Method for expanding liver cells

Abstract

The present invention relates to a method for expanding liver cells, wherein the method comprises (a) expanding the liver cells within an extracellular matrix in an expansion medium, wherein the expansion medium comprises a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway, and preferably with the proviso that the expansion medium does not comprise nicotinamide.

Description

[0001] New PCT-Patent Application Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Vossius Ref.: AG2882 PCT Method for expanding liver cells The present invention relates to a method for expanding liver cells, wherein the method comprises (a) expanding the liver cells within an extracellular matrix in an expansion medium, wherein the expansion medium comprises a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway, and preferably with the proviso that the expansion medium does not comprise nicotinamide. In this specification, a number of documents including patent applications and manufacturer’s manuals are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention, is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. The liver is the major metabolic internal organ of our body. The basic architectural unit of the liver is the liver lobule. Each lobule consists of hepatocytes sandwiched by sinusoidal endothelial cells. Liver lobules are roughly hexagonal with each of six corners demarcated by the presence of a portal triad (portal vein, bile duct, and hepatic artery). Hepatocytes are the major parenchymal cell type of the liver, responsible of bile and albumin production, detoxification and glucose and lipid metabolism. They function in concert with cholangiocytes (biliary epithelial cells), endothelial cells, sinusoidal endothelial cells, Kupffer cells, natural killer cells and hepatic stellate cells. This complex architecture is important for hepatic function (Campana et al., 2021, Nat Rev Mol Cell Biol, 22: 608-624). At present, rodents are the model of choice when performing toxicology and drug testing studies in drug development (Kelland, 2004, Eur J Cancer, 40:827-836). In vitro, fresh or cryopreserved mouse or rat hepatocytes or human cancer / hepatoma cell lines like HepG2 or HepaRG are used as models to perform these first toxicology / drug testing analysis (Marion et al., 2010, Methods Mol Biol, 640:261-272; Underhill, 2017, Cell Mol Gastroenterol Hepatol, 5:426-439). However, the predictive value of these models, both rodents as well as cell lines, is questionable and has resulted in failure to identify drugs that induce liver toxicity in patients, thus resulting in drug withdrawals from late preclinical stages or even from the market. In addition, human hepatocyte transplantation has proven very successful to rescue mouse models with human liver disease (e.g. FAH mutant mice) and has been proposed as a potential solution for organ donor shortage worldwide (Azuma et al., 2007, Nat Biotechnol, 25:903-910). Unfortunately, resembling the shortage of donors, there is little availability for cryopreserved human primary hepatocytes for these toxicology and cell therapy approaches. Therefore, using hepatocytes derived from stem cell sources, either pluripotent stem cells such as human induced pluripotent stem cells (iPS cells) or adult cells that acquire stem cell / progenitor features in vitro has been considered a potential suitable alternative (Avior et al., 2016, Nat Rev Mol Cell Biol, 17:170-182; Barker et al., 2010, Cell Stem Cell, 6:25-36). However, similar to human-derived liver cancer cell lines, the maturation of human iPS cell-derived hepatocytes is rather poor and inferior to that of fresh or cryopreserved primary human hepatocytes. Unfortunately, expanding and maintaining human adult hepatocytes in culture has proven very challenging. This is in part due to the fact that once in culture human hepatocytes lose their cellular polarity which results in loss of metabolic function and intracellular bile acid accumulation which eventually leads to hepatocyte death (Dhawan et al., 2010, Nat Rev Gastroenterol Hepatol, 7:288-298). The inventors recently published a method of culturing liver organoids where mouse- and human liver ductal cells can be expanded long-term generating millions of liver cells in culture even when starting from 1 single liver ductal cells (Huch et al., 2013, Nature, 494:247-250 and 2015, Cell, 160:299-312). The methods were provided in WO 2010 / 090513, WO 2012 / 014076, WO 2012 / 168930 and WO2015 / 173425, where Huch is co-inventor. Although these methods are able to generate hepatocyte- like cells in culture upon media change, the formed hepatocyte -like cells lack the correct apical cell polarity, do not form bile canaliculi structures and performed very poorly on the functional characteristics of adult hepatocytes, namely metabolism, detoxification, bile acid and albumin production, and can only be maintained for 7 days in culture (Huch et al.2015, Cell, 160:299-312). In 2018, hepatocyte-derived liver organoids were established by Hu et al., and are anticipated as a new source of hepatocytes. Although these methods advantageously allow the expansion of epithelial liver cells, there are limited to either mouse adult hepatocytes or human embryonic cells, but cannot provide longer-term expansion of functional human adult hepatocytes for longer than 1 passage. In addition, the fetal hepatocytes lack the cellular polarity of mature hepatocytes and hence the formation of proper bile canaliculi structures and network. Therefore, it would be advantageous to increase the length of time that adult human adult polarized hepatocytes can be expanded, especially for toxicology and pharmacological studies as well as cellular sources for cell therapy transplantation. It is, therefore, an object of the present invention to provide a method for increasing the expansion time of liver cells such as human adult hepatocytes that retain hepatocyte polarity and liver cell functions, such as metabolic and detoxifying functions. Accordingly, the present invention relates in a first aspect to a method for expanding liver cells, wherein the method comprises (a) expanding the liver cells within an extracellular matrix in an expansion medium, wherein the expansion medium comprises a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway, and preferably with the proviso that the expansion medium does not comprise nicotinamide. The method for expanding liver cells is an ex vivo or in vitro method, being carried out outside of the human or animal body. The term “expanding” liver cells as used herein refers to the propagation of the liver cells by cell division (or cell proliferation). A liver cell refers to any cell that has been obtained from or isolated from liver tissue or is derived from liver tissue (such as liver cell lines) or has been matured into a liver cell from a progenitor cell. Liver cells can be divided into four basic cell types: hepatocytes, stellate fat storing cells (also called ITO cells), Kupffer cells and liver endothelial cells. Hepatocytes make up about 80% of the cells in the liver. In accordance with a preferred embodiment the liver cells are hepatocytes. Hepatocytes are polyhedral epithelial cells, with large round centrally located nuclei (2 or more). In the liver they are grouped in interconnected plates that are arranged into thousands of small polyhedral lobules. They store glucose in the form of glycogen, also vitamin B12, folic acid and iron and participate in the turnover and transport of lipids. They synthesize some of the plasma proteins (albumin, α and β globulins, prothrombin, fibrinogen), metabolize / detoxify fat, participate in the turnover of steroid hormones, regulate cholesterol level and secrete bile. Ito cells (stellate cells) are cells of mesenchymal origin located in the perisinusoidal space of the liver lobules. Their cytoplasm contains large lipid droplets used to store vitamin A. Kupffer cells are specialized stellate macrophages. In the liver they adhere to the sinusoidal endothelium (in the lumen of the sinusoid), mainly near portal areas (=portal triads). They clear the blood of ingested bacterial pathogens that may enter portal blood from the gut, remove aged erythrocytes, free heme for re-use, act as antigen-presenting cells in adaptive immunity, and secret cytokines and chemokines that recruit and expand the population of other proinflammatory cells in the liver. Sinusoidal endothelial cells in the liver form the wall of the blood vessels (sinusoids) that carry blood throughout the liver. They form a single layer with spaces between each cell known as fenestra, that allow an efficient flow of essential materials to pass from the blood to hepatocytes and vice versa. They are rich in lysosomal enzymes needed for degrading endocytosed material. The liver cells to be expanded by the method of the invention may have been obtained by any suitable method. Preferably they are obtained from a human liver and so are, in the case of hepatocytes, primary human hepatocytes (PHHs). However, primary hepatocytes from non-human animals are also envisaged for use in the invention, for example, non-human mammals such as mouse, rabbit, rat, pig, cow, sheep, horse, dog, cat, and monkey. In some embodiments, PHHs were isolated by two-step collagenase perfusion, for example, as described in Lee et al. (2013, J Vis Exp, 79:50615), Kegel et al. (2016, J Vis Exp, 109:e53069) and Damm et al. (2019, EXCLI J, 18:1071-1091). In some embodiments, collagenase and elastase perfusion were used to obtain the liver cells for use in the invention. In some cases, hepatocytes were obtained from a cryopreserved source from a commercial provider. The