCM-HH. [Figure 17] Figure 1 shows the effect of antiviral therapy on HBV infection. Freshly isolated HLCM-HH (lot: JFC) were cultured with dHCGM (FM) or CMHH (CM) as described in Figure 13. HLCM-HH incubated with either FM or CM for 7 days were then inoculated with hepatitis B virus (HBV) (genotype A) at 5 Geq / cell using a method described in a previous study (PMID: 25791527). HBV-infected cells were cultured with FM or CM for an additional 14 days, with medium changes every 2 days, with or without entecavir (ETV) (250 nM). At the end of the study, culture supernatants were subjected to viral DNA extraction using the QIAamp MinElute Virus Spin Kit (Qiagen), followed by TaqMan probe-based one-step RT-qPCR analysis for HBV genome quantification. Bars represent the mean ± SD; *** indicates p<0.0001 by one-way ANOVA and Tukey's multiple comparison test. [Figure 18]This figure shows the effect of CMHH on the abiotic metabolic pathway of HH. Freshly isolated HLCM-HH (lot: JFC) were cultured with dHCGM (FM) or CMHH (CM) as described in Figure 13. HLCM-HH incubated with either FM or CM for 7 days were then subjected to evaluation of the indicated CYP enzymes using the P450-Glo assay (Promega). Bars represent the mean ± SD of expression levels relative to freshly isolated HLCM-HH; ** and *** indicate p<0.001 and p<0.0001 by unpaired t-test, respectively. [Figure 19]

[0049] Figure 13 shows the biological functions of CM obtained from various cell types. CM was expanded in HLCM-HH, HepG2, HuH7, 293 cells, and primary mouse hepatocytes using the protocol described in Figure 13. It was then applied to HLCM-HH cultures for 7 days and subsequently analyzed by light microscopy. The arrows indicate bile canaliculi formed between hepatocytes, which have a polygonal-cuboidal appearance, a characteristic of terminally differentiated human hepatocytes. [Figure 20] Figure 1 shows the effect of CM on HLCM-HH. HLCM-HH cultured for 7 days with CM expanded with the indicated cell types were subjected to RT-qPCR analysis to evaluate the indicated genes. Bars represent the relative expression levels compared to those in freshly isolated HLCM-HH (CYP2C9, 2D6, ALDH2, and OATP1B1) or HLCM-HH cultured with fresh dHCGM (FM) (TGFB1, TGFB2, Cyr61, CTGF, and CK19). Statistical analysis was performed using Tukey's multiple comparison test; *P<0.01, **P<0.001, ***P<0.0001 vs. HLCM-HH. [Figure 21]Figure 1 shows the effect of CM expanded with various cell types on cell fate markers in HH. HLCM-HH obtained from two different donors (JFC and BD195) or CMHH expanded with commercially available cryopreserved primary human hepatocytes from three different donors (SMC, JIY, and TAK) were used in 7-day cultures of freshly isolated HLCM-HH according to the protocol described in Figure 13. The morphological organization of the cells was then evaluated by light microscopy. Arrows indicate bile canaliculi formed between human hepatocytes, which exhibit a polygonal-cuboidal appearance, characteristic of terminally differentiated human hepatocytes. [Figure 22] Figure 13 shows the biological effects of CM expanded with HH from multiple donors. As described in Figure 13, freshly isolated HLCM-HH were cultured with either fresh dHCGM (FM), HLCM-HH (from two different donors), or CMHH expanded with primary human hepatocytes (from three different donors) for 7 days, followed by RT-qPCR analysis to detect the indicated genes. Relative expression abundance was determined by normalization with the expression abundance in freshly isolated HLCM-HH. Statistical analysis was performed using Tukey's multiple comparison test; *P<0.01, **P<0.001, ***P<0.0001. [Figure 23] Figure 14 shows a comparison of the biological effects of CM, 5Cn, and Matrigel® treatment. Freshly isolated HLCM-HH (lot: JFC) were plated as described in Figure 13 and cultured with dHCGM (FM) or CMHH (CM). Alternatively, cells were cultured in fresh dHCGM supplemented with 5C or Matrigel® at concentrations described in previous reports (PMIDs: 31023926, 16469405). Arrows indicate bile canaliculi formed between human hepatocytes, which exhibit a polygonal-cuboidal appearance, characteristic of terminally differentiated human hepatocytes. [Figure 24]Figure 23 shows a comparison of the biological effects of CM, 5Cn, and Matrigel treatment. As described in Figure 23, freshly isolated HLCM-HH (lot: JFC) were plated and cultured with fresh dHCGM, CMHH, 5C, or fresh dHCGM supplemented with Matrigel, followed by RT-qPCR analysis for relative quantification of the indicated genes. Statistical analysis was performed using Tukey's multiple comparison test; *P<0.01, **P<0.001, ***P<0.0001. [Figure 25] Figure 1 shows the effect of size-exclusion column filtration on the biological effects of CMHH. Freshly isolated HLCM-HH were cultured with either CMHH or CMHH filtered through an exclusion column with increasing size cutoffs for 7 days, followed by RT-qPCR analysis to detect the indicated genes. Relative abundance was determined by normalizing with the expression abundance of the corresponding gene in freshly isolated HLCM-HH. Statistical analysis was performed using Tukey's multiple range test; *P<0.01, **P<0.001, ***P<0.0001. [Figure 26] Figure 13 shows the compositional analysis of CMHH. In the scheme described in Figure 13, dHCGM supplemented with Matrigel® was used for relative or absolute quantification of the indicated humoral factors by measuring immunoblot signals using Image J software and ELISA, respectively. Assessment of relative abundance was performed using an equal volume of CM augmented with the indicated cell type or dHCGM supplemented with Matrigel® containing an equivalent total protein amount as CMHH. Bars represent relative protein levels relative to CMHH. [Figure 27]Figure 13 shows the biological effect of CMHH on the morphology of cryopreserved primary human hepatocytes. Commercially available primary human hepatocytes (lot: GNA, BioIVT) were cultured for 7 days in either commercially available hepatocyte culture medium (INVITROGRO HI Medium, BioIVT), fresh dHCGM, or CMHH, according to the protocol described in Figure 13. The morphological organization of the cells was then evaluated by light microscopy. Arrows indicate bile canaliculi formed between human hepatocytes, which exhibit a polygonal-cuboidal appearance, a characteristic of terminally differentiated human hepatocytes. [Figure 28] Figure 13 shows the effect of CMHH on the bile secretion mechanism of cryopreserved primary human hepatocytes. Commercially available primary human hepatocytes (lot: GNA, BioIVT) were cultured for 7 days in either commercial hepatocyte culture medium (INVITROGRO HI Medium, BioIVT), fresh dHCGM, or CMHH, according to the protocol described in Figure 13. On day 7 after seeding, the cells were washed with Hank's balanced salt solution (HBS) and incubated with HBS containing 1.25 nM carboxy-DCFDA [5(6)-carboxy-2',7'-dichlorofluorescein (CDFDA)], an MRP2-specific fluorescent substrate, at 37°C for 10 minutes. Subsequently, the cells were washed with HBSS (three times) and incubated with HBSS at 37°C for 10 minutes. The cells were then subjected to fluorescence microscopy to detect CDFDA-filled bile canaliculi (arrows). [Figure 29] Figure 13 shows the effect of CMHH on cell fate recovery and cell fate maintenance of cryopreserved primary human hepatocytes. Commercially available primary human hepatocytes (lot: GNA, BioIVT) were cultured for 7 days in either commercial hepatocyte culture medium (INVITROGRO HI Medium, BioIVT), fresh dHCGM (FM), or CMHH (CM) according to the protocol described in Figure 13. Seven days after seeding, total RNA was extracted for RT-qPCR analysis to detect the indicated genes. Relative expression abundance was determined by normalization with the expression abundance in freshly isolated HLCM-HH. Statistical analysis was performed using Tukey's multiple comparison test; *, **, and *** indicate p<0.01, p<0.001, and p<0.0001, respectively, versus CMHH. [Figure 30] Figure 13 shows the effect of CMHH on the morphology of primary dog, mouse, and rat hepatocytes. Commercially available cryopreserved primary dog, rat, and mouse hepatocytes (BioIVT) were cultured for 7 days in either commercial hepatocyte culture medium (INVITROGRO HI Medium, BioIVT), fresh dHCGM(FM), or CMHH, as described in Figure 13. The morphological organization of the cells was then assessed by light microscopy. Arrows indicate bile canaliculi formed between human hepatocytes, which exhibit a polygonal-cuboidal appearance, a characteristic of terminally differentiated hepatocytes. [Figure 31] Figure 1 shows the effect of CMHH on iHep morphology. Commercially available cryopreserved iHep (iCell Hepatocyte 2.0, FujiFilm) were thawed and cultured for 5 days using the iHep culture medium (iCell Plating Medium) provided by the manufacturer according to the manufacturer's protocol. Subsequently, they were maintained for 6 days in either the medium provided by the manufacturer (iCell Maintenance Medium), fresh dHCGM (FM), or CMHH with medium changes every 2 days. The morphological organization of the cells was then assessed by light microscopy. Arrows indicate bile canaliculi formed between human hepatocytes, which exhibit a polygonal-cuboidal appearance, characteristic of terminally differentiated human hepatocytes. [Figure 32] Figure 3 shows the effect of CMHH on iHep. Commercially available cryopreserved iHep (iCell Hepatocyte 2.0, FujiFilm) were thawed and cultured as described in Figure 31, followed by single-cell RNA sequencing analysis. Distance in the three-dimensional scatter plot represents the diversity among iHep cultured in the indicated media. Freshly isolated HLCM-HH were included in the analysis as a representative example of terminally differentiated human hepatocytes. [Figure 33]Figure 3 shows the effect of CMHH on iHep gene expression. Commercially available cryopreserved iHep (iCell Hepatocytes 2.0, FujiFilm) were thawed and cultured as described in Figure 31, followed by single-cell RNA sequencing analysis. Pie charts represent the percentage of cells with relative expression of the indicated genes. Freshly isolated HLCM-HH were included in the analysis as a representative example of terminally differentiated human hepatocytes. [Figure 34] Figure 1 shows the effect of CMHH on CLiP. CLiP cells derived from HLCM-HH were established as previously described (PMID: 31393263). After the first round of passage, CLiP cells were cultured for 7 days in fresh dHCGM(FM) or CMHH according to the protocol described in Figure 13. The expression abundance of the indicated genes was then determined by RT-qPCR analysis. Relative expression abundance was determined by normalization with the expression abundance of the corresponding gene in freshly isolated HLCM-HH. Paired tests, *, **, and ***, indicate p<0.05, p<0.01, and p<0.001, respectively. [Figure 35] Figure 1 shows the effect of CMHH pretreatment on in vivo HH expansion. Engraftment and expansion of CMHH-treated human hepatocytes. Freshly isolated HLCM-HH cells were cultured in either fresh dHCGM (control) or CMHH for 14 days and then transplanted in vivo into the spleens of 3-week-old cDNA-uPA / SCID mice (2.5 x 10 cells / animal). Serial measurements of human albumin levels in the blood were then performed on the indicated days after transplantation using a previously described method (PMID: 17761892). [Figure 36] Figure 1 shows the effect of CMHH on the morphology of cryopreserved HLCM-HH. Cryopreserved HLCM-HH were thawed and cultured in either fresh dHCGM(FM) or CMHH for 7 days with medium changes every 2 days. The morphological organization of the cells was then assessed by light microscopy. Arrows indicate bile canaliculi formed between human hepatocytes, which exhibit a polygonal-cuboidal appearance, characteristic of terminally differentiated primary hepatocytes. [Figure 37]Figure 1 shows the effect of CMHH on the recovery and fate maintenance of cryopreserved HLCM-HH. Cryopreserved HLCM-HH were thawed and cultured in either fresh dHCGM(FM) or CMHH for 7 days with medium changes every 2 days. Seven days after seeding, total RNA was extracted for RT-qPCR analysis to detect the indicated genes. Relative expression abundance was determined by normalization with the expression abundance in freshly isolated HLCM-HH. Statistical analysis was performed by Student's t-test, paired with CMHH; *, **, and *** indicate p<0.01, p<0.001, and p<0.0001, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0011] All references cited herein are incorporated by reference in their entirety as if fully set forth. Unless otherwise specified, the technical and scientific information used herein Terms are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. do. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed. , Revised, J. Wiley & Sons (New York, NY 2006);March, Advanced Organic Chemistry y Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, NY 201 3); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4th ed., C Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012) is the subject of this application. This document provides a general guide to many of the terms used in the present invention. For relevant references, see D. Lane, Antibodies: A Laboratory Manual 2nd ed. (Cold Springer) ing Harbor Press, Cold Spring Harbor NY, 2013);Kohler and Milstein, (1976) Eur. J. Immunol. 6: 511; Queen et al. U.S. Pat. No. 5,585,089; and Riechma nn et al., Nature 332: 323 (1988); U.S. Patent No. 4,946,778; Bird, Scien ce 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (19 88);Ward et al., Nature 334:544-54 (1989);Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479;Holliger P. (2005) Nat. Biotechnol. Sep;23(9):112 See 6-36.

[0012] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein. This will result in a number of variations that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials used. The terms are defined below.

[0013] The applicant's findings will be used in the field of regeneration / stem cell biology to establish hepatocyte transplantation therapy. Furthermore, the present invention is of considerable value to basic research into liver biology and pathophysiology in general. It also provides a moderate amount of benefit.

[0014] The applicant has demonstrated that the culture medium of terminally differentiated HH (hereinafter referred to as human hepatocyte conditioned medium; CMHH) Advantages for use in regenerative medicine and stem cell biology, e.g., the cellular end product of iHep These findings provide a concerted advantage to facilitate the maintenance of mature and fully mature iHep cell fates. Many humoral factors (e.g., ECM and plasma proteins synthesized by hepatocytes) contribute to In addition, CMHH provides support for liver cell biology research. However, for example, in vitro cultured HH, other species (e.g., dog, mouse, rat, etc.) Primary hepatocytes derived from humans and monkeys, hepatocytes obtained from humanized liver chimeric animals, and non-human liver cells Supports cell fate maintenance, function, and lifespan of parenchymal cells (NPCs).

[0015] In various embodiments, the CMHH is augmented with high quality HH. The term "high quality" refers to cells with high diversity and culturability. "Culturability" refers to the percentage of viable cells that have the ability to adhere to a culture dish. Viability can be determined by techniques such as trypan blue staining. Cell Quality is compromised when cells are plated at low confluency; therefore, viability and culturability In some embodiments, the methods disclosed herein involve determining both Hepatocytes cultured in this manner should be at least 95%, 90%, 80%, 70%, 60%, and In addition, it has the potential to: a) treat warm / cold ischemic injury during organ procurement; b) treat cell isolation; High quality product from cell injury / stress caused by isolation procedure and cell damage due to freeze-thaw process It is important to promote the recovery of quality HH. In some embodiments, the liver cell function is improved. It is important to optimize the biological processes of hepatocytes to better study them.

[0016] In some embodiments of the present application, the inventors have developed a HH culture medium and 1) a cell isolation procedure. and rapid and robust recovery from cell damage or stress associated with freeze-thaw cycles, Phase 1 (recovery phase), and Phase 2) functioning at a level comparable to that of the human liver. Therefore, it is suitable for various experimental applications, i.e., Phase 2 (application period) in vitro We describe a method that allows HH to be cultured in vitro.

[0017] In some embodiments, the first HH culture medium is provided for phase 1 recovery. In some embodiments, as shown in Tables 1 and 3, this first medium contains DMSO. In some embodiments, the medium is hepatocyte clonal expansion medium (dHCGM) supplemented with: dHCGM is a standard cell culture basal medium containing L-proline, insulin, and dexamethasone. , EGF and L-ascorbic acid 2-phosphate (Asc-2P), or Particularly preferred is a standard cell culture basal medium consisting essentially of HEPES, penicillin, In some embodiments, the serum is supplemented with phosphostreptomycin and serum. Fetal bovine serum (FBS) or human serum. The basal cell culture medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0018] Standard basal cell culture media are well known to those skilled in the art. Examples include, but are not limited to, DMEM, DMEM-10, Minimum Essential Medium (MEM), RPMI-1640, Iscove's Modified Dulbecco's Medium (IMDM) or William's Medium There is.

