WO2021042071A1

WO2021042071A1 - Recovery of hepatic function of cultured hepatocytes

Info

Publication number: WO2021042071A1
Application number: PCT/US2020/048841
Authority: WO
Inventors: Albert Li
Original assignee: Albert Li
Priority date: 2019-08-31
Filing date: 2020-08-31
Publication date: 2021-03-04

Abstract

Disclosed herein are reagents, methods, compositions, and kits for use in systems for "recovering" from initial de-differentiation resulting from cell culture. In some embodiments, then, this disclosure provides reagents and systems for plating cryopreserved human hepatocytes onto collagen-coated plates to establish a 100% confluent culture and replenish culture medium every two to three days to remove cellular waste products and replace spent nutrients.

Description

RECOVERY OF HEPATIC FUNCTION OF CULTURED HEPATOCYTES

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application Ser. No. 62/894,708 filed on 31 August 2019, herein incorporated by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] This disclosure relates to reagents, methods, compositions, and kits for use in systems for hepatocytes “recovering” from initial de-differentiation resulting from prolonged culture as high density, two-dimensional cultures.

BACKGROUND INFORMATION

[0003] Hepatocytes represent the “gold standard” for the evaluation of drug metabolism, and are now used extensively to study liver functions, including biochemistry, toxicology, drug metabolism, drug uptake and efflux, and pharmacology. However, it is well established that upon culturing, hepatocytes would lose hepatic functions, especially drug metabolizing enzyme activities (e.g., P450 activity has been observed to drop by 50% per day), a process commonly known as de-differentiation. As such, most applications of hepatocytes are for a relative short time duration: drug metabolism is performed using hepatocytes mostly up to four hours in culture. Recently, novel technologies have been invented to extend the hepatic functions of hepatocytes upon prolonged incubations. These technologies include the co-culturing of hepatocytes with micro-patterned mouse 3T3 cells (HepatoPac™; BioIVT Inc.), co-cultures of hepatocytes with liver stromal cells (Hurel Inc.), and 3-diminsional hepatocyte cultures (spheroids; InSphero Inc.) There is a need in the art for additional systems for maintaining hepatocytes in culture such that the cells retain liver- specific functions without necessarily co-culturing the cells or using 3-D system, although it is possible such systems could be used with the systems described herein. This invention represents a novel technology with which hepatocytes can retain differentiated, liver- specific functions, via prolonged culturing as a high density, two-dimensional culture. This novel technology represents a convenient experimental system without a need of co-culturing as required for HepatoPac™ system, and can be maintained using conventional cell culture techniques without the specialized and difficult technologies as required for spheroids. SUMMARY OF THE DISCLOSURE

[0004] The reagents and methods of this disclosure are based on the novel observation that upon prolonged culturing (> 7 days) as high density, two-dimensional cultures, human hepatocytes re-differentiate to retain liver specific functions. This disclosure provides reagents, methods, and kits for preparing hepatocyte cultures with long term maintenance of metabolic function, as well as compositions, kits, and methods comprising and/or using the same. In some embodiments, the method comprises culturing primary human hepatocytes from five to 50 days; and, selecting the primary human hepatocytes after at least about seven days of culture that recovered from de differentiation, wherein recovery is measured by hepatic functions. In some embodiments, the hepatic function is drug metabolism. In some embodiments, the hepatic function is liver biochemistry, including biosynthesis of proteins, lipids, carbohydrates, and steroids. In some embodiments, the selected hepatocytes are metabolically competent. Other embodiments are also contemplated as may be derived from this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS [0005] Figure 1 illustrates the morphology of exemplary cultured human hepatocytes prepared as disclosed herein after culture for four (4) hours, three (3) days, 21 days and 32 days. The hepatocytes were cultured as pure hepatocyte cultures without co-culturing with non-hepatocytes as a high-density monolayer (two-dimensional culture). The high cell density is critical to the recovery of hepatic functions from de-differentiation, a common criticism of cultured hepatocytes.

[0006] Figure 2 presents re-differentiation of the human hepatocytes upon culturing. Hepatic differentiation genes illustrated are transferrin, a liver- specific protein synthesized by hepatocytes, and the drug metabolizing enzymes CYP2C9, CYP2D6, and CYP3A4 (Fig. 2). Significant decreases in gene expression were observed after 2 days of culture, representing the commonly known de-differentiation of cultured hepatocytes, a major criticism of this experimental system. Upon prolonged culturing for 7 days or longer, re-differentiation was observed as demonstrated by the recovery of gene expression to that of day 0 (before de-differentiation). Thus, the data shows prolonged culturing of the hepatocytes leads to re-differentiation. The methods and reagents disclosed herein allow cultured hepatocytes to be used for studies requiring long-term culturing including drug metabolism, uptake and efflux, hepatotoxicity, pharmacology, and gene therapy, among other applications. [0007] Figure 3 presents re-differentiation of the human hepatocytes upon culturing. Hepatic differentiation genes illustrated are transferrin, a liver- specific protein synthesized by hepatocytes, and the uptake transporters NTCP and OCT1 and efflux transporters MDR1 and BSEP. The conclusions discussed above with respect to Fig. 2 are applicable to Fig. 3 as well.