method may also comprise culturing an isolated fragment of liver tissue which comprises the liver cells of said liver tissue. Methods for the isolation of PHHs are known to those of skill in the art. For example, PHHs may be isolated from a human liver using two-step collagenase perfusion. Briefly, an adult liver tissue may be washed in a cold (4-10°C) culture medium, preferably Advanced-DMEM / F12 (Invitrogen) and then, the tissue can be perfused with warm (37-39°C) perfusion solution A (NaCl, KCl, NaHCO3, NaH2PO4·2H2O, Na2HPO4·12H2O, glucose, HEPES, EGTA in distilled water) for 15-30 min. Then the tissue is preferably perfused with warm (37-39°C) perfusion solution B (NaCl, KCl, NaHCO3, NaH2PO4·2H2O, Na2HPO4·12H2O, glucose, HEPES, CaCl2, Collagenase P in distilled water) for 5-15 min. Then, tissue is transferred into petri-dish with cold (4-10°C) Williams’ E medium and can be chopped. Then, the tissue fragments can be vigorously suspended in 10 ml of cold (4-10oC) Williams’ E medium with a 10- or 25- ml pipette. Subsequently, liver tissue fragments and suspension are filtered with 100 µm cell strainer and spun at 50g for 5 min. PHHs can in this way be obtained and PHHs and retained in cold media. Then, the isolated PHHs may be precipitated. Isolated PHHs are preferably seeded in 50 µl of matrigel at an approximate ratio of 12500-50000, preferably 25000-50000 PHHs / well in 48-well plate. An extracellular matrix (ECM) refers to an intricate network composed of an array of multidomain macromolecules organized in a cell / tissue-specific manner. The main ingredients of the extracellular matrix are glycoproteins, in particular glycoproteins as secreted by the cells. The most abundant glycoprotein in the ECM of most animal cells is collagen. Collagen forms strong fibers. For instance, collagen accounts for about 40% of the total protein in the human body. In an ECM the collagen fibers can be embedded in a network woven from proteoglycans. A proteoglycan molecule consists of a small core protein with many carbohydrate chains covalently attached. In accordance with the present invention the liver cells are expanded within an ECM because the ECM facilitates the viability of the liver cells and maintains liver cell functions in vitro. In addition, The ECM ensures that the live cells upon expansion can form 3D-structures, in particular so-called organoids that have a liver tissue-like structure and enable hepatocytes to polarize and form bile canaliculi structures. An expansion medium is a medium with components that allow for and favor the propagation of the liver cells. Basal mediums that are suitable for the propagation of liver cells are known in the art and are available, for example, from Thermo Fisher (e.g. Williams Medium E with HepExtend™ Supplement (50X)) or Lonza (e.g. MM250 Hepatocyte Culture Medium). Unlike expansion media the expansion medium of the invention comprises as components that allow for and favor a superior propagation of the liver cells a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway. Wnt proteins comprise a major family of signaling molecules that orchestrate and influence a myriad of cell biological and developmental processes; see Willert and Nusse (2012, Cold Spring Harb Perspect Biol, 4:a007864). All metazoan species express Wnt genes, with the genome of mice and humans carrying 19 independent genes. Based on their primary amino acid sequence, all Wnt genes are predicted to encode secreted proteins. The defining property of Wnt proteins is a nearly invariant positioning of 22 cysteine residues, most of which are postulated to form disulfide bridges that maintain a globular secondary structure. Wnt proteins are essential cell growth factors. Several Wnt surrogates or activators (agonists) are known in the art, for example, in the form of proteins, peptides or small molecules. The term “protein” as used herein interchangeably with the term “polypeptide” describes linear molecular chains of amino acids, including single chain proteins or their fragments, containing at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting of up to 49 amino acids, whereas the term “polypeptide” (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting with increased preference of at least 15 amino acids, at least 20 amino acids at least 25 amino acids, and at least 40 amino acids. The group of peptides and polypeptides are referred to together by using the term "(poly)peptide". (Poly)peptides may further form oligomers consisting of at least two identical or different molecules. The corresponding higher order structures of such multimers are, correspondingly, termed homo- or heterodimers, homo- or heterotrimers etc.. Furthermore, peptidomimetics of such proteins / (poly)peptides where amino acid(s) and / or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention. Such functional analogues include all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides and proteins where the modification is effected, e.g., by glycosylation, acetylation, phosphorylation and similar modifications which are well known in the art. The "small molecule" as used herein is preferably an organic molecule. Organic molecules relate or belong to the class of chemical compounds having a carbon basis, the carbon atoms linked together by carbon-carbon bonds. The original definition of the term organic related to the source of chemical compounds, with organic compounds being those carbon-containing compounds obtained from plant or animal or microbial sources, whereas inorganic compounds were obtained from mineral sources. Organic compounds can be natural or synthetic. The organic molecule is preferably an aromatic molecule and more preferably a heteroaromatic molecule. In organic chemistry, the term aromaticity is used to describe a cyclic (ring-shaped), planar (flat) molecule with a ring of resonance bonds that exhibits more stability than other geometric or connective arrangements with the same set of atoms. Aromatic molecules are very stable, and do not break apart easily to react with other substances. In a heteroaromatic molecule at least one of the atoms in the aromatic ring is an atom other than carbon, e.g. N, S, or O. For all above-described organic molecules the molecular weight is preferably in the range of 200 Da to 1500 Da and more preferably in the range of 300 Da to 1000 Da. Alternatively, the "small molecule" in accordance with the present invention may be an inorganic compound. Inorganic compounds are derived from mineral sources and include all compounds without carbon atoms (except carbon dioxide, carbon monoxide and carbonates). Preferably, the small molecule has a molecular weight of less than about 2000 Da, or less than about 1000 Da such as less than about 500 Da, and even more preferably less than about 250 Da. The size of a small molecule can be determined by methods well-known in the art, e.g., mass spectrometry. The small molecules may be designed, for example, based on the crystal structure of the target molecule, where sites presumably responsible for the biological activity can be identified and verified in in vivo assays such as in vivo high- throughput screening (HTS) assays. Preferred examples of WNT surrogates and activators can be found in Table 1 of Bonnet et al. (2021, RSC Chem Biol, 2:1144-1157) which is reproduced herein: Table 1: Wnt surrogates and activators, mechanism of actions, potential therapeutic effects, and stage of development

[0002]  The most widely used and preferred class of small molecule agonists of Wnt signaling inhibits GSK3 (Glykogen Synthase Kinase 3). For example, lithium at millimolar concentrations potently inhibits GSK3 and leads to β-catenin stabilization. For example, the IC50 for LiCl inhibition of GSK-3 is about 1 mM (Snitow et al., 2021, Cells, 10:255). Several other small molecule GSK3 inhibitors with IC50 values in the nanomolar to micromolar ranges have been identified and are commercially available. A Wnt surrogate is compound, generally a protein that that can exert inside a cell the same function / trigger the same cell signalling cascades as Wnt. Wnt surrogate are, for example, known from Mia et al. (2020, Cell Stem Cell, 27:840-851) and Janda et al. (2017, Nature, 545:234–237). Preferred examples of Wnt surrogate can be taken from the above Table 1 and will be provided herein below. The Hippo signalling pathway, also known as the Salvador-Warts-Hippo (SWH) pathway, is a signaling pathway that controls organ size in animals through the regulation of cell proliferation and apoptosis; see review by Fu et al. (2022, Signal Transduct Target Ther, 7:376). In mammals, the Hippo pathway is composed of several key components, including mammalian STE20-like kinase 1 / 2 (MST1 / 2), protein Salvador homologue 1 (SAV1), MOBKL1A / B (MOB1A / B), large tumor suppressor kinase 1 / 2 (LATS1 / 2), Yes-associated protein 1 (YAP), WW-domain-containing transcription regulator 1 (TAZ), and the transcriptional enhanced associated domain (TEAD) family. YAP / TAZ are transcriptional coactivators that bind to TEAD to regulate the expression of a wide array of genes that mediate cell proliferation, apoptosis, and stem cell self-renewal. Moreover, a variety of upstream signals, such as cell polarity, mechanical cues, cell density, soluble factors and stress signals, modulate the Hippo pathway. The core of the Hippo pathway is a kinase cascade, and MST1 / 2, SAV1, LATS1 / 2, YAP, and TAZ are considered the key components. The inhibitor of the Hippo signalling pathway therefore preferably is an inhibitor of one or more of MST1 / 2, SAV1, and LATS1 / 2. Instead of an inhibitor also an activator of YAP or TAZ might be used, such as an expression vector expressing YAP