[0019] In some embodiments, the first HH culture medium is DM, as shown in Tables 2 and 3. In some embodiments, the dHMM is a hepatocyte maintenance medium supplemented with SO. Contains DMSO, standard cell culture basal medium, L-proline, insulin, and dexamethasone. and L-ascorbic acid 2-phosphate (Asc-2P), or Particularly preferred is a standard cell culture basal medium containing HEPES, penicillin, In some embodiments, the serum is supplemented with streptomycin and serum. In a preferred embodiment, the medium is a standard cell culture medium. The base medium is Dulbecco's Modified Eagle's Medium (DMEM)-10.

[0020] The medium used in the first stage of HH culture is designed to reduce the stress associated with the cell isolation and storage process. In some embodiments, the HH is at least 0.5x higher than the normal HH. 10 5 individual cells / cm 2 , preferably >1.2 x 10 5 individual cells / cm 2 Plated on a plate, This corresponds to >25% confluency immediately after cell seeding and is the first HH culture medium, e.g., dHCGM or maintained in dHMM, and scratches resulting from various isolation procedures and freeze-thaw storage processes. You will recover from the injury / stress; this recovery phase can take 4-7 days.

[0021] In some embodiments, the first HH culture medium, e.g., dHCGM or dHMM, In some embodiments, the first HH culture medium is used to maintain the cell fate of the HH. The off-target effects of DMSO, e.g., dHCGM or dHMM, may be disrupted. Not used for application.

[0022] In some embodiments, a second HH culture medium is provided for Phase 2 applications. In some embodiments, the second HH culture medium is used to establish and maintain liver function of the HH. In some embodiments, the application medium does not contain DMSO and is CYP3 In some embodiments, the expression of CYP2E1 and CYP4E1 is at physiological levels. Rather than being supplemented with DMSO, the second medium was cell culture medium supplemented with DMSO2 or contains cell culture medium supplemented with TMSO.

[0023] In some embodiments of the method, the amount of HH cultured in the second phase is C have physiological levels of expression of YP3A4 and physiological levels of expression of CYP2E1; and The amount of HH cultivated in the second phase is the same as that of alcohol, non-biological substances, vitamins, etc. Metabolizes steroids A or a combination thereof.

[0024] In some embodiments of the method, the HH promotes alcohol metabolism, and the HH promotes the metabolism of non-living organisms. To assay the performance of candidate drugs in promoting lipid metabolism or the toxicity of candidate drugs to HH. For use, the method comprises culturing in a second phase in a second cell culture medium. contacting the amount of HH with a candidate drug; and contacting the amount of HH with the candidate drug. measuring a first level of alcohol metabolism or excretion of the candidate drug by the In the presence of or after contact with a co-drug, the metabolism of alcohol or the candidate drug by the above amount of HH is and measuring a second level of the emissions.

[0025] In some embodiments, the second HH culture medium contains: Hepatocyte clonal expansion medium supplemented with DMSO2 (d2 In some embodiments, the second HH culture medium is supplemented with DMSO. Hepatocyte clonal differentiation was observed in a medium supplemented with tetramethylene sulfoxide (TMSO) rather than in a medium containing tetramethylene sulfoxide (TMSO). Growth medium (tHCGM).

[0026] In some embodiments, the second HH culture medium, e.g., d2HCGM, contains DMSO2 , standard cell culture basal medium, L-proline, insulin, dexamethasone, EGF and and Asc-2P. Particularly preferred are standard cells The culture basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In a preferred embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium ( DMEM)-10.

[0027] In some embodiments, the second HH culture medium, e.g., tHCGM, contains TMSO, standard Standard cell culture basal medium, L-proline, insulin, dexamethasone, EGF and A Particularly preferably, the standard cell culture The basal medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In one embodiment, the standard basal cell culture medium is Dulbecco's Modified Eagle's Medium (DMME). EM)-10.

[0028] In various embodiments, the second HH culture medium comprises: Hepatocyte maintenance medium supplemented with DMSO2 (d2HMM) rather than medium supplemented with DMSO In some embodiments, the second HH culture medium is a medium supplemented with DMSO. The hepatocyte maintenance medium (tHMM) is supplemented with TMSO without any ATP.

[0029] In some embodiments, the second HH culture medium, e.g., d2HMM, is DMSO2, Standard cell culture basal medium, L-proline, insulin, dexamethasone, and Asc Particularly preferred are standard cell culture media containing or consisting essentially of 2P. The medium is supplemented with HEPES, penicillin-streptomycin, and serum. In an embodiment, the serum is fetal bovine serum (FBS) or human serum. In an embodiment, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM). )-10.

[0030] In some embodiments, the second HH culture medium, e.g., tHMM, is a medium containing TMSO, standard Basal cell culture medium, L-proline, insulin, dexamethasone, and Asc-2 Particularly preferred are standard basal cell culture media, which contain or consist essentially of P. The medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, the serum is fetal bovine serum (FBS) or human serum. In this state, the standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM)- It's 10.

[0031] In some embodiments of the method, the second HH culture medium contains DMSO at a final concentration of about 140.8 mM. Contains SO2.

[0032] In some embodiments of the method, HH culture medium, e.g., dHCGM, d2HCGM or tHCGM should be added to standard cell culture basal medium at concentrations of at least about 70 mM and 3 mM, respectively. Supplemented with DMSO, DMSO2, or TMSO at 5 mM and 35 mM, the standard A typical cell culture basal medium contains L-proline, insulin, dexamethasone, EGF, Asc One or more of the following: -2P, HEPES, penicillin-streptomycin, and serum In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0033] In some embodiments of the method, HH culture medium, e.g., dHCGM, d2HCGM or and tHCGM are approximately 281.6 mM and 140.8 mM, respectively, in standard basal cell culture medium. A certain amount of DMSO, DMSO2 or T was added to reach a concentration of 70.4 mM. Standard cell culture basal medium supplemented with MSO, L-proline, insulin, dexamethasone, tazone, EGF, Asc-2P, HEPES, penicillin-streptomycin and blood In some embodiments, the serum further comprises one or more of FBS or is human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-1 It is 0.

[0034] In some embodiments of the method, the HH culture medium, e.g., dHCGM, is a standard cell culture medium. The basal medium was supplemented with an amount of DMSO to reach a concentration of approximately 2% (v / v). Standard cell culture basal media contains L-proline, insulin, dexamethasone, EGF, , Asc-2P, HEPES, penicillin-streptomycin, and serum In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0035] In some embodiments of the method, the HH culture medium, e.g., dHMM, d2HMM, or t HMM is present in standard basal cell culture medium at approximately 281.6 mM, 140.8 mM, and 140.8 mM, respectively. and an amount of DMSO, DMSO2, or TMSO to reach a concentration of 70.4 mM Standard cell culture basal medium is supplemented with L-proline, insulin, and dexamethasone. , Asc-2P, HEPES, penicillin-streptomycin, and serum In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0036] In some embodiments of the method, the HH culture medium, e.g., dHMM, is prepared using standard cell culture media. The basal medium was supplemented with a certain amount of DMSO to reach a concentration of approximately 2% (v / v). , standard cell culture basal medium contains L-proline, insulin, dexamethasone, Asc- One or more of 2P, HEPES, penicillin-streptomycin, and serum In some embodiments, the serum is FBS or human serum. In one embodiment, the standard basal cell culture medium is DMEM-10.

[0037] Methods for culturing HH for recovery and establishment of liver function after isolation and / or preservation are proposed. In a first phase, the method includes adding an amount of HH to a first cell culture medium. and removing the first cell culture medium from the amount of HH, followed by incubating the second cell culture medium. In the second phase, the amount of HH is cultured for a second period using a second cell culture medium. the first cell culture medium comprises cell culture medium supplemented with DMSO, and the second The cell culture medium was either cell culture medium supplemented with DMSO or cell culture medium supplemented with TMSO. The second cell culture medium contains DMSO and the second cell culture medium does not contain DMSO.

[0038] In another embodiment of the method, the first cell culture medium and the second cell culture medium both comprise: Contains DMSO, for example, dHCGM or dHMM.

[0039] In some embodiments of the method, the first cell culture medium contains 2% (volume / volume) DMSO and standard cell culture basal medium, the medium was supplemented with 15 μg / mL L-proline, 0. 25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 m M Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and one of 10% heat-inactivated FBS or human serum, or the second cell culture medium contained 140.8 ml of DMSO or TMSO, respectively; M or 70.4 mM and standard cell culture basal medium, and the medium contains 15 μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / m L EGF, 0.1mM Asc-2P, 20mM HEPES, 100IU / mL penicillin cillin, 100 μg / mL streptomycin, and 10% heat-inactivated FBS or human blood In a preferred embodiment, a standard cell culture basal medium is , DMEM-10.

[0040] In some embodiments of the method, the first cell culture medium contains 2% (volume / volume) DMSO and standard cell culture basal medium, the medium was supplemented with 15 μg / mL L-proline, 0. 25 μg / mL insulin, 50 nM dexamethasone, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% heat-inactivated FBS or human serum; the second cell culture medium contains DMSO, DMSO2 or TMSO at 281.6 mM, 140.8 mM, or 70.4 mM, respectively M and standard cell culture basal medium. The medium contained 15 μg / mL L-proline, 0 0.25μg / mL insulin, 50nM dexamethasone, 5ng / mL EGF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% heat-inactivated FBS or human serum. In embodiments, the standard basal cell culture medium is DMEM-10.

[0041] In some embodiments of the method, the first cell culture medium contains 2% (volume / volume) DMSO and standard cell culture basal medium, the medium was supplemented with 15 μg / mL L-proline, 0. 25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 m M Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% heat-inactivated FBS or human serum. The culture medium contained DMSO, DMSO2, or TMSO at 281.6 mM, 140. 8mM or 70.4mM and standard cell culture basal medium, the medium contains 15μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% heat-inactivated FBS or human serum; preferred In embodiments, the standard basal cell culture medium is DMEM-10.

[0042] In some embodiments of the method, the first cell culture medium contains 2% (volume / volume) DMSO and standard cell culture basal medium, the medium was supplemented with 15 μg / mL L-proline, 0. 25 μg / mL insulin, 50 nM dexamethasone, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% heat-inactivated FBS or human serum; the second cell culture medium contains DMSO, DMSO2 or TMSO at 281.6 mM, 140.8 mM, or 70.4 mM, respectively M and standard cell culture basal medium. The medium contained 15 μg / mL L-proline, 0 0.25μg / mL insulin, 50nM dexamethasone, 5ng / mL EGF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% heat-inactivated FBS or human serum. In embodiments, the standard basal cell culture medium is DMEM-10.

[0043] In some embodiments of the method, the first cell culture medium comprises 0.5% to 5% (volume / volume) DMSO, standard cell culture basal medium, 5–25 μg / mL L-proline, 0.1–0 0.5 μg / mL insulin, 10–100 nM dexamethasone, 0–10 ng / mL E GF, 0.01~1mM Asc-2P, 10~50mM HEPES, 10~300I U / mL penicillin, 10-300 μg / mL streptomycin, and 2-20% heat-insoluble The second cell culture medium contains 35 to 281.6 mM DMSO; the second contains activated FBS or human serum. SO, DMSO, or TMSO, standard cell culture basal medium, 5–25 μg / mL L -proline, 0.1-0.5 μg / mL insulin, 10-100 nM dexamethasone, 0~10ng / mL EGF, 0.01~1mM Asc-2P, 10~50mM HE PES, 10-300 IU / mL penicillin, 10-300 μg / mL streptomycin and 2-20% heat-inactivated FBS or human serum; in a preferred embodiment, the standard A typical basal cell culture medium is DMEM-10.

[0044] In some embodiments of the method, the HH is cultured in a first medium for a period of time, e.g., recovery. The phases last at least 4 days and up to 2 months. The amount of HH obtained above is characterized by cell polarity, intercellular structures characterized by bile canaliculi, and / or establishes gene expression levels comparable to control HH with liver function.

[0045] In some embodiments of the method, the first period, e.g., the recovery phase, is about 7 days. The first cell culture medium is optionally refreshed every 3 or 4 days.

[0046] In some embodiments, a kit is provided, the kit comprising a first HH culture medium, e.g. , dHCGM or dHMM, and a second HH culture medium, for example, d2HCGM, t Media containing HCGM, d2HMM, or tHMM, isolated or thawed from storage The HH obtained in step 1 can be cultured and subsequently used in experimental assays.

[0047] A kit for culturing human hepatocytes is also provided, the kit containing the first cell culture medium. a first container for carrying out the first cell culture medium, wherein the first cell culture medium is supplemented with DMSO, e.g. the first container being dHCGM or dHMM; and the second container being cell culture medium. a second container, wherein the second cell culture medium contains DMSO and / or TMSO; Supplemented and unsupplemented with DMSO, e.g., d2HCGM, d2HMM, tHCGM or a second container that is a tHMM.

[0048] Toxicity of drugs to human hepatocytes (HH) or drug metabolism and pharmacokinetics (DMP) of drugs Further provided is a method for assaying K), which method comprises using DMSO or TMSO. Culturing a quantity of HH in a medium supplemented with and not supplemented with DMSO; The agent was contacted with the above amount of HH cultured in a medium supplemented with DMSO or TMSO. and determining the level of toxicity to said amount of HH in the presence of or after contact with said agent. or measuring the amount of HH in the presence of or after contact with the drug. and measuring the level of metabolism or excretion of the drug.

[0049] In some embodiments of the assay method, the agent is ethanol, methanol, ethylene glycol, or the like. Alcohol compounds include ethanol, isopropanol, or mixtures thereof.

[0050] In some embodiments of the assay method, the contacting and measuring steps are performed in a sealed device. It will be carried out.

[0051] In some embodiments of the assay method, the level of toxicity can be measured by, but is not limited to, the above amounts. The expression or function of abiotic metabolizing enzymes, such as cytochrome P450 (CYP) enzymes, during HH This is measured by quantifying the activity.

[0052] In some embodiments of the assay method, the level of toxicity is determined by measuring the amount of acetaldehyde in the HH. Toxic metabolites of alcohol, such as hydroxybenzoates, or reduction of glutathione, activation of cell death pathways It is measured by quantifying the state.

[0053] Methods for assaying candidate agents for promoting alcohol or vitamin A metabolism The method further provides a step of: culturing a quantity of HH in a medium not supplemented with a candidate drug; The above amount of HH cultured in a medium supplemented with DMSO2 or TMSO was treated with alcohol or or vitamin A; and and measuring the level of alcohol or vitamin A metabolism by the HH. nothing.

[0054] Medium from the recovery phase (Phase 1), e.g., H in dHCGM or dHMM H was initially cultured and / or medium from the application phase (phase 2), e.g., d Culturing HH in 2HCGM, tHCGM, d2HMM, or tHMM allows for high-quality Support quality HH.

[0055] Therefore, aspects of the present invention include: a step of culturing HH in a culture medium to produce a culture medium. The culture medium was incubated with the topcoat and high-quality HH for at least 1 hour and used as conditioned medium. and collecting the CMHH.

[0056] In a specific embodiment, the HH is humanized liver chimeric mouse-derived human hepatocytes (HLCM). -HH), primary human hepatocytes (PHH), chimeric animals with humanized livers, e.g., rats, Hepatocytes obtained from mice, sheep, pigs, or monkeys, or a combination thereof. In some embodiments, the HH is PHH. In some embodiments, the HH is HLC In some embodiments, the HH is a mouse other than a mouse with a humanized liver. It is obtained from animals.

[0057] In certain embodiments, the HLCM-HH is a PHH or a previously isolated HLCM. -HH is obtained from liver obtained from a mouse injected intraspleen. HH was then injected into the spleen with HH previously isolated from PHH or humanized liver chimeric animals. Liver obtained from another animal (e.g., rat, mouse, sheep, pig, monkey, etc.) is obtained from

[0058] In some embodiments, the mice are at least 10% HLCM-HH before HLCM-HH is obtained. Human hepatocyte replacement index [based on histological examination or HH-derived factors (e.g., human albumin and or human alpha-1 antitrypsin (as indicated by blood levels of Chimeric animals with a humanized liver must contain at least 10% human liver before the HH is isolated or obtained. It has a cell replacement index.