[0008] Figure 4 illustrates the application of the long-term cultured human hepatocytes in the evaluation of drug toxicity, allowing the evaluation of time- and dose-dependent hepatotoxicity. All the drugs evaluated here are known to cause liver injuries, sometimes fatal, in human patients.

[0009] Figure 5 illustrates the application of long-termed cultured human hepatocytes in the evaluation of the efficacy and duration of efficacy of gene therapy modalities. In gene therapy, aberrant gene expression can lead to serious diseases. Numerous targets for gene therapy are represented in the liver. Illustrated here is the application of long-term cultured human hepatocytes in the evaluation of a GalNac-siRNA gene silencing modality, demonstrating >80 percent of suppressed gene expression upon transduction, with the suppression effects persisting until Day 33, and then returning to near 50% of untreated hepatocytes on Day 41.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0010] As described herein, the inventor recently discovered that upon culturing as 100% confluent cultures, human hepatocytes would “recover” from the initial de-differentiation. In some embodiments, this disclosure provides reagents and systems for plating cryopreserved human hepatocytes onto collagen-coated plates to establish a 100% confluent culture and replenish culture medium every two to three days to remove cellular waste products and replace spent nutrients. While the hepatocytes would lose liver functions very rapidly in culture, most of the liver functions would recover by around day seven. This discovery now allows hepatocytes to be applied towards the long-term applications described herein, greatly enhancing the scientific usefulness of this experimental system. In some embodiments, these hepatocytes can be used for long-term applications such as prolonged drug metabolism, chronic toxicity, viral replication, and prolonged pharmacological effects. In some embodiments, the long-term cultures are used to evaluate the effectiveness of gene therapy via the introduction of genes, RNA, siRNA, proteins, and peptides to correct genetic disorders. Disclosed and provided herein are reagents, methods, compositions, and kits for use in such systems. [0011] In certain embodiments, the “recovery” of hepatic function comprises drug metabolism function. In certain other embodiments, “recovery” of hepatic function comprises transporter- mediated drug uptake and efflux, or is liver biochemistry, including biosynthesis of proteins, lipids, carbohydrates, and steroids. In embodiments, the cultured hepatocytes are cultured for 5 to 50 days wherein selected hepatocytes are metabolically competent. In exemplary embodiments, those selected hepatocytes express CYP3A4 for at least 20, 30, 40, 43, 50, 60, 70, 80, 90, 100, or more days.

[0012] The present methods for preparing hepatocyte cultures with long-term maintenance of metabolic function leading to the selection of the primary human hepatocytes after at least about seven days of culture that recovered from de-differentiation, wherein recovery is measured by hepatic functions. Therefore, provided herein is an in vitro reagent for evaluating biological activity of a test substance, wherein the reagent is primary human hepatocytes cultured as high density, two-dimensional (monolayer) cultures for over seven (7) days, wherein the hepatocytes are not co-cultured with other cells. In preferred embodiments, the primary hepatocytes disclosed herein are not cryopreserved during the generation of the primary hepatocytes. In certain embodiments, the present primary hepatocytes with long term drug metabolizing activity are cryopreserved, thawed and then cultured for at least 5 days and up to 50 days wherein the cells go through a de-differentiation stage (e.g., loose all or most of their drug metabolizing enzyme activity) and then transition to a re-differentiated stage (e.g. gain at least 25%, or up to 100%, of their drug metabolizing activity) after culture of at least 5 days (preferably 7 days) and as measured by expression of hepatocyte drug metabolizing enzymes.

[0013] In some embodiments, the primary human hepatocytes are used for the evaluation of drug toxicity, drug metabolism, or drug-drug interactions (including inhibition and induction of drug metabolizing enzymes and transporters). In some embodiments, the evaluation can be in an “average” population of mammals, preferably human beings, thereby minimizing findings unique to an individual. In certain embodiments, the primary human hepatocytes can be used for the evaluation of pharmacology such as modulation of cholesterol synthesis, inhibition of hepatitis viral replication. In certain other embodiments, the primary human hepatocytes can be used for the evaluation of gene therapy, including introduction of genes and/or RNA to overcome liver genetic diseases. In some embodiments, the hepatocyte preparations disclosed herein can be used to study drug metabolism and pharmacokinetics (DPMK) including but not limited to metabolism, uptake, efflux and/or drug-drug interactions (e.g., induction, inhibition, time-dependent inhibition (DPMK)); toxicology (e.g., hepatotoxicity screening (acute, chronic), elimination of drug candidates with sDILI potential, species selection for safety studies); physiologically based pharmacokinetic (PBPK) modeling; genetic polymorphisms; and, other pharmacological studies. In some embodiments, exemplary gene therapy disease targets relating to the liver for which the methods and/or in vitro reagents disclosed herein can be used can include but are not limited to CN1, familial hypercholesterolemia and other lipid metabolic disorders, maple syrup urine disease, progressive familial intrahepatic cholestasis, phenylketonuria, tyrosinemia, mucopolysaccharidosis VII, AAT deficiency, OTC deficiency, Wilson's disease, glycogen storage diseases (e.g., von Gierke's disease and Pompe's disease), hyperbilirubinema, acute intermittent porphyria, citrullinemia type 1, hemophilia A and B, and oxalosis. Gene therapy-related studies can include single donor or pooled multiple donor hepatocytes; relate to transfection efficiency (e.g., delivery modality); relate to efficacy (e.g., therapeutic modality), and/or relate to duration of efficacy (e.g., evaluation of gene expression versus culture duration). Vectors commonly used for liver-related applications and can be used in conjunction with the in vitro reagents disclosed herein can include but are not limited to adeno-associated viral (AAV) vectors or GalNAc-siRNA conjugates for hepatocyte-specific asialoglycoprotein receptor (ASGPR)-mediated delivery into hepatocytes. In some embodiments, the in vitro reagents disclosed herein can be used to determine the efficacy and duration of efficacy of gene therapy modalities target human hepatocytes in vivo. The hepatocyte preparation (e.g., in vitro reagents) disclosed herein provide several advantages over current animal models including but not limited to humanized mice (expensive and inefficient, large quantity of test materials are required) and the human hepatocytes are more appropriate for studies relating to human liver-related conditions. In some embodiments, human and non-human (e.g., dog) hepatocytes can be used to select a relevant non-human cell model that can be used to study, e.g., a targeted drug and/or gene.