and / or TAZ in order to inhibitor Hippo signalling. The nature of the inhibitor is not particularly limited. The inhibitor may either inhibit a nucleic acid molecule (e.g. its expression or translation) encoding a protein (e.g. its activity or biological function) of the Hippo signalling pathway (e.g. a gene encoding MST1 / 2, SAV1, or LATS1 / 2) or protein of the Hippo signalling pathway (e.g. MST1 / 2, SAV1, or LATS1 / 2). Preferably, the expression translation, activity, biological function is reduced by at least 50%, more preferred at least 75% such as at least 90% or 95%, even more preferred at least 98%, and most preferably about 100% (e.g., as compared to the same experimental set up in the absence of the inhibitor). The efficiency of inhibition of an inhibitor can be quantified by methods comparing the level of activity in the presence of the inhibitor to that in the absence of the inhibitor. For example, the change in the amount of the nucleic acid molecule and / or the protein formed may be used in the measurement. The efficiency of several inhibitors may be determined simultaneously in high-throughput formats. High- throughput assays, independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably performed by one or more computer- controlled robotic systems including pipetting devices. In case large libraries of test compounds are to be screened and / or screening is to be performed within a short time, mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well. In case a well exhibits the expected activity, said mixture of test compounds may be de-convoluted to identify the one or more test compounds in said mixture giving rise to said activity. The inhibitor of such a nucleic acid molecule is preferably selected from a small molecule, an aptamer, a siRNA, a shRNA, a miRNA, a ribozyme, an antisense nucleic acid molecule, a CRISPR-Cas9-based construct, a CRISPR-Cpf1-based construct, a meganuclease, a zinc finger nuclease, and a transcription activator-like (TAL) effector (TALE) nuclease, and / or the inhibitor of such a nucleic acid molecule is preferably selected from a small molecule, an antibody or antibody mimetic, and an aptamer, wherein the antibody mimetic is preferably selected from affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and probodies. The term “antibody” as used in accordance with the present invention comprises, for example, polyclonal or monoclonal antibodies. Furthermore, also derivatives or fragments thereof, which still retain the binding specificity to the target, e.g. the MST1 / 2, SAV1, or LATS1 / 2, are comprised in the term "antibody". Antibody fragments or derivatives comprise, inter alia, Fab or Fab’ fragments, Fd, F(ab')2, Fv or scFv fragments, single domain VH or V-like domains, such as VhH or V-NAR-domains, as well as multimeric formats such as minibodies, diabodies, tribodies or triplebodies, tetrabodies or chemically conjugated Fab’-multimers (see, for example, Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane “Using Antibodies: A Laboratory Manual” Cold Spring Harbor Laboratory Press, 1999; Altshuler EP, Serebryanaya DV, Katrukha AG. 2010, Biochemistry (Mosc)., vol.75(13), 1584; Holliger P, Hudson PJ.2005, Nat Biotechnol., vol.23(9), 1126). The multimeric formats in particular comprise bispecific antibodies that can simultaneously bind to two different types of antigen. Non-limiting examples of bispecific antibodies formats are Biclonics (bispecific, full length human IgG antibodies), DART (Dual-affinity Re-targeting Antibody) and BiTE (consisting of two single-chain variable fragments (scFvs) of different antibodies) molecules (Kontermann and Brinkmann, 2015, Drug Discov Today, 20:838-847). The term "antibody" also includes embodiments such as chimeric (human constant domain, non-human variable domain), single chain and humanized (human antibody with the exception of non-human CDRs) antibodies. Various techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane (1988) and (1999) and Altshuler et al., 2010, loc. cit. Thus, polyclonal antibodies can be obtained from the blood of an animal following immunization with an antigen in mixture with additives and adjuvants and monoclonal antibodies can be produced by any technique which provides antibodies produced by continuous cell line cultures. Examples for such techniques are described, e.g. in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999 and include the hybridoma technique originally described by Köhler and Milstein, 1975, the trioma technique, the human B-cell hybridoma technique (see e.g. Kozbor D, 1983, Immunology Today, 4:7; Li J et al., 2006, PNAS, 103:3557) and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Alan R. Liss, Inc, 77-96). Furthermore, recombinant antibodies may be obtained from monoclonal antibodies or can be prepared de novo using various display methods such as phage, ribosomal, mRNA, or cell display. A suitable system for the expression of the recombinant (humanized) antibodies may be selected from, for example, bacteria, yeast, insects, mammalian cell lines or transgenic animals or plants (see, e.g., US patent 6,080,560; Holliger P and Hudson PJ, 2005, Nat Biotechnol, 23:11265). Further, techniques described for the production of single chain antibodies (see, inter alia, US Patent 4,946,778) can be adapted to produce single chain antibodies specific for an epitope of, for example, MST1 / 2, SAV1, or LATS1 / 2. Surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies. As used herein, the term “antibody mimetics” refers to compounds which, like antibodies, can specifically bind antigens, such as MST1 / 2, SAV1, or LATS1 / 2 in the present case, but which are not structurally related to antibodies. Antibody mimetics are usually artificial peptides or proteins with a molar mass of about 3 to 20 kDa. For example, an antibody mimetic may be selected from the group consisting of affibodies, adnectins, anticalins, DARPins, avimers, nanofitins, affilins, Kunitz domain peptides, Fynomers®, trispecific binding molecules and prododies. These polypeptides are well known in the art and are described in further detail herein below. The term “affibody”, as used herein, refers to a family of antibody mimetics which is derived from the Z- domain of staphylococcal protein A. Structurally, affibody molecules are based on a three-helix bundle domain which can also be incorporated into fusion proteins. In itself, an affibody has a molecular mass of around 6kDa and is stable at high temperatures and under acidic or alkaline conditions. Target specificity is obtained by randomisation of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain (Feldwisch J and Tolmachev V; 2012, Methods Mol Biol. 899:103-26). The term "adnectin" (also referred to as “monobody”), as used herein, relates to a molecule based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like β-sandwich fold of 94 residues with 2 to 3 exposed loops, but lacks the central disulphide bridge (Gebauer and Skerra, 2009, Curr Opinion in Chemical Biology, 13:245-255). Adnectins with the desired target specificity can be genetically engineered by introducing modifications in specific loops of the protein. The term "anticalin", as used herein, refers to an engineered protein derived from a lipocalin (Beste G et al., 1999, PNAS, 96:1898-1903; Gebauer and Skerra, 2009, Curr Opinion in Chemical Biology 13:245- 255). Anticalins possess an eight-stranded β-barrel which forms a highly conserved core unit among the lipocalins and naturally forms binding sites for ligands by means of four structurally variable loops at the open end. Anticalins, although not homologous to the IgG superfamily, show features that so far have been considered typical for the binding sites of antibodies: (i) high structural plasticity as a consequence of sequence variation and (ii) elevated conformational flexibility, allowing induced fit to targets with differing shape. As used herein, the term "DARPin" refers to a designed ankyrin repeat domain (166 residues), which provides a rigid interface arising from typically three repeated β-turns. DARPins usually carry three repeats corresponding to an artificial consensus sequence, wherein six positions per repeat are randomised. Consequently, DARPins lack structural flexibility (Gebauer and Skerra, 2009). The term “avimer”, as used herein, refers to a class of antibody mimetics which consist of two or more peptide sequences of 30 to 35 amino acids each, which are derived from A-domains of various membrane receptors and which are connected by linker peptides. Binding of target molecules occurs via the A-domain and domains with the desired binding specificity can be selected, for example, by phage display techniques. The binding specificity of the different A-domains contained in an avimer may, but does not have to be identical (Weidle UH et al., 2013, Cancer Genomics Proteomics, 10:155-168). A “nanofitin” (also known as affitin) is an antibody mimetic protein that is derived from the DNA binding protein Sac7d of Sulfolobus acidocaldarius. Nanofitins usually have a molecular weight of around 7kDa and are designed to specifically bind a target molecule by randomising the amino acids on the binding surface (Mouratou B et al., 2012, Methods Mol Biol, 805:315-331). The term “affilin”, as used herein, refers to antibody mimetics that are developed by using either gamma- B crystalline or ubiquitin as a scaffold and modifying amino-acids on the surface of these proteins by random mutagenesis. Selection of affilins with the desired target specificity is effected, for example, by phage display or ribosome display techniques. Depending on the scaffold, affilins have a molecular weight of approximately 10 or 20kDa. As