[0059] In a further embodiment, HLCM-HH, PHH, or another chimeric animal with a humanized liver. Hepatocytes from the specimen, or any combination of two or all three, are 0.5 × 10 5 pieces / cm 2 ~5×10 5 pieces / cm 2 The cells are cultured at a cell density of .

[0060] In a further embodiment, HH is expressed in HepG2 cells, Huh7 cells and mouse hepatocytes. In another embodiment, the HH comprises HLCM-HH and does not include any of the HH cells. In addition, it may contain some mouse hepatocytes and mouse non-parenchymal cells.

[0061] In a further embodiment, the HH comprises HLCM-HH, and the method further comprises the culturing step The method further includes a step of obtaining HLCM-HH before the step of obtaining HLCM-HH, wherein the step of obtaining HLCM-HH includes P From the liver of mice injected intraspleen with HH or pre-isolated HLCM-HH and isolating hepatocytes obtained from the culture, thereby obtaining HLCM-HH. Includes.

[0062] In a further embodiment, the HH obtained from mouse liver is mouse liver collagen. HH are isolated by perfusion with rhesus monkeys, and mice must be at least 10% human before the HH are isolated. It has a hepatocyte replacement index.

[0063] A further aspect of the present invention is a method for treating a pulmonary edema by administering to a patient a therapeutically effective amount of one or more humoral factors secreted by HH and A CMHH comprising a hepatocyte clonal expansion medium or a hepatocyte maintenance medium is provided.

[0064] In a further embodiment, the medium used to prepare CMHH is dHCGM , dHCGM, or tHCGM. In some embodiments, dHCGM is DM SO, standard cell culture basal medium, L-proline, insulin, dexamethasone, EGF and Asc-2P. Particularly preferred is a standard Basal cell culture medium is supplemented with HEPES, penicillin-streptomycin, and serum. In some embodiments, d2HCGM is prepared by adding DMSO2, a standard cell culture basal medium, or Contains glutathione, L-proline, insulin, dexamethasone, EGF and Asc-2P Particularly preferably, the standard cell culture basal medium is a medium containing, or consists essentially of, HEPE. In some embodiments, the serum is supplemented with S, penicillin-streptomycin, and serum. , tHCGM, TMSO, standard cell culture basal medium, L-proline, insulin, Particularly desirable are cyclohexyl 1-hydroxybenzoates (EGF), ... Preferably, the standard cell culture basal medium is HEPES, penicillin-streptomycin. In a preferred embodiment, the standard cell culture basal medium is D MEM-10. In some embodiments, the serum is FBS or human serum.

[0065] In some embodiments, the medium used to prepare CMHH is dHMM, d In some embodiments, dHMM is DMSO, standard Basal cell culture medium, L-proline, insulin, dexamethasone and Asc-2P In some embodiments, the d2HMM comprises or consists essentially of DM SO2, standard cell culture basal medium, L-proline, insulin, dexamethasone and In some embodiments, the tHM comprises or consists essentially of Asc-2P. M: TMSO, standard cell culture basal medium, L-proline, insulin, dexamethasone In a preferred embodiment, the method comprises or consists essentially of Asc-2P and Asc-2P. Therefore, standard cell culture basal media contains HEPES, penicillin-streptomycin and A particularly preferred standard cell culture basal medium is DMEM-10, which is supplemented with serum. In some embodiments, the serum is FBS or human serum.

[0066] In a further embodiment, the medium used to prepare CMHH comprises the following components: L-proline is 5 to 25 μg / mL, and insulin is 0.1 to 0.5 μg / mL. g / mL, dexamethasone is 10-100 nM, and EGF is 0-10 ng / mL, Asc-2P is 0.01 to 1 mM, and DMSO, DMSO2 or TMSO is 35 to 300 mM, HEPES is 10 to 50 mM, and penicillin The concentration of riboflavin is 10 to 300 IU / mL, and the concentration of streptomycin is 10 to 300 μg / mL. and the serum is 2 to 20% heat-inactivated FBS or human serum. -Proline is 15 μg / mL, insulin is 0.25 μg / mL, and Samethasone was 50 nM, and EGF was dHCGM, d2HCGM, or tHCG. 5 ng / mL in dHMM or 0 ng / mL in dHMM, d2HMM, or tHMM Asc-2P was 0.1 mM, and DMSO, DMSO2, or TMSO was 0.1 mM. The concentrations of HEPES and HCl were 281.6 mM, 140.8 mM, and 70.4 mM, respectively. 0 mM, penicillin 100 IU / mL, streptomycin 100 μg / mL and serum, eg, FBS or human serum, is 10%.

[0067] In a further embodiment, the medium used to prepare the CMHH is added to a new volume and is replaced every 1 to 120 hours.

[0068] A further aspect of the invention provides CMHH prepared by the method of the invention.

[0069] In some embodiments, the CMHH comprises dHCGM or dHMM, Or dHMM was used to measure HLCM-HH, PHH, or a combination thereof for at least 1 hour. The cells were cultured for 1 hour.

[0070] In a further embodiment, at least one HLCM-HH, PHH, or a combination thereof is administered. The dHCGM used for incubation contains L-proline, insulin, dexamethasone, and It contains standard cell culture basal medium supplemented with erythritol, EGF, Asc-2P, and DMSO. Preferably, standard cell culture basal media is HEPES, penicillin-streptomycin, In some embodiments, the serum is supplemented with FBS or human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-10. do.

[0071] In a further embodiment, at least one HLCM-HH, PHH, or a combination thereof is administered. The dHMM used for incubation contains L-proline, insulin, and dexamethasone. Particularly preferred are standard cell culture basal media supplemented with DMSO, Asc-2P, and DMSO. Standard cell culture basal media is composed of HEPES, penicillin-streptomycin and In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0072] In some embodiments, the CMHH comprises d2HCGM or d2HMM, and d2H Use CGM or d2HMM to reduce HLCM-HH, PHH, or a combination Both were cultured for 1 hour.

[0073] In a further embodiment, at least one HLCM-HH, PHH, or a combination thereof is administered. The d2HCGM used for incubation contains L-proline, insulin, dexamethasone, and Contains standard cell culture basal medium supplemented with ATP, EGF, Asc-2P, and DMSO. Particularly preferred is a standard cell culture basal medium containing HEPES, penicillin-streptomycin, In some embodiments, the serum is supplemented with FBS or human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-10 is.

[0074] In a further embodiment, at least one HLCM-HH, PHH, or a combination thereof is administered. The d2HMM used for incubation contains L-proline, insulin, dexamethasone, and The medium contains standard basal cell culture medium supplemented with ethanol, Asc-2P, and DMSO2. Alternatively, standard cell culture basal media may contain HEPES, penicillin-streptomycin, and and serum. In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-10.

[0075] In some embodiments, the CMHH comprises tHCGM or tHMM, Or, using tHMM, HLCM-HH, PHH, or a combination thereof for at least 1 hour. The cells were cultured for 1 hour.

[0076] In a further embodiment, at least one HLCM-HH, PHH, or a combination thereof is administered. The tHCGM used for incubation contains L-proline, insulin, dexamethasone, and It contains standard cell culture basal medium supplemented with ATP, EGF, Asc-2P, and TMSO. Preferably, standard cell culture basal media is HEPES, penicillin-streptomycin, In some embodiments, the serum is supplemented with FBS or human serum. In a preferred embodiment, the standard cell culture basal medium is DMEM-10. do.

[0077] In a further embodiment, at least one HLCM-HH, PHH, or a combination thereof is administered. The tHMM used for incubation was supplemented with L-proline, insulin, and dexamethasone. Particularly preferred are standard cell culture basal media supplemented with TMSO, Asc-2P, and TMSO. Standard cell culture basal media is composed of HEPES, penicillin-streptomycin and In some embodiments, the serum is FBS or human serum. In a preferred embodiment, the standard basal cell culture medium is DMEM-10.

[0078] A further aspect of the invention provides a combination comprising CMHH and an extracellular matrix. In some embodiments, the extracellular matrix is ​​Matrigel®, Angelica Angew. A gelatinous protein secreted by EHS mouse sarcoma cells Mixtures of fibronectin, collagen, vitronectin, and laminin These include, but are not limited to, individual extracellular matrices.

[0079] A further aspect of the present invention is a method for producing a CMHH of the present invention in combination with forskolin, SB431542, D A combination containing five chemicals: APT, IWP2, and LDN193189. do.

[0080] In various embodiments, CMHH is used for liver cell biology studies. A major current limitation in cell biology research is the lack of stable in vitro expression of terminally differentiated HH. For example, cryopreserved PHHs are not suitable for most pharmaceuticals. Industry is looking to develop new drugs for drug safety screening and drug metabolism and pharmacokinetics ( It is the main tool used in DMPK research.

[0081] Aspects of the present invention include terminally differentiated PHH, HLCM-HH, and other chimeras with humanized livers. Hepatocytes obtained from animals (e.g., rats, sheep, pigs, monkeys), other species, e.g., Primary hepatocytes from mice, rats, and monkeys, and / or cryopreserved P HH, HLCM-HH, and other chimeric animals with humanized livers (e.g., rats, sheep, and hamsters) Hepatocytes obtained from other species, e.g., dogs, mice, rats, and monkeys CMHH for use in culturing primary hepatocytes is provided.

[0082] In some embodiments, the biological effect of CMHH is to stimulate the proliferation of hepatic stellate cells (HSCs), hepatic sinusoids, Hepatic non-endothelial cells such as endothelial cells (LSECs), and hepatic macrophages (Kupffer cells) Supports the function of stromal cells (NPCs).

[0083] A further aspect of the present invention is to provide a method for producing a humanized liver comprising administering to a subject a patient having terminally differentiated HH, HLCM-HH, or another humanized liver. Hepatocytes obtained from chimeric animals (e.g., rats, sheep, pigs, monkeys), as well as PH H, and / or primary hepatocytes from other species, e.g., dog, mouse, rat, and monkey In some embodiments, the provided CMHH is used to culture the CMHH. HH includes terminally differentiated in vitro cultured HH, HLCM-HH, and humanized liver. Hepatocytes obtained from other chimeric animals (e.g., rats, sheep, pigs, monkeys) with and PHH, and / or primary livers from other species, e.g., dog, mouse, rat, and monkey. Prevents cell dedifferentiation.

[0084] Further embodiments of the present invention include HH, HLCM-HH, and other chimeric animals with humanized livers ( Hepatocytes obtained from animals such as rats, sheep, pigs, and monkeys, as well as PHHs and / or or transdifferentiation of primary hepatocytes from other species, such as dog, mouse, rat, and monkey. Provides CMHH to be used to prevent

[0085] A further aspect of the present invention is to provide a method for the preparation of cryopreserved HH, HLCM-HH, and other humanized livers. Hepatocytes obtained from chimeric animals (e.g., rats, sheep, pigs, monkeys), as well as P HH, and / or primary hepatocytes from other species, e.g., dog, mouse, rat, and monkey provide CMHH for use in promoting recovery.

[0086] A further aspect of the present invention is the prevention of de novo cell death from stress / injury associated with the cell acquisition process. Isolated HH, HLCM-HH, and other chimeric animals with humanized livers (e.g., rats, Hepatocytes obtained from PHH, and / or other species, e.g., sheep, pig, monkey). For example, it is used to promote the recovery of primary hepatocytes from dogs, mice, rats, and monkeys. Provide CMHH.

[0087] A further aspect of the present invention is to protect HH, HLCM-H from cellular stress associated with cryopreservation. H, derived from another chimeric animal (e.g., rat, sheep, pig, monkey) with a humanized liver. Hepatocytes from PHH, and / or other species, e.g., dog, mouse, rat, and We provide CMHH, which is used to protect primary hepatocytes derived from monkeys and other mammals, against thawing. This could further support the recovery of

[0088] A further aspect of the present invention is to provide a method for the maintenance and function of NPC function and morphology. In some embodiments, the CMHH is used to treat NPCs. Suspending human hepatocytes for use in co-culture and / or cryopreservation Provided for use in

[0089] A further aspect of the invention is the production of spheroids, organoids or 3D cultures, such as In addition, to develop organ-chip formats, we have developed HH, HLCM-HH, and other organ-chips with humanized livers. Hepatocytes obtained from chimeric animals (e.g., rats, sheep, pigs, monkeys), and PH H, and / or primary hepatocytes from other species, e.g., dog, mouse, rat, and monkey The present invention provides CMHH for use in supporting / enhancing the function and lifespan of a mammal.

[0090] A further aspect of the present invention is to use HH, HLCM-HH, humanized Hepatocytes obtained from the liver of another chimeric animal (e.g., rat, sheep, pig, monkey), and PHH, and / or from other species, e.g., dog, mouse, rat, and monkey CMHH for use in enhancing the utility of primary hepatocytes is provided.

[0091] Further embodiments of the present invention include HH, HLCM-HH, and other chimeric animals with humanized livers ( Hepatocytes obtained from animals such as rats, sheep, pigs, and monkeys, as well as PHHs and / or or cell fate maintenance of primary hepatocytes from other species (e.g., dog, mouse, rat, and monkey). and providing a CMHH for use in supporting the maintenance of DMPK; for various types of liver cell biology research, including drug screening, infectious and metabolic diseases This greatly enhances the usefulness of these cells.

[0092] In various embodiments, CMHH includes PHH and HLCM-HH and humanized liver. It is used to maintain the cell fate of HH obtained from another chimeric animal with liver; thereby, HH is a multidisciplinary team focused on various types of liver diseases, including DMPK, drug screening, infectious diseases, and metabolic diseases. This greatly enhances the utility of these cells for cell biology research.

[0093] Methods are further provided for assaying the toxicity of drugs to HH or the DMPK of drugs. The method was performed on samples supplemented with DMSO or TMSO and on samples not supplemented with DMSO. Incubating a quantity of HH in fresh CMHH; contacting the amount of HH cultured in CMHH supplemented with or measuring the level of toxicity to said amount of HH after contact with said agent; is the metabolism or excretion of the drug by the above amount of HH in the presence of or after contact with the drug. and measuring the level.

[0094] In some embodiments of the assay method, the agent is ethanol, methanol, ethylene glycol, or the like. Alcohol compounds include ethanol, isopropanol, or mixtures thereof.

[0095] In some embodiments of the assay method, the contacting and measuring steps are performed in a sealed device. It will be carried out.

[0096] In some embodiments of the assay method, the level of toxicity can be measured by, but is not limited to, the above amounts. The expression or function of abiotic metabolizing enzymes, such as cytochrome P450 (CYP) enzymes, during HH This is measured by quantifying the activity.

[0097] In some embodiments of the assay method, the level of toxicity is determined by measuring the amount of acetaldehyde in the HH. Toxic metabolites of alcohol, such as hydroxybenzoates, or reduction of glutathione, activation of cell death pathways It is measured by quantifying the state.

[0098] Methods for assaying candidate agents for promoting alcohol or vitamin A metabolism The method further provides a step of: culturing a quantity of HH in CMHH that is not supplemented with a candidate drug; The above amount of HH cultured in CMHH supplemented with DMSO2 or TMSO was contacting the sample with choline or vitamin A; in the presence or contact of a candidate drug; After contact, the level of alcohol or vitamin A metabolism by the above amount of HH is measured. Includes the following:

[0099] In various embodiments, CMHH is used in regenerative medicine and stem cell biology. MHH is an essential factor that enables the final step of iHep maturation into terminally differentiated human hepatocytes In some embodiments, the CMHH is used in the preparation of a hepatocyte transplant therapy, For example, mass production of mature human hepatocytes derived from iPS cells (potential substitutes for LT) Used for.

[0100] A further aspect of the present invention is the use of iHep (stem cell derived hepatocytes) to terminally differentiated hepatocytes. The present invention provides CMHH for use in facilitating further maturation of stem cells (progenitor cells).

[0101] A further aspect of the present invention is the preparation of iHep (stem cell derived hepatocytes) into cholangiocytes or other cell types. The present invention provides CMHH for use in preventing differentiation of mitochondrial cells (mitochondrial progenitor cells).