[0014] In some embodiments, cryopreserved human hepatocytes from single or multiple donors (pooled donor hepatocytes) are used for the establishment of the long-term cultures. In some embodiments, cryopreservation of hepatocytes, in some embodiments pooled hepatocytes, can be accomplished using any of the methods available in the art, preferably any of those disclosed in U.S. Pat. No. 9,078,430 B2 (Albert Li). [0015] The test compounds used in the present disclosure include, but are not limited to drugs, drug candidates, biologicals, food components, herb or plant components, proteins, peptides, oligonucleotides, DNA and RNA. In embodiments, the test compound is a drug, a drug candidate, an industrial chemical, an environmental pollutant, a pesticide, an insecticide, a biological chemical, a vaccine preparation, a cytotoxic chemical, a mutagen, a hormone, an inhibitory compound, a chemotherapeutic agent or a chemical. In certain embodiments, the drug or drug candidate is selected from the group consisting of an organic compound, an inorganic compound, a hormone, a growth factor, a cytokine, a reception, an antibody, an enzyme, a peptide, an aptamer or a vaccine. The test compound can be either naturally-occurring or synthetic, and can be organic or inorganic. A person skilled in the art will recognize that the test compound can be added to the in vitro reagent present in the cell culture medium in an appropriate solvent or buffer.

[0016] In certain embodiments, the cells and/or in vitro reagent may be used with a cell culture vessel that is a multi-well plate, such as a 6-well; 12-well; 24-well; 48-well, 96-well; 384-well, 1536-well plate or any combination thereof. In embodiments, the cells may be cultured in a cell culture vessel with a single well plate, while in some embodiments a multi well plate can be used. In embodiments, the cell culture vessel can comprise polyethylene terephthalate (PET), polycarbonate, polystyrene, polypropylene, nylon, Mylar, stainless steel, wire mesh, aluminum, synthetic mesh, plastic, or paper.

[0017] The cells of interest in this disclosure (e.g., those included in the in vitro reagent) are primary hepatocytes that are preferably not co-cultured with other cells. In some embodiments, the human primary hepatocytes can be cultured with mouse, rat, monkey, dog, mammals, avian or non-mammalian cells. In preferred embodiments, the primary hepatocytes are human primary hepatocytes. (See, Li, A. P. (2007) Human hepatocytes: isolation, cryopreservation and applications in drug development. Chemico-biological interactions, 168(1), 16-29). Freshly prepared hepatocytes can be obtained by standard methods from a human or animal liver. (See, Hewitt, Nicola J., et al. "Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transport, clearance, and hepatotoxicity studies." Drug metabolism reviews 39.1 (2007): 159-234). [0018] Hepatic metabolism is known to be the major determinant of metabolism toxicity. P450 and non-P450 phase 1 oxidation enzyme pathways are responsible mostly for the bioactivation of relatively inert parent compounds to reactive (toxic/carcinogenic/mutagenic) metabolites. Phase 2 conjugating pathways are responsible mostly for the biotransformation of toxic parent compounds or metabolites to less toxic compounds. Both phase 1 and phase 2 pathways are present in the hepatocytes - the key hepatic cell type responsible for hepatic metabolism. The hepatocytes prepared as described herein may be used in assays such as these or measuring such pathways.

[0019] In certain embodiments, the hepatocytes can be recombinant cells, wherein for example, the cells have been engineered to comprise drug metabolizing enzymes. In certain embodiments, the metabolically competent cells are engineered to contain cytochrome P450 isoforms. See, Doehmer, et ah, 1992. Biotransformation of caffeine and theophylline in mammalian cell lines genetically engineered for expression of single cytochrome P450 isoforms. Biochemical pharmacology, 43(2), pp.225-235; and, Donato, et al. (2004). Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cells expressing individual human P450 enzymes. Drug Metabolism and Disposition, 32(7), 699-706.