used herein, the term affilin also refers to di- or multimerised forms of affilins (Weidle UH et al., 2013, Cancer Genomics Proteomics, 10:155-168). A “Kunitz domain peptide” is derived from the Kunitz domain of a Kunitz-type protease inhibitor such as bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). Kunitz domains have a molecular weight of approximately 6kDA and domains with the required target specificity can be selected by display techniques such as phage display (Weidle et al., 2013, Cancer Genomics Proteomics, 10:155-168). As used herein, the term "Fynomer®" refers to a non-immunoglobulin-derived binding polypeptide derived from the human Fyn SH3 domain. Fyn SH3-derived polypeptides are well-known in the art and have been described e.g. in Grabulovski et al. (2007, JBC, 282: 3196-3204; WO 2008 / 022759; Bertschinger et al., 2007, Protein Eng Des Sel, 20:57-68; Gebauer and Skerra, 2009, Curr Opinion in Chemical Biology, 13:245-255, or Schlatter et al., 2012, MAbs 4:1-12). The term “trispecific binding molecule” as used herein refers to a polypeptide molecule that possesses three binding domains and is thus capable of binding, preferably specifically binding to three different epitopes. The trispecific binding molecule is preferably a TriTac. A TriTac is comprised of three binding domains being designed to have an extended serum half-life and be about one-third the size of a monoclonal antibody. As used herein, the term "probody" refers to a protease-activatable antibody prodrug. A probody consists of an authentic IgG heavy chain and a modified light chain. A masking peptide is fused to the light chain through a peptide linker that is cleavable by tumor-specific proteases. The masking peptide prevents the probody binding to healthy tissues, thereby minimizing toxic side effects. Aptamers are nucleic acid molecules or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications (Osborne et. al., 1997, Current Opinion in Chemical Biology, 1:5-9; Stull & Szoka, 1995Pharmaceutical Research, 12:465-483). Nucleic acid aptamers are nucleic acid species that normally consist of (usually short) strands of oligonucleotides. Typically, they have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Peptide aptamers are usually peptides or proteins that are designed to interfere with other protein interactions inside cells. They consist of a variable peptide loop attached at both ends to a protein scaffold. This double structural constraint greatly increases the binding affinity of the peptide aptamer to levels comparable to an antibody's (nanomolar range). The variable peptide loop typically comprises 10 to 20 amino acids, and the scaffold may be any protein having good solubility properties. Currently, the bacterial protein Thioredoxin-A is the most commonly used scaffold protein, the variable peptide loop being inserted within the redox-active site in the wild protein, the two cysteins lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, but the most widely used is currently the yeast two-hybrid system. Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival those of the commonly used biomolecules, in particular antibodies. In addition to their discriminatory recognition, aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications. Non-modified aptamers are cleared rapidly from the bloodstream, with a half-life of minutes to hours, mainly due to nuclease degradation and clearance from the body by the kidneys, a result of the aptamers' inherently low molecular weight. Unmodified aptamer applications currently focus on treating transient conditions such as blood clotting, or treating organs such as the eye where local delivery is possible. This rapid clearance can be an advantage in applications such as in vivo diagnostic imaging. Several modifications, such as 2'-fluorine-substituted pyrimidines, polyethylene glycol (PEG) linkage, fusion to albumin or other half life extending proteins etc. are available to scientists such that the half-life of aptamers can be increased for several days or even weeks. As discussed, the above-described small molecule, antibody or antibody mimetic and aptamer can specifically bind to the protein of the Hippo signalling pathway. This binding may block the signalling properties of the protein of Hippo signalling pathway. In this case the small molecule, antibody or antibody mimetic and aptamer are also referred to as blocking small molecule, antibody or antibody mimetic and aptamer. A blocking small molecule, antibody or antibody mimetic and aptamer blocks interactions of the protein of the Hippo signalling pathway with other cellular components of the Hippo signalling pathway. In accordance with the present invention, the term "small interfering RNA (siRNA)", also known as short interfering RNA or silencing RNA, refers to a class of 18 to 30, preferably 19 to 25, most preferred 21 to 23 or even more preferably 21 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome. siRNAs naturally found in nature have a well-defined structure: a short double-strand of RNA (dsRNA) with 2-nt 3' overhangs on either end. Each strand has a 5' phosphate group and a 3' hydroxyl (-OH) group. This structure is the result of processing by dicer, an enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. siRNAs can also be exogenously (artificially) introduced into cells to bring about the specific knockdown of a gene of interest. Essentially any gene for which the sequence is known can thus be targeted based on sequence complementarity with an appropriately tailored siRNA. The double-stranded RNA molecule or a metabolic processing product thereof is capable of mediating target-specific nucleic acid modifications, particularly RNA interference and / or DNA methylation. Exogenously introduced siRNAs may be devoid of overhangs at their 3' and 5' ends, however, it is preferred that at least one RNA strand has a 5'- and / or 3'-overhang. Preferably, one end of the double-strand has a 3'-overhang from 1 to 5 nucleotides, more preferably from 1 to 3 nucleotides and most preferably 2 nucleotides. The other end may be blunt-ended or has up to 6 nucleotides 3'- overhang. In general, any RNA molecule suitable to act as siRNA is envisioned in the present invention. The most efficient silencing was so far obtained with siRNA duplexes composed of 21-nt sense and 21- nt antisense strands, paired in a manner to have a 2-nt 3'- overhang. The sequence of the 2-nt 3' overhang makes a small contribution to the specificity of target recognition restricted to the unpaired nucleotide adjacent to the first base pair (Elbashir et al., 2001, Nature, 411:494-498). 2'- deoxynucleotides in the 3' overhangs are as efficient as ribonucleotides, but are often cheaper to synthesize and probably more nuclease resistant. Delivery of siRNA may be accomplished using any of the methods known in the art, for example by combining the siRNA with saline and administering the combination intravenously or intranasally or by formulating siRNA in glucose (such as for example 5% glucose) or cationic lipids and polymers can be used for siRNA delivery in vivo through systemic routes either intravenously (IV) or intraperitoneally (IP) (Fougerolles et al., 2008, Curr Opin in Pharmacol, 8:280-285; Lu et al., 2008, Methods in Mol Biol, 437:93-107). A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA uses a vector introduced into cells and utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match the siRNA that is bound to it. si / shRNAs to be used in the present invention are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA / RNA synthesizer. Suppliers of RNA synthesis reagents are Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, CO, USA), Pierce Chemical (part of Perbio Science, Rockford, IL, USA), Glen Research (Sterling, VA, USA), ChemGenes (Ashland, MA, USA), and Cruachem (Glasgow, UK). Most conveniently, siRNAs or shRNAs are obtained from commercial RNA oligo synthesis suppliers, which sell RNA-synthesis products of different quality and costs. In general, the RNAs applicable in the present invention are conventionally synthesized and are readily provided in a quality suitable for RNAi. Further molecules effecting RNAi include, for example, microRNAs (miRNA). Said RNA species are single-stranded RNA molecules. Endogenously present miRNA molecules regulate gene expression by binding to a complementary mRNA transcript and triggering of the degradation of said mRNA transcript through a process similar to RNA interference. Accordingly, exogenous miRNA may be employed as an inhibitor after introduction into the respective cells. A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is an RNA molecule that catalyses a chemical reaction. Many natural ribozymes catalyse either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyse the aminotransferase activity of the ribosome. Non-limiting examples of well-characterised small self-cleaving RNAs are the hammerhead, hairpin, hepatitis delta virus, and in vitro-selected lead-dependent ribozymes, whereas the group I intron is an example for larger ribozymes. The principle of catalytic self-cleavage has become well established. The hammerhead ribozymes are characterised best among the RNA molecules with ribozyme activity. Since it was shown that hammerhead structures can be integrated into heterologous RNA sequences and that ribozyme activity can thereby be transferred to these molecules, it appears that catalytic antisense sequences for almost any target sequence can be created, provided the target sequence contains a potential matching cleavage site. The basic principle of constructing hammerhead ribozymes is as follows: A region of interest of the RNA, which contains