[0102] A further aspect of the present invention is the use of chemically induced liver progenitors (CLs) into mature hepatocytes. The present invention provides CMHH for use in promoting regeneration of ip.

[0103] A further aspect of the invention is the use of hepatoma cells (e.g., HepaRG cells, HepG2 and The present invention provides CMHH for use in enhancing liver function in human liver cells (Huh1 and Huh7 cells).

[0104] In some embodiments, CMHH is used to enhance the liver function of HepaRG. will be done.

[0105] A further aspect of the present invention is the use of CMHH-treated iHep and and CLiP expansion / propagation. In an embodiment, the present invention relates to CMHH-treated PHH in the liver of a HLCM host mouse, and / or expansion of primary hepatocytes from other species, e.g., dog, mouse, rat, and monkey. Provide CMHH for use in enabling large / breeding.

[0106] A further aspect of the present invention is a method for the regression of chronic liver disease / liver fibrosis resulting from hepatocyte transplantation therapy. provide CMHH for use in promoting the

[0107] A further aspect of the present invention is a method for the treatment of liver cancer using CMH as an adjuvant therapy. Provide H.

[0108] Further aspects of the present invention include differentiation and cell migration of iHep, CliP, and HepaRG. CMHH is used to support life support and has the characteristics of mature hepatocytes. CMHH allows stable long-term culture of these cells without compromising DMP. K, drug screening, infectious and metabolic diseases, and various types of liver cell biology research This greatly enhances the usefulness of these cells for therapeutic purposes.

[0109] A further aspect of the present invention is the differentiation of chemically induced liver progenitor (CLip) cells. The present invention provides CMHH for use in generating

[0110] In some embodiments, CMHH is used to promote the redifferentiation of CLiPs into mature hepatocytes. It is used for this purpose.

[0111] In various embodiments, CHMM is used to identify HH, other species, e.g., dogs, mice, and , rat- and monkey-derived PHH, and liver cells obtained from humanized liver chimeric mice (HLCM). cells, or another chimeric animal with a humanized liver (e.g., rat, sheep, pig, or monkey) Cultivating and maintaining hepatocytes, which may include hepatocytes obtained from do.

[0112] In some embodiments, CMHH is used for hepatocyte transplantation into patients suffering from any type of liver disease. (such as iHep, which has been further differentiated by CMHH).

[0113] Various embodiments of the present invention have been described above in the description of the invention. Although the embodiments are described directly, those skilled in the art will appreciate that the embodiments shown and described herein are It is understood that modifications and / or variations to the specific embodiments described may be possible. Any such modifications or variations that fall within the scope of the description are also included within the scope of the description. Unless otherwise indicated, the words and phrases in the specification and claims are intended to be The words have their ordinary and accustomed meaning to those of ordinary skill in the applicable art. It is the intention of the inventors that

[0114] The foregoing description of various embodiments of the present invention known to applicant at the time of filing this application has been presented. It is intended for purposes of illustration and description. This specification is not intended to be exhaustive. However, it is not intended to limit the invention to the precise form disclosed, but rather to Many modifications and variations are possible, and the described embodiments are to be construed as illustrative, not restrictive, of the principles of the present invention and their equivalents. The present invention illustrates a practical application of the present invention and allows others skilled in the art to adapt various implementations to suit the particular use envisioned. It serves to enable the present invention to be utilized with various modifications in the embodiments. Therefore, it is not intended that the invention be limited to the particular embodiments disclosed for carrying out the invention. It is not intended to be a diagram.

[0115] Although specific embodiments of the present invention have been shown and described, based on the teachings herein, Therefore, changes and modifications may be made without departing from the present invention and its broader aspects. It is therefore the appended claims that fall within the true spirit and scope of this invention. It will be apparent to those skilled in the art that all such changes and modifications are to be included within the scope of the present invention. Generally, the term "open" as used herein is not intended to be a general term. (For example, the term "including" should be interpreted as meaning "including, but not limited to" and the term "having" should be interpreted as "having at least " and the term "includes" should be interpreted as "including, but not limited to, It will be understood by those skilled in the art that the terms "above" and "below" should be interpreted as "not limited to" and "not limiting" respectively.

[0116] As used herein, the terms "comprising" or "comprise" "(s)" is used in reference to compositions, methods, and their respective components, which are While useful in the embodiments, the inclusion of unspecified elements is embraced, whether useful or not. Generally, the term "open" is used herein to refer to the term "open" in general (e.g., For example, the term "including" should be interpreted as "including, but not limited to." and the term "having" should be interpreted as "having at least" and the term "includes" should be interpreted as "including, but not limited to" It will be understood by those skilled in the art that "including" should be interpreted as "not including" or "not including." ding, containing, or having The open-ended term "comprising," which is a synonym for the term, As used herein to describe and claim the present invention, or An embodiment may be "consisting of" or "consisting essentially of" Use alternative terms such as "consisting essentially of" It may be stated.

[0117] The singular terms "a," "an," and "the" refer to plural terms unless the context clearly indicates otherwise. Similarly, the word "or" includes the referent unless the context clearly indicates otherwise. The terms "and" and "and" are intended to encompass the methods and materials described herein. Although similar or equivalent methods and methods can be used in the practice or testing of the present disclosure, The ingredients are described below. The abbreviation "eg" comes from the Latin "exempli grati a" and is used herein to provide a non-limiting example. The word "eg" is synonymous with the term "for example."

[0118] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be used individually or in combination with other members of the group or in any combination herein. The present invention may be referenced and claimed in any combination with other elements found in the group. Members may be included in or deleted from a group for reasons of convenience and / or patentability. When either such inclusion or deletion occurs, the specification is treated as modified. The term "group" is considered herein to contain a group and, accordingly, may be used in the appended claims. satisfies the description of all Markush groups that can be

[0119] Unless otherwise specified herein, the scientific and technical terms used in connection with this application Terms shall have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The disclosure is limited to the particular methodology, protocols, and reagents described herein. It should be understood that these are not limiting and may vary as such. The terminology is for the purpose of describing particular embodiments only and is defined by the claims. It is not intended to limit the scope of the present invention, which is defined solely by the following: Definitions of common terms in biology are found in The Merck Manual of Diagnosis and Therapy, 1 9th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19- 3);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology a nd Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783 527600908);およびRobert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-5 6081-569-8);Immunology by Werner Luttmann, published by Elsevier, 2006;Janeway 's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Fra ncis Limited, 2014 (ISBN 0815345305, 9780815345305);Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Sprin g Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414 );Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing , Inc., New York, USA (2012) (ISBN 044460149X);Laboratory Methods in Enzymology : DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542);Current Protocols in M Olecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 ( ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), Jo hn E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 047114273 5, 9780471142737), the contents of which are incorporated herein by reference in their entirety. will be incorporated into

[0120] Those skilled in the art can readily identify the chemotherapeutic agent to use (see, e.g., Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning;Principles of Cancer Therapy, Chapter 85 in Harrison's Princ iples of Internal Medicine, 18th edition;Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abelo ff's Clinical Oncology, 2013 Elsevier; and Fischer DS (ed): The Cancer Chemot (See Therapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).

[0121] In some embodiments of any of the present aspects, the disclosure described herein provides a method for detecting a human cross-linked marker. The process of cloning, modifying the genetic identity of human germ cells, of the human embryo No industrial or commercial use or any substantial medical benefit to humans or animals processes that modify the genetic identity of animals that may cause suffering to others, and It does not concern the animals resulting from the process.

[0122] Other terms are defined herein in the description of various aspects of the invention.

[0123] Cited throughout this application are literature references, issued patents, published patent applications and concurrently issued patents. All patents and other publications, including pending patent applications; Describe and use the methodologies described in such publications that may be used in connection with the technology. and are expressly incorporated herein by reference for the purposes of disclosure. In this respect, only disclosures prior to the filing date of the present application are provided. shall be construed as an admission that you have no right to antedate such disclosure for any reason. Any statements as to the dates or contents of these documents should not be taken as proof that the applicant It is based on the information available and there is no representation as to the correctness of the dates or contents of these documents. does not constitute any endorsement.

[0124] The description of the embodiments of the present disclosure is not intended to be exhaustive or to represent the present disclosure in the precise form disclosed. While specific embodiments and examples of the present disclosure are not intended to be limiting, Described herein for illustrative purposes, and as will be appreciated by those skilled in the relevant art, Various equivalent modifications are possible within the scope of this disclosure. For example, a method step or function can be Although present in the given order, alternative embodiments may perform the functions in a different order, and the functions may The teachings of the disclosure provided herein may be implemented in a manner that is convenient for implementing the present invention. The various embodiments described herein may be applied to other procedures or methods. Aspects of the present disclosure can be combined to provide various embodiments. and modifications as necessary to utilize the compositions, functions, and concepts of the application. Further embodiments are possible. Furthermore, considerations of biological functional equivalence may be taken into account. This allows the protein structure to be altered without affecting the type or amount of biological or chemical action. These and other changes are within the scope of the present disclosure in view of the detailed description. All such modifications are intended to be within the scope of the appended claims. It is intended to.

[0125] Certain elements of any of the foregoing embodiments may be combined with elements in other embodiments. Furthermore, advantages associated with certain embodiments of the present disclosure include the Although these embodiments have been described, other embodiments may exhibit such advantages. Not all embodiments need exhibit such advantages to fall within the scope of this disclosure. Nothing.

[0126] Some embodiments of the technology described herein may include any of the following numbered paragraphs: It can be defined by: 1. A method for preparing conditioned medium from cultured human hepatocytes, comprising: culturing human hepatocytes in a culture medium to produce a culture medium; Culture medium incubated with human hepatocytes for at least 1 hour was collected as conditioned medium. Steps and A method comprising: 2. Human hepatocytes are derived from primary human hepatocytes (PHH), chimeric animals with humanized livers. 10. The method of paragraph 1, comprising: a hepatocyte; 3. Hepatocytes obtained from chimeric animals with humanized livers are derived from humanized liver chimeric mice. Human hepatocytes (HLCM-HH), which can be isolated from PHH or previously isolated obtained from the liver of a mouse injected intraspleen with HLCM-HH; Method 2. 4. Before obtaining HLCM-HH, mice must have a replacement index of at least 10%. The method of paragraph 3. 5. The medium is hepatocyte clonal expansion medium (dHC) supplemented with DMSO or DMSO. GM), and dHCGM is Dulbecco's modified Eagle's medium (DMEM), L-proline , insulin, dexamethasone, EGF, and L-ascorbic acid 2-phosphate (Asc 10. The method of paragraph 1, comprising, or consisting essentially of: 6. DMEM is DMEM-10, and DMEM-10 is a mixture of DMEM, HEPES, Contains or consists of penicillin-streptomycin and fetal bovine serum (FBS). 6. The method of paragraph 5, consisting essentially of 7. L-proline is 15 μg / mL in dHCGM and insulin is M, dexamethasone was 50 nM in dHCGM; EGF was 5 ng / mL in dHCGM, and Asc-2P was 0.1 mM, DMSO or DMSO2 is 2% in dHCGM, and HEPES is 20 mM in dHCGM, and penicillin at 100 IU / mL in dHCGM , streptomycin is 100 μg / mL in dHCGM, FBS is 100 μg / mL in dHCGM 6. The method of paragraph 5, wherein the soluble fraction is 10% heat-inactivated FBS in M. 8. L-proline is 5-25 μg / mL in dHCGM, and insulin is 0.1-0.5 μg / mL in dHCGM, and dexamethasone at 10-1 00 nM, EGF was 1 to 10 ng / mL in dHCGM, and Asc-2P was , 0.01 to 1 mM in dHCGM, and DMSO or DMSO2 in dHCGM The concentration of HEPES in dHCGM is 0.5% to 5%, and the concentration of penicillin is 10 to 50 mM. Phosphorus is 10-300 IU / mL in dHCGM, and streptomycin is 10-300 μg / mL in GM, and 2-20% heat-inactivated FBS in dHCGM The method of paragraph 5 is FBS. 9. Paragraphs 1-8, in which the medium is replaced with a new volume every 24-120 hours. Either one way. 10. PHH, hepatocytes obtained from chimeric animals with humanized livers, or a combination, Cell density 0.5×10 5 pieces / cm 2 ~5×10 5 pieces / cm 2Cultivated in paragraph 2 method. 11. Human hepatocytes do not include HepG2 cells, Huh7 cells, or mouse hepatocytes; Any of the methods in paragraphs 1 to 3. 12. The human hepatocytes include hepatocytes obtained from a chimeric animal having a humanized liver, and the method However, before the culturing step, a step of obtaining hepatocytes obtained from a chimeric animal having a humanized liver is performed. The step of obtaining hepatocytes from a chimeric animal having a humanized liver is carried out by using a PHH or or previously isolated hepatocytes obtained from a humanized liver chimeric animal are injected into the animal's spleen. and isolating hepatocytes from the liver of the animal, thereby producing a humanized liver. 10. The method of paragraph 1, further comprising the step of obtaining hepatocytes obtained from the chimeric animal. 13. Hepatocytes obtained from chimeric animals are purified by collagenase perfusion of the liver of chimeric animals. The chimeric animals were isolated using a 10% replacement index before hepatocytes were isolated. 13. The method of paragraph 12. 14. A cultured human liver prepared by the method described in any one of paragraphs 1 to 13. Conditioned medium derived from cells (CMHH). 15. A hepatocyte clonal expansion medium, wherein the hepatocyte clonal expansion medium is used to grow primary human hepatocytes. Hepatocytes obtained from chimeric animals with humanized livers (PHH), or a combination thereof. Conditioned medium from cultured human hepatocytes (CMHH) used for incubation for at least 1 hour. 16. One or more humoral factors secreted by human hepatocytes and hepatocyte clones Conditioned medium from cultured human hepatocytes (CMHH) containing a nutrient-rich growth medium. 17. Hepatocyte clonal growth medium contains L-proline, insulin, dexamethasone, E GF, L-ascorbic acid 2-phosphate (Asc-2P) and dimethyl sulfoxide ( Dulbecco's modified Eagle's medium supplemented with DMSO or dimethyl sulfone (DMSO). 17. The CMHH-derived conditioned medium of paragraph 15 or paragraph 16, comprising DMEM. 18. A CMHH-derived conditioned medium and extracellular matrix mixture according to any one of paragraphs 14 to 17. Combinations containing ox. 19. A CMHH-derived conditioned medium and forskolin according to any one of paragraphs 14 to 17. , SB431542, DAPT, IWP2 and LDN193189 A combination including: 20. Terminally differentiated human hepatocytes, hepatocytes obtained from chimeric animals with humanized livers, and Cultivated human hepatocytes for use in culturing cryopreserved and / or cryopreserved primary human hepatocytes - Patent Application 20090222997 (CMHH)-derived conditioned medium. 21. Bipotent hepatic progenitor cells (iHep) and / or chemically induced hepatic progenitor cells Use the genita to differentiate into terminally differentiated hepatocytes or mature hepatocytes, respectively. Conditioned medium derived from cultured human hepatocytes (CMHH) for 22. For use in co-culture with non-parenchymal cells and / or for cryopreservation Conditioned medium from cultured human hepatocytes (CMHH) for use in suspending human hepatocytes. 23. Isolation and / or preservation of human hepatocytes (HHC) for recovery and establishment of liver function H) a method for culturing the In the first phase, a certain amount of HH is cultured in a first cell culture medium for a first period of time. Top and The first cell culture medium is removed from the amount of HH, followed by the second phase of Culturing the amount of HH for a second period using a cell culture medium; Including, The first cell culture medium is a cell culture medium supplemented with dimethyl sulfoxide (DMSO). the second cell culture medium is a cell culture medium supplemented with dimethyl sulfone (DMSO). or cell culture medium supplemented with tetramethylene sulfoxide (TMSO), The method, wherein the second cell culture medium does not contain DMSO. 24. The first period is at least 4 days and up to 2 months, and in the first phase The above amount of HH cultured in the presence of cholangiocarcinoma (HC) showed cell polarity, intercellular structures characterized by bile canaliculi, and and / or establish gene expression levels comparable to control HHs exhibiting liver function, 23 ways. 25. The first period is about 7 days, and the first cell culture medium is optionally changed every 3 or 4 days. The method of paragraph 24 is newly established at the discretion of the applicant. 26. The above amount of HH cultured in the second phase is a cytochrome P450 family CYP3A4 subfamily A member 4 (CYP3A4) and cytochrome P450 family have physiological levels of expression of CYP2E1 subfamily E member 1 (CYP2E1); or The amount of HH cultivated in the second phase is free of alcohol, non-biological substances, vitamins, etc. 24. The method of paragraph 23, wherein A or a combination thereof is metabolized. 27. The first cell culture medium is a hepatocyte culture medium supplemented with DMSO, The medium contained DMSO, Dulbecco's modified Eagle's medium (DMEM), and L-proline. Insulin, dexamethasone, epidermal growth factor (EGF), L-ascorbic acid diphosphate Acid salt (Asc-2P), 4-(2-hydroxyethyl)-1-piperazineethanesulfone acid (HEPES), penicillin-streptomycin, and heat-inactivated fetal bovine serum (FB 26. The method of any one of paragraphs 23-25, including one or more of: 28. The second cell culture medium is a hepatocyte culture medium supplemented with DMSO2, The culture medium is DMSO2, DMEM, L-proline, insulin, and dexamethasone. , EGF, Asc-2P, HEPES, penicillin-streptomycin and heat inactivation 27. The method of paragraph 23 or paragraph 26, comprising one or more of the FBS. 29. A second cell culture medium containing DMSO2 at a final concentration of about 140 mM. F23 method. 30. The first cell culture medium is prepared by adding 2 (vol / vol)% DMSO, DMEM, 15 μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1mM Asc-2P, 20mM HEPES, 100IU / mL penicillin, 100 μg / mL streptomycin, and 10% heat-inactivated FBS; The second cell culture medium was prepared by adding 140 mM DMSO2, DMEM, 15 μg / mL L-Propionate, and 1% DMSO. Lorin, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EG F, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, Paragraph 2, containing 100 μg / mL streptomycin and 10% heat-inactivated FBS Method 3. 31. The first cell culture medium is a mixture of 0.5% to 5% (vol / vol) DMSO, DMEM, 5 ~25μg / mL L-proline, 0.1~0.5μg / mL insulin, 10~100 nM dexamethasone, 1–10 ng / mL EGF, 0.01–1 mM Asc-2P, 10-50mM HEPES, 10-300IU / mL penicillin, 10-300μg / mL streptomycin and 2–20% heat-inactivated FBS; The second cell culture medium is DMEM supplemented with 100-180 mM DMSO, 5-25 μg / mL mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM dexamethasone Tazone, 1–10 ng / mL EGF, 0.01–1 mM Asc-2P, 10–50 m M HEPES, 10-300 IU / mL penicillin, 10-300 μg / mL streptomycin 24. The method of paragraph 23, comprising 2-20% heat-inactivated FBS. 32. The efficacy of candidate drugs in promoting alcohol metabolism by HH or candidate drugs for HH 1. A method for use in assaying the toxicity of a co-agent, comprising: contacting the amount of HH cultured in the second phase with a candidate drug; Before contact with the candidate drug, the first test was performed to measure the metabolism of alcohol or the excretion of the candidate drug by the above amount of HH. and measuring the level of HH in the presence or after contact with the candidate agent. and measuring a second level of alcohol metabolism or excretion of the candidate drug. RAGRAPH 23 METHOD. 33. Cell culture medium supplemented with dimethyl sulfone (DMSO) or tetramethylene Contains cell culture medium supplemented with TMSO and dimethyl sulfoxide (DMSO) SO-free human hepatocyte (HH) culture for establishing and maintaining liver function Culture medium. 34. DMSO2 reaches a concentration of approximately 140 mM or approximately 2% (vol / vol) in DMEM. DMEM supplemented with L-proline, insulin, dexamethasone, EGF , Asc-2P, HEPES, penicillin-streptomycin and heat-inactivated FBS 34. The human hepatocyte culture medium of paragraph 33, further comprising one or more of: 35. 140 mM DMSO2, 15 μg / mL L-proline, 0.2 5 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / m 35. The human hepatocytes of paragraph 34, containing L-streptomycin and 10% heat-inactivated FBS. Culture medium. 36. 100–180 mM DMSO2, 5–25 μg / mL L-Propionate in DMEM Phosphorus, 0.1–0.5 μg / mL insulin, 10–100 nM dexamethasone, 1–1 0ng / mL EGF, 0.01~1mM Asc-2P, 10~50mM HEPES , 10-300IU / mL penicillin, 10-300μg / mL streptomycin and 35. The human hepatocyte culture medium of paragraph 34, comprising 2 to 20% heat-inactivated FBS. 37. A first container for storing a first cell culture medium, wherein the first cell culture medium a first container that is supplemented with DMSO; and a second container for storing a second cell culture medium, said second cell culture medium being DMS; A second container supplemented with O2 and / or TMSO but not DMSO. A kit for culturing human hepatocytes, comprising: 38. Toxicity of drugs to human hepatocytes (HH) or drug metabolism and pharmacokinetics of drugs ( 1. A method for assaying DMPK, comprising: Cultivate a certain amount of HH in medium supplemented with DMSO and medium not supplemented with DMSO. Steps and; contacting the agent with the amount of HH cultured in a medium supplemented with DMSO2; and; Measure the level of toxicity to the above dose of HH in the presence of or after contact with the drug. step, or metabolism of the drug by the above amount of HH in the presence of or after contact with the drug. or measuring the level of emissions; A method comprising: 39. The drug is ethanol, methanol, ethylene glycol, isopropanol or 39. The method of paragraph 38, wherein: is an alcohol compound including mixtures thereof. 40. The method of paragraph 39, in which the contacting and measuring steps are carried out in a sealed device Law. 41. The level of toxicity is determined by the expression or is measured by quantifying the function, the method of paragraph 38. 42. How to assay candidate drugs for promoting alcohol or vitamin A metabolism It is a law, Cultivate a certain amount of HH in medium supplemented with DMSO and medium not supplemented with DMSO. Steps and; The above amount of HH cultured in DMSO2-supplemented medium in the presence of a candidate drug was subjected to alcohol treatment. contacting the soluble fiber with ethanol or vitamin A; The presence of or contact with a candidate drug followed by the above-mentioned amount of HH was examined for alcohol or vitamin D. measuring the level of metabolism of amine A; A method comprising:

[0127] Some embodiments of the technology described herein may include any of the following numbered paragraphs: It can be defined by: 1. A method for preparing conditioned medium from cultured human hepatocytes (HH), comprising: Culturing the HH in a culture medium to produce a culture medium; Culture medium incubated with HH for at least 1 hour was collected as conditioned medium. Top and A method comprising: 2. HH is a primary human hepatocyte (PHH), a liver derived from a chimeric animal with a humanized liver. 10. The method of paragraph 1, comprising a cell, or a combination thereof. 3. Hepatocytes obtained from chimeric animals with humanized livers are derived from humanized liver chimeric mice. Human hepatocytes (HLCM-HH), which can be isolated from PHH or previously isolated obtained from the liver of a mouse injected intraspleen with HLCM-HH; Method 2. 4. Before obtaining HLCM-HH, mice must have a replacement index of at least 10%. The method of paragraph 3. 5. The medium contains neither dimethyl sulfoxide (DMSO) nor dimethyl sulfone (DMSO2). or tetramethylene sulfoxide (TMSO)-supplemented hepatocyte clonal expansion medium ( dHCGM, dHCGM, or tHCGM), where HCGM is a standard cell culture medium Foundation medium, L-proline, insulin, dexamethasone, EGF, and L-ascorbic acid 2-phosphate (Asc-2P), method. 6. The medium is hepatocyte maintenance medium (dH) supplemented with DMSO, DMSO2, or TMSO. MM, d2HMM, tHMM), where HMM is a standard cell culture basal medium, L-Pro Phosphorus, insulin, dexamethasone, and L-ascorbic acid diphosphate (Asc-2P) 10. The method of paragraph 1, comprising or consisting essentially of: 7. Standard cell culture basal media include DMEM, MEM, RPMI-1640, and IMDM. or William E. medium, Law. 8. Standard cell culture basal media is formulated with HEPES, penicillin-streptomycin, and 8. The method of paragraph 7, wherein the method is supplemented with a serum. 9. The standard cell culture basal medium is DMEM, and DMEM is DMEM-10; DMEM-10 contains DMEM, HEPES, penicillin-streptomycin and serum 8. The method of paragraph 7, comprising or consisting essentially of: 10. Paragraphs 8 or 9, wherein the serum is fetal bovine serum (FBS) or human serum. Either way. 11. dHCGM, d2HCGM or tHCGM in standard basal cell culture medium 15 μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone 281.6 mM, 14 mM, 5 ng / mL EGF, 0.1 mM Asc-2P, respectively. 0.8mM or 70.4mM DMSO, DMSO2, or TMSO, 20mM H EPES, 100 IU / mL penicillin, 100 μg / mL streptomycin, and 6. The method of paragraph 5, comprising or consisting essentially of 10% serum. 12. dHCGM, d2HCGM, and tHCGM are used in standard cell culture basal medium. 25 μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 n M dexamethasone, 1–10 ng / mL EGF, 0.01–1 mM Asc-2P, 5 0-300mM DMSO, DMSO2 or TMSO, 10-50mM HEPES, 10 to 300 IU / mL penicillin, 10 to 300 μg / mL streptomycin, and 6. The method of paragraph 5, comprising or consisting essentially of and 2-20% serum. 13. dHMM, d2HMM, and tHMM were added at 15 μg / ml in standard basal cell culture medium. mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 0.1 mM Asc-2P, 281.6 mM, 140.8 mM, or 70.4 mM, respectively DMSO, DMSO2 or TMSO, 20 mM HEPES, 100 IU / mL penicillin containing or containing 10% serum, 100 μg / mL cyclosporine, 100 μg / mL streptomycin, and 10% serum. 6. The method of paragraph 6, consisting essentially of 14. dHMM, d2HMM, and tHMM were cultured at 5-25 μL in standard basal cell culture medium. μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM dextran Samethasone, 0.01-1 mM Asc-2P, 50-300 mM DMSO, DMSO 2, TMSO, 10-50 mM HEPES, 10-300 IU / mL penicillin, 10 Containing or consisting of ~300 μg / mL streptomycin and 2–20% serum The method of paragraph 6 essentially becomes. 15. The medium is replaced with a new volume every 24 to 120 hours, paragraphs 1-1 One of the four methods. 16. PHH, hepatocytes obtained from chimeric animals with humanized livers, or a combination thereof, Cell density 0.5×10 5 pieces / cm 2 ~5×10 5 pieces / cm 2 Cultivated in paragraph 2 method. 17. HH does not contain HepG2 cells, Huh7 cells or mouse hepatocytes. Use any of the methods from Rough 1 to 3. 18. The HH comprises hepatocytes obtained from a chimeric animal having a humanized liver, and the method comprises culturing The cultivation step was preceded by a step of obtaining hepatocytes from a chimeric animal having a humanized liver. The step of obtaining hepatocytes from a chimeric animal having a humanized liver is carried out using a PHH or humanized liver. Animals injected intraspleen with previously isolated hepatocytes obtained from the simulated liver chimeric animals. and isolating hepatocytes obtained from the liver of the subject, thereby producing a chimeric liver having a humanized liver. 10. The method of paragraph 1, further comprising the step of obtaining hepatocytes obtained from the animal. 19. Hepatocytes obtained from chimeric animals are purified by collagenase perfusion of the liver of chimeric animals. and chimeric animals were isolated with a replacement index of at least 10% before the HH was isolated. The method of paragraph 18. 20. A cultured human liver prepared by the method described in any one of paragraphs 1 to 19. Conditioned medium derived from cells (CMHH). 21. The CMHH comprises a hepatocyte clonal expansion medium or a hepatocyte maintenance medium, wherein the medium is Primary human hepatocytes (PHH), hepatocytes obtained from chimeric animals with humanized livers, or CMHH was used to incubate the combination for at least 1 hour. 22. CMHH binds to one or more humoral factors secreted by human hepatocytes and 22. The CMHH of paragraph 21, comprising a hepatocyte clonal expansion medium or a hepatocyte maintenance medium. 23. Hepatocyte clonal growth medium contains L-proline, insulin, dexamethasone, E GF, Asc-2P, HEPES, penicillin, streptomycin, serum and DMS Paragraph O, including standard cell culture basal medium supplemented with DMSO2 or TMSO. Any of 20-22 CMHH. 24. Hepatocyte maintenance medium contains L-proline, insulin, dexamethasone, and Asc-2P. , HEPES, penicillin, streptomycin, serum and DMSO, DMSO2 or Any of paragraphs 20-22, including standard basal cell culture medium supplemented with TMSO That CMHH. 25. A composition comprising any one of CMHH and extracellular matrix as set forth in paragraphs 20 to 24. Match. 26. Paragraphs 20-24 of any one of CMHH and forskolin, SB431 A combination containing five chemicals: 542, DAPT, IWP2, and LDN193189 . 27. From freshly isolated terminally differentiated HH, PHH, and chimeric animals with humanized livers for use in culturing the resulting hepatocytes or primary hepatocytes from animals of other species, Any one of paragraphs 20 to 24 of the CMHH. 28. Cryopreserved terminally differentiated HH, PHH, and humanized liver-bearing chimeric animals for use in culturing hepatocytes derived from animals of other species, or primary hepatocytes derived from animals of other species. CMHH of any of the Lagraf 20-24. 29. Bipotential hepatic progenitor cells (iHep) and / or chemically induced hepatic progenitor cells Genita is a terminally differentiated hepatocyte, a hepatoma cell such as HeparRG™, or any of paragraphs 20 to 24 for use in differentiating the cells into mature hepatocytes. That CMHH. 30. Any of paragraphs 20 to 24 for use in co-culturing with non-parenchymal cells. CMHH. 31. Paragraphs 20-24 for use in suspending HH during cryopreservation. Either CMHH. 32. Culturing HH for recovery after isolation and / or storage and establishment of liver function 1. A method comprising: In the first phase, a certain amount of HH is cultured in a first cell culture medium for a first period of time. Top and The first cell culture medium was removed from the above amount of HH, followed by the second cell culture medium in two phases. Culturing the amount of HH for a second period using a culture medium; Including, The first cell culture medium comprises a cell culture medium supplemented with DMSO, and the second cell culture medium comprising cell culture medium supplemented with DMSO, DMSO2 or TMSO. 33. Culturing HH for recovery after isolation and / or storage and establishment of liver function 1. A method comprising: In the first phase, a certain amount of HH is cultured in a first cell culture medium for a first period of time. Top and The first cell culture medium is removed from the amount of HH, followed by the second phase of Culturing the amount of HH for a second period using a cell culture medium; Including, The first cell culture medium was cell culture medium supplemented with dimethyl sulfoxide (DMSO). and the second cell culture medium comprises a cell culture medium supplemented with DMSO or TMSO. , The method, wherein the second cell culture medium does not contain DMSO. 34. The first period is at least 4 days and up to 2 months, and in the first phase The above amount of HH cultured in the presence of cholangiocarcinoma (HC) showed cell polarity, intercellular structures characterized by bile canaliculi, and and / or equivalent to control HH with hepatic function of mature hepatocytes or liver tissue from healthy individuals 34. The method of paragraph 32 or 33, wherein the gene expression level of 35. The first period is about 7 days, and the cell culture medium of the first (recovery) phase is 3 or the method of paragraph 32 or 33, optionally renewed every four days. 36. The amount of HH cultured in the second phase includes, but is not limited to: Alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), cytochrome c Cytochrome P450 family 3 subfamily A member 4 (CYP3A4) and cytochrome Hepatocyte metabolic genes such as P450 family 2 subfamily E member 1 (CYP2E1) or the above amount of H cultured in the second phase. H metabolizes alcohol, non-biological substances, vitamin A, or a combination thereof; 32 or 33 ways. 37. The first cell culture medium is hepatocyte culture medium or CMHH supplemented with DMSO. The hepatocyte culture medium or CMHH was mixed with DMSO, standard cell culture basal medium, and L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin one or more of: cytochrome c phosphate-streptomycin, human serum, and / or FBS 37. The method of any one of paragraphs 32 to 36, including: 38. The second cell culture medium is supplemented with DMSO, DMSO2, or TMSO. Cell culture medium or DMSO, DMSO2, or TMSO, standard cell culture basal medium , as well as L-proline, insulin, dexamethasone, EGF, Asc-2P, and HEP ES, penicillin-streptomycin, human serum and / or heat-inactivated FBS 37. The method of any of paragraphs 32 to 36, wherein the CMHH comprises one or more of the following: 39. The first or second cell culture medium is diluted to a final concentration of approximately 2% volume / volume or 281.6 39. The method of any of paragraphs 32 to 38, comprising at least 10 mM DMSO. 40. The second cell culture medium contains DMSO2 at a final concentration of about 140.8 mM, Use one of the methods shown in Graphs 32 to 38. 41. Paragraph 3, wherein the second cell culture medium contains TMSO at a maximum concentration of about 70 mM. 39. The method according to any one of 2 to 38. 42. The first cell culture medium is a 2% (vol / vol) or The solution contained 286.1 mM DMSO, 15 μg / mL L-proline, and 0.25 μg / mL ibuprofen. thorin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 mM Asc-2P, 20mM HEPES, 100IU / mL penicillin, 100µg / mL streptomycin Contains Syn and 10% serum; The second cell culture medium was prepared by adding 281.6 mM, 1 mM, and 281.6 mM of PEG-1000 in standard basal cell culture medium. 40.8mM or 70.4mM DMSO, DMSO2 or TMSO, 15μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 n g / mL EGF, 0.1mM Asc-2P, 20mM HEPES, 100IU / m Contains 100 μg / mL penicillin, 100 μg / mL streptomycin, and 10% serum. Any of the methods from 32 to 41. 43. The first cell culture medium is a 2% (vol / vol) or The solution contained 286.1 mM DMSO, 15 μg / mL L-proline, and 0.25 μg / mL ibuprofen. Surin, 50 nM dexamethasone, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin and 10% serum Includes; The second cell culture medium was prepared by adding 281.6 mM, 14 mM, and 281.6 mM of PEG-14 HCl in standard basal cell culture medium. 0.8mM or 70.4mM DMSO, DMSO2 or TMSO, 15μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 μg 1. The method of claim 32, further comprising administering to said patient a therapeutically effective amount of 100 mg of 10 ... method. 44. The first cell culture medium is prepared by dissolving 0.5% to 5% (volume / volume) of ATP in standard cell culture medium. ) DMSO (5-25 μg / mL L-proline, 0.1-0.5 μg / mL insulin , 10-100nM dexamethasone, 0-10ng / mL EGF, 0.01-1mM Asc-2P, 10-50 mM HEPES, 10-300 IU / mL penicillin, 10 containing ~300 μg / mL streptomycin and 2–20% serum; The second cell culture medium is 50-300 mM DMSO in standard basal cell culture medium; DMSO2 or TMSO, 5–25 μg / mL L-proline, 0.1–0.5 μg / mL insulin, 10-100nM dexamethasone, 0-10ng / mL EGF, 0. 01~1mM Asc-2P, 10~50mM HEPES, 10~300IU / mL PE containing nicotine, 10–300 μg / mL streptomycin, and 2–20% serum. Any of the methods in paragraphs 32 to 41. 45. The efficacy of candidate drugs in promoting alcohol metabolism by HH or candidate drugs for HH 1. A method for use in assaying the toxicity of a co-agent, comprising: contacting the amount of HH cultured in the second phase with a candidate drug; Before contact with the candidate drug, the first test was performed to measure the metabolism of alcohol or the excretion of the candidate drug by the above amount of HH. and measuring the level of HH in the presence or after contact with the candidate agent. and measuring a second level of alcohol metabolism or excretion of the candidate drug. Any of the methods in paragraphs 32 to 44. 46. ​​Cell culture medium supplemented with DMSO2 or TMSO, and the cell culture medium is DMSO HH culture medium containing no SO for establishing and maintaining liver function in HH. 47. DMSO2 or TMSO reacted with approximately 140 The basal medium was supplemented to reach a concentration of 0.8 mM or 70.4 mM. Insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin and / or one or more of streptomycin, human serum, and / or heat-inactivated FBS. 47. The HH culture medium of paragraph 46, further comprising: 48. 140.8 mM DMSO2, 15 μg / mL in standard cell culture basal medium L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1mM Asc-2P, 20mM HEPES, 100IU / mL penicillium phosphate, 100 μg / mL streptomycin and 10% serum, or 47 HH culture medium. 49. 70.4 mM TMSO, 15 μg / mL L- in standard cell culture basal medium Proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL E GF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin , 100 μg / mL streptomycin and 10% serum, 47 HH culture medium. 50. 50–200 mM DMSO2 or TMSO in standard basal cell culture medium; 5–25 μg / mL L-proline, 0.1–0.5 μg / mL insulin, 10–10 0 nM dexamethasone, 1-10 ng / mL EGF, 0.01-1 mM Asc-2P , 10-50mM HEPES, 10-300IU / mL penicillin, 10-300μg / mL streptomycin and 2 to 20% serum, any of paragraphs 46 to 49 The HH culture medium. 51. A first container for storing a first cell culture medium, wherein the first cell culture medium a first container that is supplemented with DMSO; and a second container for storing a second cell culture medium, said second cell culture medium being DMS; A second container that is supplemented with 0, DMSO2, and / or TMSO A kit for culturing HH, comprising: 52. A first container for storing a first cell culture medium, wherein the first cell culture medium a first container that is supplemented with DMSO; and a second container for storing a second cell culture medium, said second cell culture medium being DMS; A second container supplemented with O2 and / or TMSO but not DMSO. A kit for culturing human hepatocytes, comprising: 53. Toxicity of drugs to HH or drug metabolism and pharmacokinetics (DMPK) of drugs A method of transmitting Cultivate a certain amount of HH in medium supplemented with DMSO and medium not supplemented with DMSO. Steps and; contacting the agent with the amount of HH cultured in a medium supplemented with DMSO2; and; Measure the level of toxicity to the above dose of HH in the presence of or after contact with the drug. step, or metabolism of the drug by the above amount of HH in the presence of or after contact with the drug. or measuring the level of emissions; A method comprising: 54. The drug is ethanol, methanol, ethylene glycol, isopropanol or 54. The method of paragraph 53, wherein the alcohol compound includes a mixture thereof. 55. Paragraph 53 or paragraph 54, in which the contacting and measuring steps are carried out in a sealed device There are 54 ways. 56. The level of toxicity is, but is not limited to, the cytochrome P450 in the above amount of HH. It is measured by quantifying the expression or function of enzymes that metabolize non-biological substances, such as CYP enzymes. any of the methods set out in paragraphs 53 to 55. 57. The level of toxicity is higher than the toxic equivalents of alcohol, such as acetaldehyde in the above amount of HH. by quantifying metabolic products, or glutathione reduction, and activation of cell death pathways. Measured by any of the methods set out in paragraphs 53 to 55. 58. How to assay candidate drugs for promoting alcohol or vitamin A metabolism It is a law, Cultivate a certain amount of HH in medium supplemented with DMSO and medium not supplemented with DMSO. Steps and; The above amount of HH cultured in DMSO2-supplemented medium in the presence of a candidate drug was subjected to alcohol treatment. contacting the compound with ethanol or vitamin A; and The presence of or contact with a candidate drug followed by the above-mentioned amount of HH was examined for alcohol or vitamin D. measuring the level of metabolism of amine A; A method comprising: 59. The medium used is a HH-derived conditioned medium according to any one of paragraphs 20 to 24. , any of the methods set forth in paragraphs 32 to 58. [Example]