[0020] The term “culture condition” encompasses cells, media, factors, time and temperature, atmospheric conditions, pH, salt composition, minerals, etc. Cell culturing is typically performed in a sterile environment mimicking physiological conditions, for example, at 37°C in an incubator containing a humidified 92-95% air/5-8% CO2 atmosphere. In embodiments, the cell culture temperate is a range from 33-40°C. Cell culturing may be carried out in nutrient mixtures containing undefined biological fluids such a fetal calf serum, or media that is fully defined and serum free. A variety of culture media are known in the art and are commercially available.

[0021] In embodiments, the primary hepatocytes are cultured under cell culture conditions replicate physiological conditions as much as possible. The term “physiological conditions” as used herein is defined to mean that the cell culturing conditions are very specifically monitored to mimic as closely as possible the natural tissue conditions for hepatocytes in vivo.

[0022] Exemplary of pharmaceutical agents suitable for this disclosure are those described in “The Pharmacological Basis of Therapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996), Ninth edition, under the sections: Drugs Acting at Synaptic and Neuroeffector Junctional Sites; Drugs Acting on the Central Nervous System; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions; Drugs Affecting Renal Function and Electrolyte Metabolism; Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; Drugs Affecting Uterine Motility; Chemotherapy of Parasitic Infections; Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases; Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming Organs; Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology, all incorporated herein by reference. Also included are toxins, and biological and chemical warfare agents, for example see Somani, S. M. (Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992). While many samples will comprise compounds in solution, solid samples that can be dissolved in a suitable solvent may also be assayed. Samples containing test compounds of interest include environmental samples, e.g., ground water, sea water, or mining waste; biological samples, e.g., lysates prepared from crops or tissue samples; manufacturing samples, e.g., time course during preparation of pharmaceuticals; as well as libraries of compounds prepared for analysis; and the like. Samples of interest include test compounds being assessed for potential therapeutic value, e.g., drug candidates from plant or fungal cells.

[0023] Various methods can be utilized for quantifying the presence of selected metabolism markers. Liquid chromatography (LC), mass spectrometry (MS), and their combination (LC/MS- MS) are routinely used for the quantification of metabolites. For non-LC/MS measurement of the amount of a molecule that is present, a convenient method is to label the molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, or enzymatically active. Fluorescent and luminescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluore scent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to autofluoresce, e.g., by expressing them as green fluorescent protein chimeras inside cells (for a review, See Jones et al. (1999) Trends Biotechnol. 17(12):477-81).

[0024] Output variables may be measured by immunoassay techniques such as, immunohistochemistry, radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA) and related non-enzymatic techniques. These techniques utilize specific antibodies as reporter molecules that are particularly useful due to their high degree of specificity for attaching to a single molecular target. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these. For toxicity assays, outputs can include measurement of cell viability such as enzyme release, cellular ATP contents, reactive oxygen species formation, decrease of reduced glutathione, protein synthesis, protein contents, DNA contents, dye exclusion, dye inclusion, and cell detachment. For pharmacological assays, specific disease target related assays can be used. For genotoxicity assays, endpoints measured may include DNA damage, chromosomal aberration, mutant generation, and induction of DNA repair. In embodiments, the results of screening assays may be compared to results obtained from a reference compound, concentration curves, controls (with and without metabolically competent cells), etc. The comparison of results is accomplished by the use of suitable deduction protocols, A1 systems, statistical comparisons, etc.

[0025] A database of reference output data can be compiled. These databases may include results from known agents or combinations of agents, as well as references from the analysis of cells treated under environmental conditions in which single or multiple environmental conditions or parameters are removed or specifically altered. A data matrix may be generated, where each point of the data matrix corresponds to a readout from an output variable, where data for each output may come from replicate determinations, e.g., multiple individual cells of the same type. The readout may be a mean, average, median or the variance or other statistically or mathematically derived value associated with the measurement. The output readout information may be further refined by direct comparison with the corresponding reference readout. The absolute values obtained for each output under identical conditions will display a variability that is inherent in live biological systems and also reflects individual cellular variability as well as the variability inherent between individuals.

[0026] In some embodiments, the method for hepatocyte recovery can include the following steps: 1) isolating human hepatocytes (e.g., from each of multiple donors) and culturing the cells for multiple days, ranging from five to 40 (5 - 40) days, preferably at least seven days; 2) at multiple days during the culture (e.g., 5, 7, 14, 21, 28, 35, 42 days), the hepatocytes are collected and gene expression of liver specific genes are quantified; 3) the hepatocyte lots (each lot represent hepatocytes from a donor or day of culture) that are able to fully recover from de-differentiation as determined by recovery of hepatic gene expression are identified as lots for use in long-term culturing and the evaluation of specific drug properties that require long-term hepatocyte cultures. In some embodiments, each donor sample can lead to multiple samples (e.g., 500 vials per donor) and used for multiple experiments including but not limited to evaluation of metabolism of slowly metabolized chemicals (e.g., seven (7) day metabolism), chronic drug toxicity, prolonged pharmacological effects, as well as special evaluation of the stability of modified gene expression using gene therapy modalities such as down-regulating expression of specific genes siRNA, and/or aiding in the selection of siRNA molecules for stable down-regulation of hepatic genes. In some embodiments, the genes for which expression can be measured can include one or more, preferably at least two to all, of CYP2C9, CYP2D6, CYP3A4, Na+-taurocholate co-transporting polypeptide (NTCP), organic cation transporter 1 (OCT1), multi-drug resistance 1 (MDR1), bile salt export pump (BSEP), p450, and/or transferrin, and/or other genes and/or the like (e.g., albumin).