the GUC (or CUC) triplet, is selected. Two oligonucleotide strands, each usually with 6 to 8 nucleotides, are taken and the catalytic hammerhead sequence is inserted between them. The best results are usually obtained with short ribozymes and target sequences. A recent development, also useful in accordance with the present invention, is the combination of an aptamer, recognizing a small compound, with a hammerhead ribozyme. The conformational change induced in the aptamer upon binding the target molecule can regulate the catalytic function of the ribozyme. The term “antisense nucleic acid molecule”, as used herein, refers to a nucleic acid which is complementary to a target nucleic acid. An antisense molecule in accordance with the invention is capable of interacting with the target nucleic acid, more specifically it is capable of hybridizing with the target nucleic acid. Due to the formation of the hybrid, transcription of the target gene(s) and / or translation of the target mRNA is reduced or blocked. Standard methods relating to antisense technology have been described (see, e.g., Melani et al., 1991, Cancer Res, 51:2897-2901). CRISPR / Cas9, as well as CRISPR-Cpf1, technologies are applicable in nearly all cells / model organisms and can be used for knock out mutations, chromosomal deletions, editing of DNA sequences and regulation of gene expression. The regulation of the gene expression can be manipulated by the use of a catalytically dead Cas9 enzyme (dCas9) that is conjugated with a transcriptional repressor to repress transcription a specific gene. Similarly, catalytically inactive, "dead" Cpf1 nuclease (CRISPR from Prevotella and Francisella-1) can be fused to synthetic transcriptional repressors or activators to downregulate endogenous promoters. Alternatively, the DNA-binding domain of zincfinger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) can be designed to specifically recognize a gene or its promoter region or its 5`-UTR thereby inhibiting the expression of the gene. Inhibitors provided as inhibiting nucleic acid molecules that target a gene or a regulatory molecule involved in gene expression are also envisaged herein. Such molecules, which reduce or abolish the expression of a target gene or a regulatory molecule include, without being limiting, meganucleases, zinc finger nucleases and transcription activator-like (TAL) effector (TALE) nucleases. Such methods are described in Silva et al., 2011, Curr Gene Ther, 11:11-27; Miller et al., 2011, Nature biotech, 29:143- 148, and Klug, Annu Rev Biochem.2010, 79:213-231. In accordance with the final proviso of the first aspect the expansion medium preferably does not comprise nicotinamide. Nicotinamide (Niacinamide; Cas No. 98-92-0) is a form of vitamin B3, a poly (ADP-ribose) polymerase (PARP) inhibitor, and the primary precursor of NAD+. Nicotinamide is often used in cell culture media because it generally promotes cell survival and differentiation (see, for example, Meng et al. (2018), Stem Cell Reports, 11:1347-1356). In the examples herein below a synergistic effect was observed when combining the activation of WNT and the inhibition of the Hippo signalling pathway for expanding liver cells. The cultured cells and the resulting organoids expanded for a longer time and increased passage number as compared to prior expansion methods. In addition, the combination of the activation of WNT and the inhibition of the Hippo signalling pathway also resulted in the expansion of human hepatocellular carcinoma organoid cultures derived from highly differentiated tumors. In a second step it was surprisingly found that if not only an WNT activator and a Hippo signaling pathway are added to the expansion medium but in addition nicotinamide is omitted from the expansion medium, the cultured cells and the resulting organoids expanded for an even longer time. The omittance of nicotinamide improved organoid formation efficiency by nearly 10-fold and sustained long-term expansion for at least 3 months in a serial passage. This was totally unexpected because it was believed that nicotinamide in the expansion medium is favorable factor and not hindering factor. In the prior art the attempt to expand well-differentiated human hepatocellular carcinoma organoid cultures failed completely (see Broutier et al., 2017, Nat Med, 23:1424-1435 and van Tienderen et al., 2021, Cancer Cell, 40:226-230) and only with the method of the present invention it is for the first time possible to expand them. In vitro liver cell culture models are of importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. For this reason, the method of the present invention constitutes an important contribution to the art. It is also shown in the appended examples that the expanded liver cells as obtained by the method of the invention present features of liver zonation. Pericentrally zonated genes, such as CYP2E1 and GLUL (Glutamine Synthase, GS), as well as some periportally zonated genes, such as ALB, or ASGR2 are highly expressed. Furthermore, expanded liver cells as obtained by the method of the invention secret high levels of albumin, better than the standard protocol of culturing hepatocytes in 2D-monoculture. Also, the cultured cells display cytochrome p450 activity. This shows that the expanded liver cells as obtained by the method of the invention closely resemble in vivo liver cells (hepatocytes). Hence, the method for expanding liver cells is preferably a method for expanding liver cells that express one or more zonated genes, such as CYP2E1 and GLUL (Glutamine Synthase, GS) and / or one or more periportally zonated genes, such as ALB, or ASGR2. Similarly, the method for expanding liver cells is preferably a method for expanding liver cells that secret albumin and / or display cytochrome p450 activity. The therapeutic potential expanded liver cells as obtained by the method of the invention is further underlined by the data in the examples in a mouse model for human liver monogenic diseases. Remarkably, after transplantation into the mouse model in vivo, both expanded and differentiated hepatocyte organoids as obtained by the method of the invention retained their hepatic function and were able to rescue the lethal phenotype of the mice upon transplantation. These results demonstrate that the hepatocytes as obtained by the method of the invention are a suitable cellular source for liver cell therapy, thereby having the potential of avoiding in the future the need of liver transplants. From only one donor from which cryopreserved hepatocytes could be obtained, several patients could be transplanted at once when using the method of the invention. In accordance with a preferred embodiment the method of the first aspect comprises after step (a) (b) isolating the expanded liver cells, wherein the expanded liver cells are preferably in the form of organoids. As discussed herein above, the liver cells are expanded in an ECM in an expansion medium. Over time the liver cells organize themselves into organoids in the ECM. Liver cells or organoids can be isolated from the ECM, for example, by centrifugation (e.g. at about 50 g for about 5 min) followed by mechanical disruption of the ECM and / or in the presence of an enzyme (e.g. trypsin). The term “about” as used herein refers with increasing preference to ±20%, ±10%, and ±5% of each respective value. In accordance with a further preferred embodiment the method of the first aspect comprises before step (a) (a’) adding the liver cells into the extracellular matrix. The ECM is generally preformed, so that the liver cells can be embedded into the ECM. The cells can be fixed in the ECM by paraformaldehyde (e.g. about 4% paraformaldehyde for about 30 or about 20 minutes at room temperature), acetone, methanol or a mixture of any combination of the foregoing. In accordance with a further preferred embodiment the method of the first aspect comprises after step (a) and, if present, before step (b) (b’) differentiating the expanded liver cells in a differentiation medium that comprises a Wnt surrogate or activator and does not comprise an inhibitor of the Hippo signalling pathway. The expansion of liver cells has to be held distinct from the differentiation of liver cells. During expansion the number of liver cells increase via proliferation but the differentiation stage of the liver cell remains the same or essentially the same. On the other hand, during differentiation the liver cells further acquire a mature liver cell fate. Accordingly, the differentiation medium is a medium that favors a mature liver cell fate. Several media for hepatic differentiation are commercially available; see Toba et al., 2020, PLoS One, 15: e0229654. To such media the Wnt surrogate or activator can be added. The differentiation medium does not comprise an inhibitor of the Hippo signalling pathway. The cells are preferably kept in the differentiation medium for about 5 says to about 15 days and more preferably for about 7 days to about 9 days. It is demonstrated in the appended examples that the Wnt surrogate or activator promotes a mature liver cell fate while, on the contrary, the inhibitor of the Hippo signalling pathway facilitates the expansion of adult liver cells at the expenses of preventing a mature liver cell fate. Following treatment with differentiation medium, the cells reduced the expression of the embryonic and cholangiocyte markers AFP and KRT19, respectively. Mature markers, markers for xenobiotic metabolism, lipid metabolism and detoxifying enzymes were highly upregulated, almost to the same levels as fresh isolated human hepatocytes. Immunofluorescence analysis revealed that the cells have acquired the right apical polarity, with expression