[0128] The following examples are provided to better illustrate the claimed invention, To the extent that specific materials are mentioned, the scope of the disclosure should not be construed as limiting the scope of the disclosure. This is for illustrative purposes only and is not intended to limit the invention. Without the exercise of inventive faculty, equivalent or counter means may be produced without departing from the scope of the invention. It is possible to develop new materials.

[0129] [Example 1] Composition and regimen of the two-step culture method for HH. Maintaining hepatocyte cell fate and liver function requires a two-step culture method, with phase 1 being , a phase that promotes recovery from cell damage that occurs during the cell isolation procedure and freeze-thaw process. This phase involves in vitro culture for at least 4 days. Preferably, the incubation period is 7 days. During Phase 1, the "HH Recovery Medium" (Tables 1 and 3) Using this method, HH has been shown to inhibit cell polarity and bile canaliculi, among other things. In addition to morphological recovery, phase 1 also restored the normal intercellular structure. It was also possible to restore hepatocyte characteristics at the protein expression level. The robustness of the culture medium was rigorously compared with other publicly available culture media for HH, demonstrating that the method: These conditions [HCM™ Hepatocyte Culture Medium Bulletkit or HMM™ Hepatocyte basal medium, both manufactured by LONZA, or CELLARTIS (registered trademark) manufactured by TAKARA Substantially more effective than commercially available hepatocyte culture media such as POWER™ Primary Hepatocyte Medium The culture conditions in Phase 1 were found to be superior to those in in vitro culture (Figure 1). We also found that the cell fate of the HHs can be maintained for up to two months. I. Culture conditions for hepatocytes, including but not limited to alcohol metabolism and drug metabolism We found that the metabolic function of the cerebrospinal fluid was substantially impaired (Fig. 2 and Fig. 3). The culture conditions that allow for cell fate maintenance and differentiation compromise the versatility of HH as an experimental tool. Indicates that.

[0130] [Table 1]

[0131] [Table 2]

[0132] [Table 3]

[0133] We explored the mechanism by which phase 1 culture conditions interfere with the basic metabolic functions of HH and investigated the mechanisms underlying the disruption of HH by the inclusion of ATP in the culture medium. DMSO, which is present in the blood, was identified as the cause (Figures 2, 3, and 4). Because withdrawal of DMSO from the culture medium results in rapid deterioration of hepatocyte function and morphology, However, we found that this was not feasible (Figure 5). Culturing HH without DMSO during Phase 1 This facilitates recovery from cell injury and stress caused by cell isolation / preservation procedures. We also found that this is unlikely to occur (Figure 6).

[0134] A substitute for DMSO (i.e., a substitute that replaces DMSO) was sought, and the substitute was found to have a beneficial effect on liver cells. They are not inhibitory to cellular metabolic functions, yet are capable of maintaining terminally differentiated HH cell fate. Among the many organic compounds tested (Table 3; Figures 7 and 8), DMSO DMSO2, the oxidized form of DMSO, was identified as a desirable substitute. Phase 2 culture (7 days and thereafter) in medium containing DMSO but not DMSO This allows HH to metabolize alcohol at a level similar to that of the human liver (Fig. 2 and (Fig. 4) Phase 2 culture medium (containing DMSO2) showed in vivo effects on alcoholic liver disease. It has shown potential to function as a platform for in vitro research.

[0135] Therefore, HH is a promising candidate for Phase 2 (e.g., DMPK and various liver disease studies) Establish culture conditions that can exhibit basic hepatocyte functions, and these conditions are Phase 1 The cell fate of HH was not impaired during the incubation period (days 0 to 7). See Tables 4 and 6. It refers to light.

[0136] [Table 4]

[0137] [Table 5]

[0138] [Table 6]

[0139] In addition, HH cultured in Phase 1 culture medium (containing DMSO) showed a significant increase in CYP3A4 and The expression of CYP2E1 and CYP2E1 was increased to supraphysiological levels, and the cells were cultured in a medium containing DMSO. We found that the HH strongly interfered with the metabolic functions of these enzymes (Figs. 6 and 9). These enzymes are central players in the study of drug metabolism and drug toxicity, so DM The SO-containing medium is a versatile tool for in vitro culture of HH as a research tool for DMPK. In contrast, HH cultured in DMSO2-containing medium showed no metabolic activity of non-biological substances. In this study, the HH cells cultured in DMSO showed significantly superior activity to those cultured in DMSO (Fig. 3). DMSO2-containing medium allowed researchers to study DMPK in vitro. These results suggest that cell culture media containing DMSO2 may be commercially available. Overall, the results show that the technology has great potential for

[0140] Overall, culture conditions suitable for HH recovery (phase 1) are consistent with the characteristics of terminally differentiated HH. Although DMSO helps restore and maintain HH activity, it is not a potent inhibitor of many enzyme activities in HH. The inhibitory effect of HH limits its versatility as a research tool. These include, but are not limited to, alcohol metabolic pathways and non-biological substance metabolic pathways. This has demonstrated the usefulness of HH in the study of alcoholic liver disease (ALD) and DMPK. are blocked, respectively (Figs. 2, 3, 4 and 9).

[0141] [Table 7]

[0142] [Example 2] Use of DMSO2-containing media for the study of ALD. Phase 1 medium containing DMSO inhibits two major alcohol metabolic pathways, i.e., AD The researchers found that the most common cause of chronic liver disease (CLD) was is also a significant cause of ALD (almost 50% of CLD in the United States) for which there is no effective therapeutic intervention. However, there are no methods (culture conditions) that allow the recovered HH to be used in research on ALD. Therefore, establishing suitable culture conditions for ALD research has enormous commercial potential. It is a technological advancement that has

[0143] In the search for DMSO substitutes, a large number of organic compounds were screened. The list (Table 7) is based on molecular size, solubility in cell culture media, and stability at standard temperatures. Qualitative (melting and boiling temperatures), handling (no significant chemical hazards) and selected based on characteristics they share with DMSO, including but not limited to cost and These chemicals were added to cell culture medium as a substitute for DMSO and were then used to treat recovered HH. The highest concentration that does not cause direct toxicity was assayed first. The recovered HHs (7 days old) were cultured at the same concentration for 7 days, with the medium being renewed every 3-4 days. On days 14 and 21, the cells were further cultured for 14 days. The cells were subjected to morphological and cell fate evaluation (Fig. 7 and Fig. 8).

[0144] Of those tested, only TMSO and DMSO2 were as effective as DMSO-containing medium. These are two conditions that are capable of maintaining morphological structures and marker gene expression at the same level ( 7 and 8). Follow-up experiments showed that TMSO-containing medium was significantly superior to DMSO-containing medium and alcohol-containing medium. It was found that DMSO2 was equally inhibitory to metabolic pathways (Fig. 10). The rich medium allows HH to consume ethanol at a level similar to the capacity of the human liver. (Figure 4).

[0145] The study focused on performing in vitro alcohol treatment on HH using a closed device. We also found that ethanol is important for the liver because it is highly volatile. It evaporates rapidly, at a rate much faster than the cells can metabolize it. Ethanol-treated HH cultured in soil showed significant ALD only when a sealed device was applied. The use of sealed devices to study ALD results in the accumulation of lipid droplets (LDs), a phenotype. This is another aspect of technological innovation.

[0146] To culture HH, replace DMSO with DMSO (i.e., DMSO Once the cells have achieved full recovery (by replacing with DMSO-containing medium), For approximately 4 days, preferably 7 days, HH metabolizes alcohol (ethanol). This allows HH to grow in a DMSO2-containing medium (Fig. 2, Fig. 3, and Fig. 4). Others that are more relevant to human toxicology include alcohol, ethylene glycol, and isopropanol. This allows us to test the metabolism and toxicity of various types of alcohol. This allows HH to metabolize vitamin A (which is also metabolized by ADH). Therefore, HH can be used to study vitamin A biology. The application of sealed apparatus is essential for ALD studies (or any metabolic studies using highly volatile chemicals). This will be the default experimental condition (Figure 12).