[0027] In some embodiments, this disclosure provides reagents, methods, and kits for preparing hepatocyte cultures with long term maintenance of metabolic function, as well as compositions, kits, and methods comprising and/or using the same. In some embodiments, the method comprises: a) culturing primary human hepatocytes from five to 50 days, preferably at least seven (7); and, b) selecting the primary human hepatocytes after at least about seven days of culture that recovered from de-differentiation, wherein recovery is measured by hepatic functions. In some embodiments, the primary human hepatocytes are not co-cultured with other cells. In some embodiments, the hepatic function is drug metabolism. In some embodiments, the hepatic function is transporter-mediated drug uptake and efflux. In some embodiments, the hepatic function is liver biochemistry, including biosynthesis of proteins, lipids, carbohydrates, and steroids. In some embodiments, the selected hepatocytes are metabolically competent. In some embodiments, the hepatocytes selected in step b) are subsequently frozen at a temperature of -10°C to about -175°C. In some embodiments, the hepatocytes selected in step b) maintain CYP3A4 gene expression for up to 20, 30, 40, 43, 50, 60, 70, 80, 90, 100, or more days.

[0028] In some embodiments, this disclosure provides compositions comprising the hepatocytes produced by the methods herein, and optionally at least one excipient, further optionally wherein said excipient is cell culture medium comprising a cryoprotectant. In some embodiments, the compositions and/or the hepatocytes are cryopreserved. [0029] In some embodiments, as illustrated in Fig. 1, this disclosure provides methods for culturing hepatocytes as pure hepatocytes, without co-culturing with non-hepatocytes, as a high- density monolayer (in a two-dimensional culture system). The high cell density is critical to the recovery of hepatic functions from de-differentiation, a common criticism of cultured hepatocytes. As used herein, “high density” means, in reference to a 24-well plate (e.g., collagen-coated), an initial seeding of at least about 250,000 hepatocytes per well that eventually grows to at least 80% confluence. In preferred embodiments, the hepatocytes are cultured in Williams Medium E (available from Sigma-Aldrich) comprising ITS Liquid Media Supplement comprising recombinant human insulin, human transferrin, and sodium selenite (ITS; available from Millipore Sigma), which is referred to herein as “modified Williams E medium”. Those of ordinary skill in the art would understand that in some embodiments other initial seeding amounts can be used depending on the type of culture well being used (e.g., a 12-well plate can be seeded with a proportionally greater number of cells, while a 48-well plate can be seeded with a proportionally lesser number of cells).

[0030] In some embodiments, de-differentiation of the primary hepatocytes can be indicated by the decrease in gene expression of at least one of CYP2C9, CYP2D6, CYP3A4, NTCP, OCT1, MDR1, BSEP, p450, and/or transferrin (and/or other genes such as albumin) to near-zero to zero detectable expression after an initial culturing time period (e.g., two days). Upon prolonged culturing for seven days or longer, re-differentiation can be indicated by the recovery of gene expression of such genes (e.g., as compared to day two of culture). In preferred embodiments, recovery of gene expression is an increase to greater than zero, preferably an increase to at least about 25% or more (e.g., 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100%) of the original amount of expression (e.g., to pre-culture levels), such increased gene expression being indicative of re-differentiation. In some embodiments, re differentiation of the primary human hepatocytes can be determined by the expression of at least about 25% greater level(s) of at least one of CYP2C9, CYP2D6, CYP3A4, NTCP, OCT1, MDR1, BSEP, p450, and/or transferrin (and/or other genes such as albumin) after seven days in culture as compared to the level of expression of such gene(s) after culture for two days. In some such embodiments, as illustrated in Figs. 2 and 3, this disclosure provides methods for re-differentiating human hepatocytes upon culturing. Hepatic differentiation genes illustrated are transferrin, a liver- specific protein synthesized by hepatocytes, and the drug metabolizing enzymes CYP2C9, CYP2D6, and CYP3A4 (Fig. 2). Hepatic differentiation genes illustrated in Fig. 3 are transferrin, a liver- specific protein synthesized by hepatocytes, and the uptake transporters NTCP and OCT1 and efflux transporters MDR1 and BSEP. Significant decreases in gene expression were observed after two days of culture, representing the commonly known de-differentiation of cultured hepatocytes, a major criticism of this experimental system. As shown in Figs. 2-3, upon prolonged culturing for seven days or longer, re-differentiation was observed as demonstrated by the recovery of gene expression to that of day 0 (before de-differentiation). Thus, the data shows prolonged culturing of the hepatocytes leads to re-differentiation. The methods and reagents disclosed herein allow cultured hepatocytes to be used for studies requiring long-term culturing including drug metabolism, uptake and efflux, hepatotoxicity, pharmacology, and gene therapy, among other applications.

[0031] As shown in Fig. 4, the long-term cultured human hepatocytes produced as disclosed herein can be used in the evaluation of drug toxicity, allowing the evaluation of time- and dose- dependent hepatotoxicity. All the drugs evaluated here are known to cause liver injuries, sometimes fatal, in human patients.