of bile canaliculi marker at the apical side of two adjacent hepatocytes, while E-cadherin was present basolaterally. Hepatic differentiated PHH organoids also presented mature hepatic functions including cytochrome p450 activities and albumin secretion. RNAseq analysis revealed that the differentiated organoids clustered together with the freshly isolated hepatocytes and far away from cholangiocyte organoids published in Huch et al. (2013 and 2015) RNAseq analysis indicates that bile acid metabolism as well as fatty acid metabolism are all similar to primary human liver tissue. Hence, the method of the invention results in cells that very closely resemble primary human liver tissue, in particular freshly isolated primary hepatocytes. Freshly isolated primary hepatocytes are the gold standard in analyzing drug metabolism, liver toxicity and drug-mediated responses in vitro. The method of the invention has the capacity to reduce the need for freshly isolated primary hepatocytes by expanding and optionally differentiating them before using them for the discussed in vitro tests. In accordance with a preferred embodiment of the first aspect, the cells are expanded (i) for at least 5 passages, preferably at least 10 passages and most preferably at least 15 passages; and / or (ii) for at least 50 days, preferably at least 100 days and most preferably at least 200 days. Donors of all ages and genders have been tested with no differences in expansion or differentiation potential. In this connection it is of note that to best knowledge of the inventors the method of the first aspect makes it possible for the first time to expand liver cells – without further differentiation – for least 10 passages and a fortiori at least 15 passages and / or at least 100 days and a fortiori at least 200 days. This expansion time is longer as compared to the prior art methods being described in WO 2012 / 168930 and WO 2012 / 014076, WO 2015 / 173425 and US 202 / 0298485. For example, Figure 2 shows that it allows human hepatocytes to be differentiated as opposed to attached cells or even previous patented organoids as in US202 / 0298485, which used Wnt inhibition techniques. In accordance with another preferred embodiment of the first aspect, the Wnt surrogate or activator is SZN-1326, SZN-413 or a water-soluble and Wnt receptor frizzled (FZD)-specific surrogate Wnt agonist, preferably a water-soluble FZD-LRP5 / LRP6 heterodimerizer. SZN-1326 is a bi-specific full-length human antibody that directly modulates Wnt signaling in target tissue by binding to particular Frizzled and LRP receptors that are highly expressed in intestinal crypts. It acts by targeting Frizzled-5 (FZD5) and Low-density lipoprotein receptor-related protein 6 (LRP6); see https: / / www.surrozen.com / programs / szn-1326. SZN-413 is a bi-specific antibody targeting Fzd4-mediated Wnt signaling and low-density lipoprotein receptor-related protein 5 (LRP5): it has been designed using Surrozen's SWAP technology (Nguyen et al., 2022, Transl Vis Sci Technol, 11:19). A water-soluble and Wnt receptor frizzled (FZD)-specific surrogate comprises a Fzd binding module being fused to a Wnt antagonist. The water-soluble FZD-LRP5 / LRP6 heterodimerizer is most preferred because it is used in the appended examples (see Miao et al., 2020, Cell Stem Cell, 27:840-851 and Janda et al., 2017, Nature. 2017, 545:234–237). The water-soluble FZD-LRP5 / LRP6 heterodimerizer be the Fzd binding module (B12, scFv) linked to the C-terminal domain of human Wnt antagonist DKK1. This C-terminal domain, which is sufficient for Wnt inhibition and binds Lrp5 / 6 with moderate affinity (20–70 nM) is preferably linked to B12 and scFv, through a flexible Gly-Ser polypeptide linker (these fusions are referred to as B12-DKK1c and scFv- DKK1c throughout) of 0–15 amino acids, and 5 amino acids, respectively. The water-soluble FZD-LRP5 / LRP6 heterodimerizer can also be Designed Repeat Protein Binder Fz7 / 8 (DRPB_Fz7 / 8) as the Fzd binding module fused with high-affinity Lrp5 / 6 receptor binder DKK1c, again preferably through a flexible Gly-Ser polypeptide linker. In accordance with further preferred embodiment of the first aspect, the inhibitor of the Hippo signalling pathway is an activator of the yes-associated protein (YAP) / WW-domain-containing transcriptional regulator (TAZ) and / or an inhibitor of the large tumor suppressor kinase 1 / 2 (LATS1 / 2). As discussed herein above, YAP, TAZ and LATS1 / 2 are among the core components of the Hippo pathway and are therefore the targets according to this preferred embodiment. Among this list LATS1 / 2 is preferred since it is the target of the Hippo signalling pathway inhibitor as used in the appended examples. In accordance with preferred embodiment of the first aspect, the inhibitor of the Hippo signalling pathway is selected from one or more of TRULI, TDI-011214, TDI-011241, TDI-011536, TM-25659, XMU-MP-1, I3MT-3 IHMT-MST1-58, and SBP-3264, PY-60, GA-017, TT-10, MA-5 and VT02956. Within the above list TRULI and TDI-011536 are preferred and TRULI is most preferred. The one or more are with increasing preference two or more, three or more, four or more, and five or more. TRULI inhibits both LATS1 and LATS2 with a IC50 of 0.2 nM, suppresses YAP phosphorylation, induces cell proliferation in several cell lines and tissues, and promotes the initial stages of proliferative regeneration of the sensory receptors in the inner ear. TRULI has the CAS No.1424635-83-5. TDI-011214, TDI-011241 and TDI-011536 are derivatives of TRULI being described in Kastan et al. (2022); PNAS; 119(28):e2206113119 have the structures: TM-25659 has the CAS-Number 260553-97-7 and the formula C30H28N8. XMU-MP-1 has the CAS-Number 2061980-01-4 and the formula C17H16N6O3S2. I3MT-3 has the CAS-Number 459420-09-8 and the formula C17H14N2O2S. IHMT-MST1-58 has the CAS-Number 2414484-25-4 and the formula C21H22N6O3S. SBP-3264 has the CAS-Number 2869148-13-8 and the formula C19H20ClN5O, PY-60 has the CAS-Number 2765218-56-0 and the formula C16H15N3O2S. GA-017 has the CAS-Number 2351906-74-4 and the formula C18H21N3O4. TT-10 has the CAS-Number 2230640-94-3 and the formula C11H10FN3OS2. MA-5 has the CAS-Number 1354707-41-7 and the formula C18H13F2NO3. VT02956 has the formula C22H23N5O and is In accordance with a further preferred embodiment of the first aspect, the expansion medium further comprises one or more of (i) a Wnt activator, preferably Wnt, Norrin or a GSK-inhibitor, wherein the GSK inhibitor is preferably a GSK3b inhibitor and is most preferably CHIR99021, tideglusib, AL-001, dronabinol, elraglusib, AP-001, AP-1, IB-AD, MD-053; (ii) a FGFR2 / FGFR4 agonist, wherein the FGFR2 / FGFR4 agonist is preferably FGF and is most preferably FGF10 and / or FGF7; (iii) a Rho-kinase inhibitor, wherein the Rho-kinase inhibitor is preferably an inhibitor of ROCK1 or ROCK2 and is most preferably selected from Y-27632, RKI-1447, DJ4, H-1152, Chroman 1, Ripasudil, GSK269962A, Fausdil, belumosudil, OPL-0401, TDI-01, zelasudil, NRL-1049, ANG-4201, CVT-100069, CVT-100077, KD-045, REDX-10616, RXC-008, TRX-101; (iv) a TGF-inhibitor, wherein the TGF-inhibitor is preferably an inhibitor of ALK4, ALK5 or AKL7 and is most preferably A83-01, SB-431542, SB-5225334 LY364947, SS-208, SNJ 2511, EW-7197, SB505124, RepSox, GW788388, SD-208, Galunisertib, EW-7197, or LY2109761; (v) B-27 Supplement, wherein the B-27 Supplement is preferably without retinoic acid; (vi) N-acetylcysteine (viii) a growth factor, preferably EGF and / or HGF; (ix) R-Spondin (RSPO) conditioned media supplement (preferably prepared from cell transfectant stably secreting RSPO) or RSPO protein, wherein RSPO is preferably RSPO1, RSPO2, RSPO3 or RSPO4; and (x) Gastrin. The one or more are with increasing preference two or more, three or more, four or more, five or more six or more, seven or more, and all ten. An expansion medium with the above ten ingredients and in addition a Wnt surrogate or activator and that comprises an inhibitor of the Hippo signalling pathway is used in the appended examples. Acting as intercellular signals, Wnt proteins regulate the proliferation of cells. Wnt signals are active in numerous contexts, initially in early development and later during the growth and maintenance of various tissues. In comparison to other growth factors, Wnt signals have several unique properties, including a short range of action. Thereby, Wnts predominantly mediate signaling locally, between neighboring cells. In addition, Wnt signals give shape to tissues as cells are proliferating. This is a consequence of the ability of Wnt signaling to confer polarity and asymmetry to cells. As currently understood, Wnt proteins bind to receptors of the Frizzled and LRP families (e.g. FZD receptors) on the cell surface. Wnts are not the only ligands of the FZD receptors. The cysteine-knot protein Norrin, encoded by the NDP gene, can also bind and activate Wnt receptors. CHIR99021 is 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile. The Cas-Number is 252917-06-9. Tideglusib has the CAS-Number 865854-05-3 and the formula C19H14N2O2S. AL-001 (Latozinemab) is a recombinant humanized anti-Sortilin monoclonal antibody. Dronabinol is a generic name for the molecule of delta-9-tetrahydrocannabinol. Elraglusib has the CAS--Number 1034895-42-5 and the formula C22H13FN2O5. FGF10 and FGF7 are the fibroblast growth factors 10 and 7. Rho-associated kinases ROCK1 and ROCK2 are serine / threonine kinases that are downstream targets of the small GTPases RhoA, RhoB, and RhoC. ROCKs are involved in diverse cellular activities including actin cytoskeleton organization, cell adhesion and motility, proliferation and apoptosis, remodeling of the extracellular matrix