[0147] [Example 3] Use of DMSO2-containing media in drug toxicity and DMPK studies. DMPK studies serve as crucial milestones in the drug development process. This is one of the best applications of in vitro cultured HH. Regulatory agencies have adopted HH for preclinical screening of newly developed compounds prior to clinical studies. Therefore, HH is a valuable tool for preclinical research screening. To fulfill its role, it has a fully functional drug metabolism pathway at a level comparable to that of the human liver. HH cultured in DMSO-containing medium and DMSO2-containing medium In a study investigating the similarities and differences between DMSO-containing media and non-biological Overall, it was found to upregulate genes involved in substance uptake, metabolism, and excretion For example, studies have shown that the ATPase inhibitors involved in the metabolism of over 50% of drugs showed a significant upregulation of CYP3A4, the most important drug-metabolizing enzyme involved in The study found that DMSO strongly inhibits the metabolic activity of CYP3A4, and We showed that supraphysiological induction may reflect the degree of inhibition (Fig. 3 and Fig. 9) Such reciprocal changes are also seen in CYP2E1 (Figures 3 and 9), and DM We further demonstrated that conditions using SO-containing medium are not suitable for DMPK studies.

[0148] Therefore, the study demonstrated that cells cultured in DMSO2-containing medium could secrete CYP2 at physiological levels. The expression abundance of P3A4 and CYP2E1 was demonstrated, and the cells were cultured in DMSO2. We demonstrated that HH maintains physiological relevance in terms of drug metabolic pathways (Figures 3 and 9). Furthermore, the effect of DMSO2 on the metabolic activity of these enzymes was not observed in DMSO-containing substantially less than in the medium (Figs. 3 and 9).

[0149] The study showed that cells cultured in DMOS2 had reduced drug-drug interactions and drug-metabolizing enzyme induction. For example, one of the classic anti-seizure (epileptic) drugs, Carbamazepine, which is a CYP3A4 inducer, is also known to induce CYP3A4. Observations show that HH cultured in DMSO2 but not in DMSO is metabolized by A4. reproduced CYP3A4 induction in a dose-dependent manner during 7 days of carbamazepine treatment. Similar results were observed with rifampicin. In addition, DMSO, but not DMSO2-containing medium, HH maintained in a medium containing CYP2E1 expressed at supraphysiological levels, and We observed a lack of CYP2E1 induction by ethanol and isoniazid. (Figure 9).

[0150] Taken together, the present findings suggest that DMSO2-containing media may be an effective in vivo treatment for DMPK studies. This indicates that the versatility of HH cultured in vitro is greatly expanded. MSO2-containing media provide an improved platform for in vitro studies of non-biological metabolism. The sensitivity of the DMSO-containing medium is not exceeded by the culture conditions of the DMSO-containing medium. This allows for detection of xenobiotic metabolizing enzyme induction with much higher sensitivity. The culture conditions provide an experimental platform for the detection of drug toxicity occurring over long-term culture. DMSO2-containing medium culture conditions are useful for preventing drug-drug interactions (multiple drug administration). This provides improved experimental conditions for detecting toxicity due to

[0151] [Example 4] Production of conditioned medium from in vitro cultured human hepatocytes and its inherent biological effects The discovery of CMHH was made possible by this technology in the production of HLCM. , it takes approximately 10 5 PHH and / or HLCM-H H is required, which is injected into the spleen of a host mouse (such as the uPA-SCID strain). This was achieved by transplanting endogenous mouse hepatocytes overexpressing a toxic transgene (uPA). There was a strong increase in PHH in the liver of host mice that did not proliferate as a result of competition with human hepatocytes. (After PHH transplantation, endogenous mouse hepatocytes are reduced; whereas, in the host without PHH transplantation, In mice, mouse hepatocytes can proliferate and do not decrease. After a week, the host mouse's liver will be replaced by up to 95% human hepatocytes. As a result, 2–3 × 10 cells were obtained from one HLCM liver. 8 Human hepatocytes were obtained and This will enable mass production of HLCM-HH. We will provide a stable supply of HLCM-HH, which will be used in the mass production of H. In addition, The advantages of HLCM-HH over PHH in CMH production are its viability and culturability. (Sugahara G. et al., Semin Liver Dis. 2020 May;40(2):189-212). Therefore, the use of HLCM-HH has economic advantages over the use of PHH.

[0152] Materials and Methods: HLCM was used to assess the extent of human hepatocyte proliferation, i.e., replacement, in the liver. Serum / blood human albumin levels are subjected to quality assessment of the RI. It was shown that the results were in good agreement with those of H with blood albumin levels higher than 10 mg / mL. LCMs are considered to have a high RI (>70%). HLCMs with a high RI are considered to have a high RI (>70%). The isolation of human hepatocytes by a method based on enzyme perfusion is preferred. Following evaluation of the cell density and viability, HLCM-derived human hepatocytes (HLCM-HH) were 0.5~5×10 5 individual cells / cm 2(or 2.1-4.2×10 in some implementations) 5 individual cells / cm 2 ) and collagen [with or without other types of extracellular matrix (ECM)] Plated onto coated cell culture flasks / dishes. HH or HepaRG cells were cultured in collagen-coated cell culture flasks / dishes. [The HEPARG™ cell line retains many of the characteristics of primary human hepatocytes. HEPARG™ cells are an immortalized hepatocyte cell line that possesses a terminally differentiated and easily differentiated These cells are provided in a convenient frozen format. HCGM supplemented with analogs or equivalents of dHCGM, d2HCGM , dHMM, or d2HMM) media (Tables 8 to 10) (dHCGM containing DMSO (e.g., at least 21 days, at least 1 month) The cells are cultured for 4, 5, 6, 7, or 8 weeks, or for about 2, 3, 4, 5, or 6 months. After 4 days (preferably 7 days) of cell seeding, the culture medium is replaced every 24-48 hours. The medium incubated with the cells was collected and stored as human hepatocyte-conditioned medium (CMHH). The biological effects of CMHH are unique and potent, as discussed in the following sections. do.

[0153] [Table 8]

[0154] [Table 9]

[0155] [Table 10]

[0156] The biological effects of CMHH were evaluated using HLCM-HH, and the effects were compared with those of CMH The cellular structure of HH incubated with H showed reconstruction of polygonal-cuboidal cellular morphology and In the liver tissue of healthy individuals, evidenced by the formation of bile canaliculi between the cells and intimate contacts between the cells. This demonstrates that the cellular structure of terminally differentiated human hepatocytes is well reproduced (Figure 13). In addition, we used a fluorescent substrate of MRP2, one of the transporters involved in the bile secretion mechanism. The function of the bile canaliculi formed in the CMHH-treated HH was confirmed (Fig. 14). HH treated with HH significantly upregulated hepatocyte marker genes, cholangiocytes / liver progenitor cells. Downregulation of cell marker genes and T, pathways known to impair cell fate in HH. We found that GF-β / Yap signaling was inhibited (Fig. 15). , HHs cultured in CMHH, but not in the new dHCGM, are hepatotropic viral pathogens. Hepatitis B virus (HBV) is a type of hepatitis that causes hepatitis B. Response to steroid drugs (Figures 16 and 17), and enhanced function of abiotic metabolic pathways (Fig. 18) showed that HH treated with CMHH produced authentic terminally differentiated human hepatocytes. Finally, we further demonstrated that DMSO2-containing hepatocyte culture medium (d CMHH grown in HCGM (HCGM) showed similar levels of cytotoxicity compared to those prepared in DMSO-containing medium. It was found that the bell possessed the biological effects (Figure 15).

[0157] In vitro cultured conventional hepatoma cell lines (HepG2, Huh7 cells) , as well as conditioned medium from 293 cells and primary mouse hepatocytes, all of which It lacks the biological effects seen with CMHH (Figures 19 and 20). In contrast, HL C expanded in CM-HH (2 different donors) or PHH (3 different donors) All M cells have a mature, final stage, including a polygonal-cuboid appearance of mature mononuclear or binuclear cells. They have the ability to maintain the distinctive appearance of differentiated human hepatocytes and ensure close cell-cell contact. In addition, HLCM-HH (from two different donors) ) or PHH (three different donors) all showed a high hepatocyte marker Upregulation of genes (CYP2C9, CYP2D6, OATP1B1, and ALDH2) / maintenance, and dedifferentiation / transdifferentiation genes (TGFβ1, TGFβ2, Cyr61, C The IL-16-16 agonist was able to support the suppression of TGF-β and CK19 (Figure 22).

[0158] Matrigel® and five chemical combinations (i.e., 5C), A proposed methodology to enhance the maturation and function of in vitro cultured hepatocytes There were several (Xiang C., Science. 2019 Apr 26;364(6438):399-402). Compare This product (CMHH) is substantially more potent in maintaining the appearance characteristic of mature HH. Furthermore, this gene expression analysis revealed that the effect of CMHH was Rigel® or 5C [five chemicals: forskolin (FSK); adenyl acid cyclase activator), SB431542 (SB43; TGF-β inhibitor), DAP T (Notch inhibitor), IWP2 (Wnt inhibitor), and LDN193189 (BM This analysis revealed that the effects of the α-glucanase inhibitor (α-glucanase inhibitor) were significantly different from those of the α-glucanase inhibitor (α-glucanase inhibitor) (Figure 24). Matrigel® and 5C biosynthesis in preventing dedifferentiation / transdifferentiation of HH It was revealed that the biological effect was weaker than that of CMHH.

[0159] The humoral factors present in CMHH were also analyzed. First, the CMHH was filtered through a size exclusion column. Analysis of the CM revealed key biological activities of CMHH involved in the cell fate and functional maintenance of HH. demonstrate that sexual composition is largely mediated by protein molecules secreted by HH (Figure 25). Evaluation of protein molecules contained in CMHH was performed using Matrigel®. The high abundance of LAMC2, FGA, SERPINA3, and TGM2 is in stark contrast to In addition, CMHH revealed the amount of A2MG, A1AT, and albumin. Contains secreted proteins specific to hepatocytes, most of which include, but are not limited to, possesses hepatocellular protective properties.

[0160] Taken together, CM consists of a combination of extracellular matrix and secreted proteins specific to HH. Hepatokines in HH confer specialized biological functions to in vitro cultured human hepatocytes. Present.

[0161] [Example 5] Application of CMHH for recovery and maintenance of primary hepatocytes The effect of CMHH on the recovery and maintenance of primary human hepatocytes (PHH) in vitro The biological effects were tested. In general, the viability, culturability and lifespan of primary hepatocytes were , which vary substantially between lots and donors; therefore, optimal cell seeding density is As a result, the original properties are lost. It was recognized as a research tool with limited usefulness and a rapidly expiring shelf life. The biological effects of CMHH on cell recovery and cell fate maintenance were examined. To do this, thaw the cryopreserved HLCM-HH and transfer it to either CMHH or new dHCGM. After 7 days of incubation, the cell structure was analyzed by light microscopy and the The bile secretion mechanism and the expression abundance of hepatocyte marker genes were evaluated (Figure 27, Figure 28 and Figure 29). Human hepatocytes from stress and injury associated with the freeze-thaw process. We observed a strong recovery; incubation with CMHH resulted in the development of terminally differentiated human hepatocytes. Support the reconstruction of appropriately designed structures, functions, and expression abundance of marker genes, which are characteristic of , which is in clear contrast with the new dHCGM and the culture medium supplied by the manufacturer. is.

[0162] For the recovery and maintenance of primary mouse, rat, and dog hepatocytes in vitro The biological effects of CMHH on primary hepatocytes from these species were examined. The cell lifespan is substantially lower than that of HLCM-HH; therefore, cell seeding at optimal cell density is This is rarely achieved, resulting in the loss of the original properties. Incubation of rat and dog primary hepatocytes with CMHH resulted in morphological changes in the primary hepatocytes. Demonstrates a powerful biological effect in maintaining characteristics of appearance and function (Figure 30).

[0163] The powerful effects of CMHH may not only be due to the maintenance of HH cell fate and function. Further research is being conducted on stem cell-derived hepatocyte-like cells, i.e., iHep (induced hepatocyte-like cells). This may provide the necessary environment for differentiation of the CM into morphological configurations. The effects of HH were examined. Similar to the effects on PHH and HLCM-HH, CMHH Treatment promotes the formation of bile canaliculi between human hepatocyte-like cells, which have a polygonal-cuboidal appearance. (Figure 31). Effect of CMHH on iHep characteristics by single-cell RNA sequencing. The classical dimension reduction method, principal component analysis (PCA), was evaluated (Figures 32 and 33). , the dimension between HLCM-HH treated with CMHH and iHep treated with iHep culture medium This demonstrates that the variability is much lower than previously estimated.

[0164] We will also examine whether CMHH promotes the redifferentiation of Clip cells. Incubation with MHH significantly increased hepatocyte survival compared to cells cultured in fresh dHCGM. The significant upregulation of marker genes (Fig. 34) indicates that CMHH is involved in cell fate maintenance. It has also been shown that the cells have not only endogenous properties but also hepatocyte-inducing differentiation properties.

[0165] To confirm the favorable and robust effects of CMHH on cell fate maintenance in human hepatocytes, To investigate this, in vivo studies were performed. The human hepatocytes cultured with the humanized liver chimeric mice were transplanted into the host mice, and engraftment and The extent of the increase in hepatocyte activity and the degree of its proliferation were monitored. , and serum human albumin levels revealed comparable engraftment on day 2 after transplantation (Figure 3 5). However, at later time points, 14 and 21 days after transplantation, CMHH-treated HH , and showed a stronger increase in blood human albumin levels than dHCGM-treated HH. The proliferation potential within the liver of HLCM host mice is a proxy for the quality of primary hepatocytes. The results of this in vivo study contribute to the accumulation of further evidence for the biological effects of CMHH. Give it a layer of texture.

[0166] Finally, the usefulness of CMHH for the recovery and fate maintenance of cryopreserved HLCM-HH For this purpose, cryopreserved HLCM-HH were thawed and The cells were seeded under the optimized conditions shown in and cultured in the presence of CMHH or the new dHCGM. As expected, CMHH treatment resulted in morphological and structural changes similar to those of terminally differentiated hepatocytes. The expression abundance of hepatocyte marker genes was greatly improved in the control group (Figures 36 and 37).

[0167] In summary, this study demonstrated that CMHH is a culture medium that strongly supports the recovery and maintenance of primary hepatocytes. These effects were observed whether the cells were freshly isolated or cryopreserved. Regardless of primary human hepatocytes as well as HLCM-HH, mouse, rat, and dog In addition, CMHH is not specific to primary hepatocytes. Promoting further maturation of LiPs may be an alternative to liver transplantation using stem cell-derived hepatocytes. This is likely to contribute to the future development of transplant therapy.

Claims

1. 1. A method for preparing conditioned medium from cultured human hepatocytes (HH), comprising: Culturing the HH in a culture medium to produce a culture medium; The culture medium incubated with the HH for at least 1 hour was used as the conditioned medium. A step of collecting A method comprising:

2. The HH may be a primary human hepatocyte (PHH), a liver obtained from a chimeric animal having a humanized liver, or 10. The method of claim 1, comprising administering to a subject a therapeutically effective amount of ...

3. The hepatocytes obtained from the chimeric animal having a humanized liver are used to produce a humanized liver chimeric mouse. and the HLCM-HH is derived from a human hepatocyte (HLCM-HH), and the HLCM-HH is derived from a human hepatocyte (PHH) or a human hepatocyte (HLCM-HH) derived from a human hepatocyte (HLCM-HH) in advance. isolated HLCM-HH obtained from the liver of a mouse injected into the spleen. The method according to claim 2.

4. Before the HLCM-HH was obtained, the mice had a replacement index of at least 10%.

4. The method of claim 3, comprising:

5. The medium may contain dimethyl sulfoxide (DMSO), dimethyl sulfone (DMSO2), or or tetramethylene sulfoxide (TMSO)-supplemented hepatocyte clonal expansion medium ( dHCGM, d2HCGM or tHCGM), wherein the HCGM is a standard cell culture medium. Basal medium, L-proline, insulin, dexamethasone, EGF and L-ascorbic acid 10. The method of claim 1, comprising or consisting essentially of phosphonic acid 2-phosphate (Asc-2P). The method described.

6. The medium is a hepatocyte maintenance medium (dH) supplemented with DMSO, DMSO2, or TMSO. MM, d2HMM, tHMM), wherein the HMM is a standard cell culture basal medium, L- Proline, insulin, dexamethasone and L-ascorbic acid diphosphate (Asc- 2P).