[0032] As illustrated in Fig. 5, the long-term cultured human hepatocytes produced as disclosed herein can be used in the evaluation of the efficacy and duration of efficacy of gene therapy modalities. In gene therapy, aberrant gene expression can lead to serious diseases. Numerous targets for gene therapy are represented in the liver. Illustrated here is the application of long-term cultured human hepatocytes in the evaluation of a GalNac-siRNA gene silencing modality, demonstrating >80 percent of suppressed gene expression upon transduction, with the suppression effects persisting until Day 33, and then returning to near 50% of untreated hepatocytes on Day 41.

[0033] In some embodiments, this disclosure provides an in vitro reagent for evaluating biological activity of a test substance, wherein the reagent is a cryopreserved mixture comprising: primary human hepatocytes with long-term drug metabolizing enzyme activity up to 50 days of culturing, wherein the hepatocytes are not co-cultured with other cells; and, a cell culture medium, optionally comprising a cryoprotectant. In some such embodiments, the in vitro reagent and/or primary human hepatocytes are prepared as described above and/or in Example 1. In some such embodiments, the in vitro reagent and/or primary human hepatocytes are used for the evaluation of drug toxicity, drug metabolism, and drug-drug interactions (including inhibition and induction of drug metabolizing enzymes and transporters. In some such embodiments, the in vitro reagent and/or primary human hepatocytes are used for the evaluation of pharmacology such as modulation of cholesterol synthesis, inhibition of hepatitis viral replication. In some such embodiments, the in vitro reagent and/or primary human hepatocytes are used for the evaluation of gene therapy, including introduction of genes and/or RNA to overcome liver genetic diseases. In some such embodiments, the drug or drug candidate is selected from the group consisting of an organic compound, an inorganic compound, a hormone, a growth factor, a cytokine, a reception, an antibody, an enzyme, a peptide, a NSAID, an aptamer, and/or a vaccine. In some such embodiments, the test substance is a protein, peptide, RNA, siRNA, and DNA. In some such embodiments, the in vitro reagent and/or primary human hepatocytes are cultured and used to evaluate metabolism of slowly metabolized chemicals, chronic drug toxicity or prolonged pharmacological effects. In some embodiments, this disclosure provides methods for using such an in vitro reagent and/or primary human hepatocytes. In some embodiments, this disclosure provides methods for preparing such an in vitro reagent by combining primary human hepatocytes with long-term drug metabolizing enzyme activity up to 50 days of culturing, wherein the hepatocytes are not co-cultured with other cells; and, a cell culture medium comprising a cryoprotectant. In some embodiments, the methods further comprise cryopreserving the primary human hepatocytes. In some embodiments, the methods further comprise thawing the cryopreserved primary human hepatocytes. In some embodiments, the methods further comprise further comprising using the primary human hepatocytes in an assay.

[0034] In some embodiments, this disclosure provides products (e.g., compositions, in vitro reagents) comprising single or multiple donor hepatocyte preparations as, in some embodiments, pre-plated cultures. In some embodiments, the products can be provided in multi- well plate format (e.g., 6-, 12-, 24-, 48-, 96-, 128-, 256-, or 384-well plate format). In some embodiments, human hepatocyte spheroids of a single donor can be provided in 384-well format (e.g., one spheroid per well, about 2000 cells per spheroid). In some embodiments, the pre-plated cells can be covered with cell culture medium (e.g., modified Williams E medium), sealed and shipped pre-warmed (e.g., using a warming gel). In some embodiments, the hepatocyte preparations disclosed herein (e.g., in vitro reagents and/or products) can be used to study drug uptake, metabolism, DDI, hepatotoxicity, and/or gene therapy, or other uses disclosed herein and/or as may be otherwise apparent to the skilled artisan.

[0035] In some embodiments, this disclosure provides methods for preparing hepatocyte cultures with long term maintenance of metabolic function by: culturing primary human hepatocytes for a time period of from five to 50 days; and, selecting the primary human hepatocytes after at least about seven days of culture that recovered from de-differentiation, wherein recovery is measured by one or more hepatic functions; as well as primary human hepatocytes produced by such methods, and/or compositions comprising the same. In preferred embodiments, the primary human hepatocytes are not co-cultured with other cells. In preferred embodiments, the cells are cultures as a high-density monolayer two-dimensional culture. In preferred embodiments, the primary human hepatocytes produced by such methods are re-differentiated. In some embodiments, including some preferred embodiments, the primary human hepatocytes produced by these methods express CYP2C9, CYP2D6, CYP3A4, NTCP, OCT1, MDR1, BSEP, p450, and/or transferrin, and/or other genes and/or the like (e.g., albumin), at levels characteristic of re differentiated hepatocytes. In preferred embodiments, the one or more hepatic functions is drug metabolism, and can be transporter-mediated drug uptake and/or efflux, and/or liver biochemistry, including but not limited to the biosynthesis of proteins, lipids, carbohydrates, and steroids. In preferred embodiments, the primary human hepatocytes produced by these methods are metabolically competent. In some embodiments, the hepatocytes produced by these methods express P450, uptake transporters, and/or efflux transporters, for at least 7 or more days. In some embodiments, this disclosure provides in vitro reagent(s) for evaluating biological activity of a test substance, wherein the reagent is a cryopreserved mixture comprising: primary human hepatocytes with long-term drug metabolizing enzyme activity up to 50 days of culturing (preferably produced as disclosed herein), wherein the hepatocytes are not co-cultured with other cells; and, a cell culture medium comprising a cryoprotectant. In some embodiments, such in vitro reagent(s) can be used for the evaluation of drug toxicity, drug metabolism, or drug-drug interactions, including inhibition and induction of drug metabolizing enzymes and transporters. In some embodiments, such in vitro reagent(s) can be used for the evaluation of pharmacology such as modulation of cholesterol synthesis, or inhibition of hepatitis viral replication. In some embodiments, such in vitro reagent(s) can be used for the evaluation of gene therapy, including introduction of genes and/or RNA to overcome liver genetic diseases. In some embodiments, the drug or drug candidate to be studied using such in vitro reagent(s) can be selected from the group consisting of an organic compound, an inorganic compound, a hormone, a growth factor, a cytokine, a reception, an antibody, an enzyme, a peptide, a NSAID, an aptamer and a vaccine. In some embodiments, the test substance can be a protein, peptide, RNA, siRNA, and DNA. In some embodiments, the primary human hepatocytes (e.g., in vitro reagent(s)) can be cultured and used to evaluate metabolism of slowly metabolized chemicals, chronic drug toxicity or prolonged pharmacological effects. Thus, this disclosure also provides methods for using such in vitro reagent(s). In some embodiments, such methods can include combining primary human hepatocytes with long-term drug metabolizing enzyme activity up to 50 days of culturing, wherein the hepatocytes are not co-cultured with other cells; and, a cell culture medium comprising a cryoprotectant. In some embodiments, such methods can include cry opreserving the primary human hepatocytes, thawing the cryopreserved primary human hepatocytes, and using the primary human hepatocytes in an assay. Other embodiments are also contemplated as would be understood by those of ordinary skill in the art from this disclosure.

[0036] The terms “about”, “approximately”, and the like, when preceding a list of numerical values or range, refer to each individual value in the list or range independently as if each individual value in the list or range was immediately preceded by that term. The terms mean that the values to which the same refer are exactly, close to, or similar thereto. Optional or optionally means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. Ranges may be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about or approximately, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Ranges (e.g., 90-100%) are meant to include the range per se as well as each independent value within the range as if each value was individually listed. All references cited within this disclosure are hereby incorporated by reference in their entirety. EXAMPLES

[0037] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to use the embodiments provided herein, and are not intended to limit the scope of the disclosure nor are they intended to represent that the Examples below are all of the experiments or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by volume, and temperature is in degrees Centigrade. It should be understood that variations in the methods as described can be made without changing the fundamental aspects that the Examples are meant to illustrate.

[0038] Example 1: Recovery of hepatic function of cultured hepatocytes [0039] Major hepatic functions of cultured hepatocytes would decrease after two days in culture, a phenomenon that is well-established. However, it was also found that continued maintenance of the culture led to “recovery” of the hepatic functions. Results with hepatocytes obtained from seven human livers are shown below for the following key hepatic functions:

1) P450: P450 isoforms are the most important enzymes responsible for drug metabolism. Via culturing the hepatocytes as high density monolayer cultures, P450 gene expression decreased on Day 2 of culturing and returned to levels on Day 0 (before de-differentation), with gene expression in some lots of human hepatocytes maintained for 40 days in culture. This phenomenon occurs for several of the human hepatocyte lots (Fig. 2).

2) Transferrin: Transferrin is an important protein, mainly synthesize by the liver, with a key function of iron transport into cells and tissues, especially for hemoglobin synthesis. Similar maintenance of transferrin gene expression was observed in the 100% confluent monolayer hepatocyte cultures (Fig. 2).

3) Uptake and efflux transporters: Transporters are responsible for drug uptake into the hepatocytes while efflux transporters are responsible for the bile excretion of drugs and metabolites. Bile salt efflux via BSEP, if inhibited, would lead to liver injuries due to accumulation of the toxic bile salts. Both uptake and efflux transporters were shown to recover upon prolonged culturing for over 7 days (Fig. 3). [0040] Example 2. Application in the evaluation of drug toxicity. Hepatocytes prepared as disclosed herein can be used to study hepatotoxicity. Figure 4 illustrates results of the treatment of human hepatocytes with toxic drugs for multiple time durations, with 15-day treatment representing prolonged treatment. The toxic drugs were found to demonstrate time- and dose- dependent hepatotoxicity. All the drugs evaluated here are known to cause liver injuries, sometimes fatal, in human patients.

[0041] Example 3. Application in the evaluation of gene therapy modalities. Figure 5 illustrates the application of long-termed cultured human hepatocytes in the evaluation of the efficacy and duration of efficacy of gene therapy modalities. In gene therapy, aberrant gene expression can lead to serious diseases. Numerous targets for gene therapy are represented in the liver. Illustrated here is the results of the transduction of long-term cultured human hepatocytes with a GalNac-siRNA gene silencing modality for the HPRT gene, demonstrating >80 percent of suppressed gene expression upon transduction, with the suppression effects persisting until Day 33, and then a return to near 50% of untreated hepatocytes on Day 41.