7. The standard cell culture basal medium is Dulbecco's Modified Eagle's Medium (DMEM), a minimal essential Essential medium (MEM), RPMI-1640, Iscove's modified Dulbecco's medium (IMDM) or The method according to claim 5 or 6, wherein the medium is selected from the group comprising William's E medium.

8. The standard cell culture basal medium contains HEPES, penicillin-streptomycin and 8. The method of claim 7, wherein the cells are supplemented with serum and erythrocytes.

9. The standard cell culture basal medium is DMEM, and the DMEM is DMEM-10. The DMEM-10 contains DMEM, HEPES, penicillin-streptomycin, and 8. The method of claim 7, comprising or consisting essentially of erythrocytes and serum.

10. 10. The method of claim 8 or 9, wherein the serum is fetal bovine serum (FBS) or human serum. The method according to any one of claims 1 to 5.

11. dHCGM, d2HCGM or tHCGM is one of the standard cell culture basal media 5 μg / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone , 5 ng / mL EGF, 0.1 mM Asc-2P, 281.6 mM, 140 . 8mM or 70.4mM DMSO, DMSO2, or TMSO, 20mM HE PES, 100 IU / mL penicillin, 100 μg / mL streptomycin, and 1 6. The method of claim 5, comprising or consisting essentially of 0% serum.

12. dHCGM, d2HCGM, tHCGM in the standard cell culture basal medium 5 μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM Dexamethasone, 1-10 ng / mL EGF, 0.01-1 mM Asc-2P, 50 ~300mM DMSO, DMSO2 or TMSO, 10-50mM HEPES, 1 0-300 IU / mL penicillin, 10-300 μg / mL streptomycin, and 6. The method of claim 5, comprising or consisting essentially of 2-20% serum.

13. dHMM, d2HMM, and tHMM were added at 15 μg / ml in the standard basal cell culture medium. L L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 0.1 m M Asc-2P, 281.6 mM, 140.8 mM or 70.4 mM D, respectively MSO, DMSO or TMSO, 20 mM HEPES, 100 IU / mL penicillin containing or consisting of phosphate, 100 μg / mL streptomycin, and 10% serum The method of claim 6 consisting essentially of

14. 5-25 μg of dHMM, d2HMM, and tHMM in the standard basal cell culture medium / mL L-proline, 0.1 to 0.5 μg / mL insulin, 10 to 100 nM dextromethorphan Methasone, 0.01-1 mM Asc-2P, 50-300 mM DMSO, DMSO2 , TMSO, 10-50 mM HEPES, 10-300 IU / mL penicillin, 10- 300 μg / mL streptomycin, and 2-20% serum, or The method of claim 6, wherein the solubility is qualitative.

15. 15. The method of claim 1, wherein the medium is replaced with a new volume every 24 to 120 hours.

1. The method according to claim 1.

16. The PHH, the hepatocytes obtained from the chimeric animal having a humanized liver, or the tissue The combined cell density was 0.5 x 10 ５ pieces / cm ２ ~5 x 10 ５ pieces / cm ２ Cultivated in, claim The method according to item 2.

17. 10. The method of claim 1, wherein the HH does not comprise HepG2 cells, Huh7 cells, or mouse hepatocytes. The method according to any one of claims 1 to 3.

18. the HH comprises hepatocytes obtained from a chimeric animal having a humanized liver, Before the culturing step, the hepatocytes obtained from the chimeric animal having a humanized liver are obtaining said hepatocytes from said chimeric animal having a humanized liver. The method further comprises the steps of: (1) transferring previously isolated hepatocytes obtained from PHH or humanized liver chimeric animals to the animal; isolating hepatocytes from the liver of said animal injected intraspleen, The method further comprises obtaining the hepatocytes from the chimeric animal having a more humanized liver. The method of claim 1 .

19. The hepatocytes obtained from the chimeric animal are and the chimeric animals are isolated by perfusion, and the chimeric animals are at least 10% 19. The method of claim 18, wherein the replacement index is

20. Cultured human hepatocytes (CM) prepared by the method according to any one of claims 1 to 19. Conditioned medium derived from HH.

21. CMHH comprises a hepatocyte clonal expansion medium or a hepatocyte maintenance medium, and the medium Humanized hepatocytes (PHH), hepatocytes or their combinations obtained from chimeric animals with humanized livers CMHH used to incubate the mixture for at least 1 hour.

22. The CMHH comprises one or more humoral factors secreted by human hepatocytes and hepatocytes.

22. The CMHH of claim 21, comprising a cell clonal expansion medium or a hepatocyte maintenance medium.

23. The hepatocyte clonal growth medium contains L-proline, insulin, dexamethasone, EG F, Asc-2P, HEPES, penicillin, streptomycin, serum, and DMSO 20 to 22, comprising a standard cell culture basal medium supplemented with DMSO or TMSO.

23. The CMHH according to any one of claims 22.

24. The hepatocyte maintenance medium contains L-proline, insulin, dexamethasone, Asc-2P, HEPES, penicillin, streptomycin, serum and DMSO, DMSO2 or 23. Any one of claims 20 to 22, comprising a standard basal cell culture medium supplemented with TMSO. CMHH as described in

25. A combination comprising the CMHH according to any one of claims 20 to 24 and an extracellular matrix.

26. CMHH according to any one of claims 20 to 24, forskolin, and SB43154 2, a combination containing five chemicals: DAPT, IWP2, and LDN193189.

27. Newly isolated terminally differentiated HH, PHH, and humanized livers were obtained from chimeric animals. for use in culturing hepatocytes from animals of the same species or primary hepatocytes from other species.

25. A CMHH according to any one of claims 20 to 24.

28. Derived from cryopreserved chimeric animals with terminally differentiated HH, PHH, and humanized livers 2. For use in culturing hepatocytes or primary hepatocytes from animals of other species. 0-24. A CMHH according to any one of claims 0-24.

29. Hepatic bipotential progenitor cells (iHep) and / or chemically induced liver progenitors The cells were then transformed into terminally differentiated hepatocytes, hepatoma cells such as HeparRG™, and 25. The method of claim 20, wherein the method is for use in differentiating a hepatocyte into a mature hepatocyte. Featured on CMHH.

30. A method according to any one of claims 20 to 24 for use in co-culturing with non-parenchymal cells CMHH.

31. 25. The method according to claim 20, for use in suspending HH during cryopreservation. The CMHH described in paragraph .

32. A method for culturing HH for recovery and establishment of liver function after isolation and / or preservation There was, In the first phase, a quantity of HH is cultured in a first cell culture medium for a first period of time. Top and The first cell culture medium is removed from the amount of HH, followed by the second phase culturing said amount of HH for a second period of time using the second cell culture medium; Including, The first cell culture medium comprises a cell culture medium supplemented with DMSO, and the second cell The method wherein the culture medium comprises cell culture medium supplemented with DMSO, DMSO2, or TMSO. 。

33. A method for culturing HH for recovery and establishment of liver function after isolation and / or preservation There was, In the first phase, a quantity of HH is cultured in a first cell culture medium for a first period of time. Top and The first cell culture medium is removed from the amount of HH, followed by the second phase culturing said amount of HH for a second period of time using the second cell culture medium; Including, The first cell culture medium is a cell culture medium supplemented with dimethyl sulfoxide (DMSO). wherein the second cell culture medium is a cell culture medium supplemented with DMSO or TMSO. Including the earth, The method, wherein said second cell culture medium does not contain DMSO.

34. The first period is at least 4 days and at most 2 months, and the first phase The amount of HH cultured in the above-mentioned medium is characterized by cell polarity, bile canaliculi, and intercellular structures. and / or a control HH that exhibits the hepatic function of mature hepatocytes or liver tissue of a healthy individual. The method of claim 32 or 33, wherein the gene expression levels of the gene, such as:

35. the first period of time is about 7 days, and the first (recovery) phase cell culture medium comprises:

34. The method of claim 32 or 33, optionally refreshed every 3 or 4 days.

36. The amount of HH cultured in the second phase may include, but is not limited to, Alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), cytochrome P450 family 3 subfamily A member 4 (CYP3A4) and cytochrome P Hepatocyte metabolic genes such as 450 family 2 subfamily E member 1 (CYP2E1) or the amount of the enzyme cultured in the second phase.

3. The method of claim 2, wherein the HH metabolizes alcohol, non-biological substances, vitamin A, or a combination thereof. 2 or 33.

37. the first cell culture medium is a hepatocyte culture medium supplemented with DMSO or CMHH; The hepatocyte culture medium or CMHH may contain DMSO, standard cell culture basal medium, and L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPES, one or more of nicotinic acid, streptomycin, human serum, and / or FBS The method of claims 32 to 36, including a number.

38. The second cell culture medium is supplemented with DMSO, DMSO2, or TMSO. Culture medium or DMSO, DMSO2, or TMSO, standard cell culture basal medium; and L-proline, insulin, dexamethasone, EGF, Asc-2P, HEPE S, penicillin-streptomycin, human serum and / or heat-inactivated FBS The method according to any one of claims 32 to 36, which is a CMHH comprising one or more of: Law.

39. The first or second cell culture medium has a final concentration of about 2% volume / volume or 281.6 m 39. The method of any one of claims 32 to 38, comprising M DMSO.

40. the second cell culture medium contains the DMSO2 at a final concentration of about 140.8 mM; The method according to any one of claims 32 to 38.

41. 3. The method of claim 32, wherein the second cell culture medium contains TMSO at a maximum concentration of about 70 mM.

9. The method according to any one of claims 8 to 8.

42. The first cell culture medium is 2% (vol / vol) of a standard basal cell culture medium or 286.1 mM DMSO, 15 μg / mL L-proline, 0.25 μg / mL insulin Phosphorus, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 mM Asc-2P, 2 0 mM HEPES, 100 IU / mL penicillin, 100 μg / mL streptomycin containing 10% serum and 10% ethanol; The second cell culture medium comprises 281.6 mM of each of: 140.8 mM or 70.4 mM DMSO, DMSO2 or TMSO, 15 μ g / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1mM Asc-2P, 20mM HEPES, 100IU / 100 μg / mL penicillin, 100 μg / mL streptomycin, and 10% serum.

42. The method of any one of claims 32 to 41.

43. The first cell culture medium is 2% (vol / vol) of a standard basal cell culture medium or 286.1 mM DMSO, 15 μg / mL L-proline, 0.25 μg / mL insulin Phosphorus, 50 nM dexamethasone, 0.1 mM Asc-2P, 20 mM HEPES, 1 Contains 100 IU / mL penicillin, 100 μg / mL streptomycin, and 10% serum. fruit; The second cell culture medium comprises 281.6 mM of each of: 140.8 mM or 70.4 mM DMSO, DMSO2 or TMSO, 15 μ g / mL L-proline, 0.25 μg / mL insulin, 50 nM dexamethasone, 0 1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 100 42. The method according to claim 32, comprising: μg / mL streptomycin and 10% serum. The method described in paragraph .

44. The first cell culture medium is 0.5% to 5% (volume / volume) of a standard cell culture medium. DMSO, 5-25 μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM dexamethasone, 0-10 ng / mL EGF, 0.01-1 mM A sc-2P, 10-50 mM HEPES, 10-300 IU / mL penicillin, 10- containing 300 μg / mL streptomycin and 2-20% serum; The second cell culture medium is 50-300 mM DMS in a standard basal cell culture medium. 0, DMSO2 or TMSO, 5-25 μg / mL L-proline, 0.1-0.5 μ g / mL insulin, 10-100 nM dexamethasone, 0-10 ng / mL EGF, 0.01-1mM Asc-2P, 10-50mM HEPES, 10-300IU / m Contains L penicillin, 10-300 μg / mL streptomycin, and 2-20% serum The method according to any one of claims 32 to 41.

45. Performance of candidate drugs in promoting alcohol metabolism by HH or candidate drugs for HH 10. A method for assaying the toxicity of a compound comprising: contacting said amount of HH cultured in said second phase with said candidate drug. and metabolism of alcohol by the HH in the amount before contact with the candidate drug or the candidate drug. measuring a first level of excretion of the agent in the presence or after contact with the candidate agent; A second level of metabolism of the alcohol or excretion of the candidate drug by the amount of HH is measured. The method of any one of claims 32 to 44, comprising the steps of:

46. and a cell culture medium supplemented with DMSO or TMSO, wherein the cell culture medium is DMS. An O-free HH culture medium for establishing and maintaining liver function in HH.

47. The DMSO2 or TMSO was added in standard basal cell culture medium at a concentration of about 140. The basal medium was supplemented to reach a concentration of 8 mM or 70.4 mM, and the basal medium was supplemented with L-proline. Phosphorus, insulin, dexamethasone, EGF, Asc-2P, HEPES, penicillin streptomycin, human serum, and / or heat-inactivated FBS.

47. The HH culture medium of claim 46, further comprising:

48. 140.8 mM DMSO2, 15 μg / mL L-Protein in standard cell culture basal medium Lorin, 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EG F, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 48. The method of claim 46 or 47, comprising 100 μg / mL streptomycin and 10% serum. HH culture medium as described.

49. 70.4 mM TMSO, 15 μg / mL L-proline in standard cell culture basal medium 0.25 μg / mL insulin, 50 nM dexamethasone, 5 ng / mL EGF, 0.1 mM Asc-2P, 20 mM HEPES, 100 IU / mL penicillin, 10 48. The method of claim 46 or 47, comprising 0 μg / mL streptomycin and 10% serum. HH culture medium.

50. 50-200 mM DMSO or TMSO, 5-2 5 μg / mL L-proline, 0.1-0.5 μg / mL insulin, 10-100 nM Dexamethasone, 1-10 ng / mL EGF, 0.01-1 mM Asc-2P, 10 ~50 mM HEPES, 10-300 IU / mL penicillin, 10-300 μg / mL 50. The method according to any one of claims 46 to 49, comprising streptomycin and 2 to 20% serum. HH culture medium as described above.

51. A first container for storing a first cell culture medium, the first cell culture medium being DMS a first container refilled with O; and a second container for storing a second cell culture medium, said second cell culture medium being DMS; A second container supplemented with 0, DMSO2 and / or TMSO A kit for culturing HH, comprising:

52. A first container for storing a first cell culture medium, the first cell culture medium being DMS a first container refilled with O; and a second container for storing a second cell culture medium, said second cell culture medium being DMS; A second container supplemented with O2 and / or TMSO but not DMSO. A kit for culturing human hepatocytes, comprising:

53. Assaying drug toxicity or drug metabolism and pharmacokinetics (DMPK) of drugs on HH A method of Cultivate a certain amount of HH in medium supplemented with DMSO2 and medium not supplemented with DMSO. Steps and contacting the agent with said amount of HH cultured in medium supplemented with DMSO2; and; determining the level of toxicity to said amount of HH in the presence of or after contact with said agent; measuring said amount of HH in the presence of said agent or after contact with said agent. measuring the level of metabolism or excretion of said drug by A method comprising:

54. The agent is ethanol, methanol, ethylene glycol, isopropanol or 54. The method of claim 53, wherein the alcohol compound comprises a mixture thereof.

55. 53. The method of claim 52, wherein the contacting and measuring steps are performed in a sealed device.

54. The method according to claim 54.

56. The level of toxicity includes, but is not limited to, the amount of cytochrome P450 ( Quantifying the expression or function of enzymes that metabolize non-biological substances, such as CYP enzymes The method according to any one of claims 53 to 55,

57. The level of toxicity is determined by the toxic metabolism of alcohol, such as acetaldehyde, in the amount of HH. The reduction of glutathione and the activation of cell death pathways are measured by quantifying the product or glutathione reduction. The method according to any one of claims 53 to 55, wherein

58. A method for assaying candidate agents for enhancing the metabolism of alcohol or vitamin A. So, Cultivate a certain amount of HH in medium supplemented with DMSO2 and medium not supplemented with DMSO. Steps and The amount of HH cultured in DMSO2-supplemented medium in the presence of a candidate drug was alcoholized. contacting the soluble fiber with ethanol or vitamin A; The amount of alcohol or vitamin D produced by HH in the presence of or after contact with a candidate drug is measuring the level of metabolism of amine A; A method comprising:

59. The medium used is a HH-derived conditioned medium according to any one of claims 20 to 24. Item 32. The method of any one of Items 32 to 58.

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