[0042] The data presented in these examples demonstrates that hepatocytes prepared as described herein can be used in DPMK studies (e.g. drug metabolism, drug-drug interactions); toxicology studies (e.g., hepatotoxicity screening); and pharmacological studies (e.g., gene therapy). Those skilled in the art can devise many modifications and other embodiments within the scope and spirit of this disclosure and its embodiments. The disclosed embodiments, examples and experiments are not intended to limit the scope of the disclosure nor to represent that the experiments below are all or the only experiments performed. Indeed, variations in the materials, methods, drawings, experiments examples and embodiments described may be made by skilled artisans without changing the fundamental aspects of this disclosure and its embodiments. Any of the disclosed embodiments can be used in combination with any other disclosed embodiment.Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. It should be understood that variations in the methods as described may be made without changing the fundamental aspects that the experiments are meant to illustrate.

Claims

CLAIMS What is claimed is:

1. A method for preparing hepatocyte cultures with long term maintenance of metabolic function, the method comprising: a) culturing primary human hepatocytes for a time period of from five to 50 days, optionally at least seven days; and, b) selecting the primary human hepatocytes after at least about seven days of culture that recovered from de-differentiation, wherein recovery is measured by hepatic functions.

2. The method of claim 1, wherein the primary human hepatocytes are not co-cultured with other cells.

3. The method of claim 1 or 2 wherein the culturing of step a) is as a high-density monolayer two-dimensional culture.

4. The method of any preceding claim wherein the primary human hepatocytes of step b) are re differentiated.

5. The method of any preceding claim, wherein following step b) the primary human hepatocytes express CYP2C9, CYP2D6, CYP3A4, NTCP, OCT1, MDR1, BSEP, p450, and/or transferrin at levels characteristic of re-differentiated hepatocytes.

6. The method of any preceding claim wherein the primary human hepatocytes after culture for at least day seven days express at least about 25% greater level(s) of at least one of CYP2C9, CYP2D6, CYP3A4, NTCP, OCT1, MDR1, BSEP, p450, and/or transferrin as compared to the level of expression after culture for two days.

7. The method of claim 1 or 2, wherein hepatic function is drug metabolism.

8. The method of any preceding claim, wherein the hepatic function is transporter- mediated drug uptake and/or efflux.

9. The method of any preceding claim, wherein the hepatic function is liver biochemistry, including biosynthesis of proteins, lipids, carbohydrates, and steroids.

10. The method of any preceding claim, wherein the hepatocytes selected in step b) are metabolically competent.

11. The method of any preceding claim wherein the hepatocytes selected in step b) express P450 for at least 7 or more days.

12. The method of any preceding claim wherein the hepatocytes selected in step b) express uptake transporters for at least 7 or more days.

13. The method of any preceding claim wherein the hepatocytes selected in step b) express efflux transporters for at least 7 or more days.

14. The method of any preceding claim, wherein the primary human hepatocytes of step a) were previously cryopreserved.

15. An in vitro reagent for evaluating biological activity of a test substance, wherein the reagent is a mixture comprising: a. primary human hepatocytes with long-term drug metabolizing enzyme activity up to 50 days of culturing, wherein the hepatocytes are not co-cultured with other cells; and, b. a cell culture medium.

16. The reagent of claim 15, wherein the primary human hepatocytes are prepared according to claim 1.

17. The reagent of claim 15 or 16, wherein the primary human hepatocytes and cell culture medium are in wells of a multi- well cell culture plate.

18. The reagent of claim 15-17, wherein the primary human hepatocytes are used for the evaluation of drug toxicity, drug metabolism, or drug-drug interactions, including inhibition and induction of drug metabolizing enzymes and transporters.

19. The reagent of any one of claims 15-18, wherein the primary human hepatocytes are used for the evaluation of pharmacology such as modulation of cholesterol synthesis, or inhibition of hepatitis viral replication.

20. The reagent of any one of claims 15-19, wherein the primary human hepatocytes are used for the evaluation of gene therapy, including introduction of genes and/or RNA to overcome liver genetic diseases.

21. The reagent of any one of claims 15-20, wherein the drug or drug candidate is selected from the group consisting of an organic compound, an inorganic compound, a hormone, a growth factor, a cytokine, a reception, an antibody, an enzyme, a peptide, a NSAID, an aptamer and a vaccine.

22. The reagent of any one of claims 15-21, wherein the test substance is a protein, peptide, RNA, siRNA, and DNA.

23. The reagent of any one of claims 15-22, wherein the primary human hepatocytes are cultured and used to evaluate metabolism of slowly metabolized chemicals, chronic drug toxicity or prolonged pharmacological effects.

24. A method comprising using the in vitro reagent of any one of claims 15-23.

25. A method for preparing the in vitro reagent of any one of claims 15-23, the method comprising combining primary human hepatocytes with long-term drug metabolizing enzyme activity up to 50 days of culturing, wherein the hepatocytes are not co-cultured with other cells; and, a cell culture medium.

26. The method of claim 25, wherein the primary human hepatocytes were previously cryopreserved and thawed prior to the up to 50 days of culturing.

27. The method of any one of claims 25 or 26, further comprising using the primary human hepatocytes in an assay.