and smooth muscle cell contraction. Y-27632 has the CAS-Number146986-50-7 and the formula C14H21N3O. RKI-1447 has the CAS-Number 1342278-01-6 ad the formula C16H14N4O2S. DJ4 is the compound H-1152 has the CAS-Number 451462-58-1 and the formula C16H21N3O2S. Chroman 1 has the CAS-Number 1273579-40-0 and has the formula C24H28N4O4. Ripasudil has the CAS-Number 887375-67-9 and the formula C15H23ClFN3O4S. GSK269962A has the CAS-Number 850664-21-0 and the formula C29H30N8O5. Fausdil has the CAS-Number 103745-39-7 and the formula C14H17N3O2S. Belumosudil has the CAS-Number 911417-87-3 and the formula C26H24N6O2. Zelasudil has the CAS-Number 2365193-22-0 and the formula C22H21F2N7O. NRL-1049 (BA-1049) has the CAS-number 1973494-16-4 and formula C16H21N3O2S. ANG-4201 is a RCOK2 inhibitor being described Ali et al., Novel Rho associated coiled kinase 2 (ROCK2) inhibitor reduces steatosis and fibrosis in mice model of liver disease. FASEB Journal.2020. HA-1100 has the CAS-number 155558-32-0 and formula C14H17N3O3S·HCl·xH2O. KD-045 is a ROCK inhibitor described in Kumar et al. Engineered cord blood megakaryocytes evade killing by allogeneic T-cells for refractory thrombocytopenia. Frontiers in Immunology.2022 REDX-10616 is a ROCK2 selective inhibitor being described in the abstract of the conference "Integrated Pathways of Disease in NASH and NAFLD” Offer et al. ROCK2 inhibitors for the treatment of fibrosis, DOI: 10.13140 / RG.2.2.25059.81448. RXC-008 is a rock-inhibitor being available from available from redxpharma (see https: / / www.redxpharma.com / our-pipeline / rxc008-gi-targeted-rock-inhibitor / ). TRX-101 isa rock-inhibitor being described in Novel Rho Kinase Inhibitor for Systemic Sclerosis, Wright, Susan C. at https: / / grantome.com / grant / NIH / R43-AR064625-01A1. ALK4, ALK5 and AKL7 are transforming growth factor-beta type I receptors. A83-01 has the CAS-number 909910-43-6 and the formulaC25H19N5S.SB-431542 has the CAS-number 301836-41-9 and the formulaC22H16N4O3. SB-5225334 has the CAS-number 356559-20-1 and the formula C21H21N5. LY364947 has the CAS-number 396129-53-6 and the formula C17H12N4. SS-208 has the CAS-number 2245942-72-5 and the formula C13H11Cl2N3O4. SNJ 2511 has the CAS-number 446859-33-2 and the formula C17H13N5. EW-7197 has the CAS-number 1352608-82-2 and the formula C22H18FN7. SB505124 has the CAS-number 694433-59-5 and the formula C20H21N3O2. RepSox has the CAS-number 446859-33-2 and the formula C17H13N5. GW788388 has the CAS-number 452342-67-5 and the formula C25H23N5O2. SD-208 has the CAS-number 627536-09-8 and the formula C17H10ClFN6. Galunisertib has the CAS-number 700874-72-2 and the formula C22H19N5O. EW-7197 has the CAS-number 1352608-82-2 and the formula C22H18FN7. LY2109761 has the CAS-number 700874-71-1 and the formula C26H27N5O2. B-27 supplement is a defined yet complex mixture of antioxidant enzymes, proteins, vitamins, and fatty acids that are combined in optimized ratios to support neuronal survival in culture. The original serum- free neuronal culture supplement formula developed by Dr. Gregory Brewer and colleagues is described in Brewer et al., J Neuroscience Res 35: 567-576, 1993 and Brewer and Cotman, Brain Res 494: 65- 74, 1989. EGF and / or HGF are the epidermal growth factor (EGF) and the hepatocyte growth factor (HGF). 3D organoid culture systems are increasingly employed as powerful tools for the study of human diseases. R-Spondin proteins are a family of cysteine-rich, thrombospondin type I repeats containing proteins that activate both the WNT / β-catenin and WNT / PCP pathways. R-Spondin-1 Conditioned Media Supplement is a potent and inexpensive alternative to purified recombinant RSPO1 protein for organoid cultures. A R-Spondin Conditioned Media Supplement can be generated, for example, from a 293T cell transfectant stably secreting mouse R-spondin-1 tagged with an influenza hemagglutinin (HA) epitope at the N-terminus and a murine IgG2a Fc fragment at the C-terminus. Among RSPO1, RSPO2, RSPO3 and RSPO4, RSPO1 is preferred. The concentration of CHIR99021 in the expansion medium (EM) and / or differentiation medium (DM) is preferably about 0.25 µM – about 10µM and most preferably about 3µM. The concentration of FGF10 and / or FGF7 in the EM is preferably about 10 ng / ml – 1µg / ml and most preferably about 100 ng / ml. The concentration of the most preferred Rho-kinase inhibitor Y-27632 in the EM and / or DM is preferably about 0.5 µM – about 50µM and most preferably about 10µM. The concentration of the most preferred TGF-inhibitor A83-01 in the EM and / or DM is preferably about 0.1 µM – about 20 µM and most preferably about 2 µM. The concentration of N-acetylcysteine in the EM and / or DM is preferably about 0.25 mM – about 20 mM and most preferably about 1.25 mM. The concentration of gastrin in the EM is preferably about 0.2 nM – about 20 nM and most preferably about 10 nM. The concentration of EGF and / or HGF (preferably both are present) in the EM and / or DM is preferably about 1 ng / ml – 100 ng / ml and most preferably about 50 ng / ml. The concentration of the most preferred RSPO protein RSPO1 in the EM and / or DM is preferably about 1% to about 50% v / v and most preferably about 15% v / v. In accordance with another preferred embodiment of the first aspect, the differentiation medium, if present, further comprises one or more of (i) a Wnt activator, preferably Wnt, Norrin or a GSK-inhibitor, wherein the GSK inhibitor is preferably a GSK3b inhibitor and is most preferably selected from CHIR99021 (6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5- methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), tideglusib, AL-001, dronabinol, elraglusib, AP-001, AP-1, and IB-AD, MD-053; (ii) a FGFR2 / FGFR4 agonist, wherein the FGFR2 / FGFR4 agonist is preferably a Pan-FGFR inhibitor and is most preferably FGF19; (iii) a Rho-kinase inhibitor, wherein the Rho-kinase inhibitor is preferably an inhibitor of ROCK1 or ROCK2 and is most preferably selected from Y-27632, RKI-1447, DJ4, H-1152, Chroman 1, Ripasudil, GSK269962A, Fausdil, belumosudil, OPL-0401, TDI-01, zelasudil, NRL-1049, ANG-4201, CVT-100069, CVT-100077, KD-045, REDX-10616, RXC-008, and TRX-101; (iv) a TGF-inhibitor, wherein the TGF-inhibitor is preferably an inhibitor of ALK4, ALK5 or AKL7 and is most preferably A83-01, SB-431542, SB-5225334 LY 364947, SS-208 or SNJ 2511; (v) B-27 Supplement, wherein the B-27 Supplement is preferably without retionic acid; (vi) N-acetylcysteine; (vii) a growth factor, preferably EGF and / or HGF; (viii) Dexamethasone; (ix) R-Spondin (RSPO) conditioned media supplement (preferably prepared from cell transfectant stably secreting RSPO) or RSPO protein, wherein RSPO is preferably RSPO1, RSPO2, RSPO3 or RSPO4. The one or more are with increasing preference two or more, three or more, four or more, five or more six or more, seven or more, eight of more, and all nine. A differentiation medium with the above nine ingredients an in addition a Wnt surrogate or activator but without an inhibitor of the Hippo signalling pathway is used in the appended examples. The concentration of dexamethasone in the DM is preferably about 0.25 µM - about 20 µM and most preferably about 1.6 µM or about 3 µM. The concentration of FGF19 in the DM is preferably about 1000ng / ml - about 1ng / ml and most preferably about 100 ng / ml. In accordance with a further preferred embodiment of the first aspect the extracellular matrix (i) comprises one or more and preferably all of collagen (preferably collagen type IV), entactin, perlecan (preferably heparan sulfate proteoglycan), and laminin; and / or (ii) is a reconstituted basement membrane derived from extracts of mammalian cells, preferably mouse cells, and most preferably Engelbreth-Holm-Swarm mouse tumor cells. In the examples of the application Matrigel is used as the ECM. Matrigel is the trade name for the solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells produced by Corning Life Sciences. Matrigel resembles the laminin / collagen IV-rich basement membrane extracellular environment found in many tissues and is used by cell biologists as a substrate (basement membrane matrix) for culturing cells. In accordance with a yet further preferred embodiment of the first aspect the concentration of the inhibitor of the Hippo signalling pathway in the expansion medium is with increasing preference at least 0.01 nM, at least 0.05 nM, at least 0.50 nM, and at least 10 nM, or is in the range of about 0.05 nM to about 50 uM and preferably 3 µM. In accordance with another preferred embodiment of the first aspect the concentration of Wnt surrogate in the expansion medium and / or the differentiation medium is about 0.05 nM to 50 nm and preferably 0.5nM. The above concentrations of the inhibitor of the Hippo signalling pathway (in the EM) and / or the Wnt surrogate are based on the concentrations as used in the EM and DM in the appended examples. The present invention relates in a second aspect to a liver cell expansion medium that comprises a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway as defined herein above in connection with the first aspect of the invention. Also describes herein is a liver cell differentiation medium that comprises a Wnt surrogate or activator but does not comprise an inhibitor of the Hippo signalling pathway as defined herein above in connection with the first aspect of the invention. The present invention relates in a third aspect to a liver cells expansion kit that comprises (i) a liver cells expansion medium comprising a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway as defined herein above in connection with the first aspect of the invention, and (ii) a liver cells differentiation medium comprising a Wnt surrogate or activator and does not comprise an inhibitor of the Hippo signalling pathway as defined herein above in connection with the first aspect of the invention. The present invention relates in a fourth aspect to the use of the expansion medium of the second aspect or the kit of the third for expanding liver cells (and optionally also subsequently differentiating the expanded liver cells). The above definitions and preferred embodiments of the first aspect of the invention apply mutatis mutandis to the second to fourth aspect of the invention as far as being amenable therewith. This in particular holds true for the preferred ingredients of the EM and DM as well as the preferred concentrations of the ingredients of the EM and DM. In accordance with a preferred embodiment all above aspects of the invention the liver cells are (i) adult liver cells; (ii) mammalian liver cells, preferably human liver cells; (iii) liver tumor cells, preferably liver cancer cells; and / or (iv) liver epithelial or mesenchymal cells, preferably hepatocytes. In the appended examples hepatocytes from human adults were expanded, both normal and cancerous cells. The liver epithelial or mesenchymal cells, preferably hepatocytes can be culture alone or co-cultured with other types of liver cells, noting the different types of liver cells have been described above. As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise. Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1, a dependent claim 2 referring back to claim 1, and a dependent claim 3 referring back to both claims 2 and 1, it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1, of claims 4, 2 and 1, of claims 4, 3 and 1, as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed. The figures show: Figure 1. Efficiency of primary human hepatocyte organoids (PPH-organoid) derivation. Top, table describing the patients’ demographics, method of isolation, cell viability, medium used and passage obtained. Note that organoid derivation depends on the isolation method, viability at start of the culture and medium conditions and not on the demographics of the patient nor the patients’ diagnostic. Figure 2. Schematic diagram of primary human hepatocyte (PHH) isolation and culture method. Figure 3. Generation and characterization of PHH organoids. (A) Freshly isolated primary human hepatocytes (PHHs) were cultured in expansion medium for 137 days, as described in methods. Representative brightfield images are shown. P, passage. Scale bars, 50 µm and 100 µm. (B) Expanded PHH organoids were collected and characterized by immunofluorescence staining. Representative immunofluorescence stainings are shown. Left, staining for hepatocyte markers (MRP2, green and HNF4A, magenta), middle, staining for proliferation (KI67, magenta) and hippo signalling (YAP, green) markers. Right, staining for HNF4a (Magenta) and Ki67 (green) indicates that the proliferating cells are bona-fide hepatocytes. Nuclei were counterstained with DAPI. Scale bars, 50 µm. Figure 4. Generation and characterization of liver cancer organoids. (A) Freshly isolated primary liver cancer cells were cultured in MM + Wnt S, MM + Wnt S + TRULI or MM + Wnt S + TDI-011536 for 15 days as indicated in methods. Representative pictures of cultures collected at day 5 after seeding. Scale bars, 100 µm. (B) Gene expression levels of hepatocyte, hepatoblast, and hepatocellular carcinoma (HCC) markers analyzed by RT-qPCR. Figure 5. Transcriptional analysis of PHH organoids. PHH cells and corresponding PHH organoids cultured for several weeks were collected and processed for RNAseq. A) and (B) Unsupervised hierarchical cluster analysis of WNT signaling and YAP dependent up regulated gene profiles in PHHs and PHH organoid cultures (passage 0) in MM + Wnt S and MM + Wnt S + TRULI. The color scale indicates gene expression in log2 scale. #, donor. (C) Heatmap of hepatocyte, hepatoblast and cholangiocyte specific gene expression in PHH organoids and human cholangiocyte organoids expanded for the indicated passages (P, passage). The color scale indicates gene expression in log2 scale. (D) Gene expression levels of hepatocyte / hepatoblast (ALB, AFP, HNF4A, CD13 and CYP1A2) and cholangiocyte (KRT19, EpCAM and KI67) markers in PHHs, PHH organoids (PHH-Orgs) and human cholangiocyte organoids (hChol-Orgs) analyzed by qRT-PCR. Graph presents mean ± SEM from n=4 independent human donors, experiments were performed in duplicate. Statistical analysis was performed using Mann-Whitney test, **p<0.01, ***p<0.001. (E) Hierarchical cluster of global gene expression profiles in PHHs, PHH organoids and human cholangiocyte organoids. #, independent donor Figure 6. Characterization of differentiated PHH organoids. (A) Representative bright filed images of PHH organoids in expansion (PHH-Orgs_Expansion) or hepatic differentiation conditions (PHH- Orgs_Hep-Diff). Scale bars, 50 µm. Please note the more polygonal shape morphology of the cells upon differentiation. (B) Representative immunofluorescence stainings for the hepatocyte apical marker CD13 (bile canaliculi) and the cytoplasmic hepatocyte marker CYP2E1 in PHH-Orgs expanded for at least 4 weeks and cultured in differentiation medium as described in methods. Scale bars, 50 µm. (C) PHHs, human cholangiocyte organoids (hChol-Orgs) and PHH organoids expanded for the indicated passages from the same donors were cultured in expansion medium (PHH_Orgs) or in differentiation medium (PHH-Orgs_Hep-Diff), collected and processed for RNAseq. Heatmaps for the differentially expressed genes related to mature hepatocyte functions in PHHs, PHH-Orgs, PHH-Orgs_Hep-Diff and are shown. Scale bar indicates gene expression in log2 scale. (D) Principal component analysis (PCA) plot shows clustering of transcriptomic profiles of PHH-Orgs and PHH-Orgs_Hep-Diff with PHHs. (E) and (F) Cytochrome P450 activities and human albumin secretion are measured in hChol-Orgs, PHH- Orgs_Expand, PHH-Orgs_Hep-Diif and PHHs. Data are measured from 5 independent human donors and is presented as mean ± SEM. Figure 7. Primary human hepatocytes (PHHs) expand long-term when grown as human hepatocyte organoids (h-HepOrg) under conditions of high Wnt, low Hippo signalling and no nicotinamide. a. Ingenuity Pathway Analysis (IPA) of several publicly available datasets as indicated in the figure, used to identify signaling pathways involved in hepatocyte proliferation. Bar plots show IPA pathway activity z-score for each selected pathway. Dots represent the different datasets. b. Representative brightfield images of primary h-HepOrg cultured in the indicated media at day 10 of culture (P0). Scale bar, 500 μm (top); magnification, 100 μm (bottom). c. Representative brightfield images of patient-derived primary h-HepOrg serially expanded and cultured long-term in h-HepOrg-EM2 (MM+WntS+TRULI w / o Nic). Scale bar, 100 μm (left), 1 mm (right). P, passage; d, day. d. Organoid formation efficiency of h-HepOrg cultured with the indicated media. Graph represents ± SEM from n = 4-5 donors (biological replicates). Dot colour, same donor. Two-way ANOVA with Tukey Test for multiple comparisons. e. Serial expansion (1 : 2) of h-HepOrg from indicated donors. Graph indicates the expansion potential of h-HepOrg at the indicated media. Dot, passage. Note that for these donors, we check their expandability beyond 10 passages. As detailed in the graph and in Supplementary Table 2, under EM1 the cultures exhibit lower expansion potential, with none of them reaching beyond passage 10. Also, note that for the donors expanded in EM2 that reached passage 10, we stopped culturing them at time of submission. Figure 8. Establishment of the culture conditions to expand Human hepatocyte organoids (h-HepOrg) long-term while retaining their genetic stability over time. a. H&E staining of h-HepOrg grown in h-HepOrg-EM2 medium shows that the h-HepOrg grow as solid structures in vitro. Two representative images from n=3 independent experiments are shown. b. h-HepOrg cultured in the presence of Wnt and either TRULI or TDI-011536 show lumen formation after several passages under TDI-011536 treatment. Representative images of n=3 independent experiments are shown. Scale bar, 1mm (left), 50 μm (middle), 200 μm (right). P, passage. c. Graph showing the expansion potential of h-HepOrg from multiple donors in EM1 (complete) and in EM1 with the removal of individual components as indicated. Note that removal of Nic increased the longevity of the cultures, for all the donors tested. This medium, is subsequently called h-HepOrg-EM2. d. Chromosome analysis of h-HepOrg expanded for long or short time in culture. Representative image of a chromosome spread is shown (top). Graph shows the number of chromosomes from h-HepOrg- EM2 at different passages from multiple donors indicating the maintenance of genetic stability over time. P, passage. e. Cryopreserved h-HepOrg grown in EM2 can be recovered from cryopreservation without exhibiting any signs of loss of expansion potential. Representative brightfield images of h-HepOrg after freezing and thawing are shown. Scale bar, 500 μm (top), 100 μm (bottom). P, passage. Figure 9. Human hepatocyte organoids retain in culture the expression of zonated genes from in vivo tissue a. We performed RNAseq analysis on HepOrg expanded in EM2 (MM+WntS+TRULI w / o Nicotinamide) and at passage 2-4 the cultures were differentiated using the DM medium. Seven days after the start of the differentiation protocol, the organoids were harvested and processed for RNAseq analysis. The list of differentially expressed genes (DEG) between DM and EM2 was generated and overlapped with the gene list of pericentrally or periportally zonated genes from MacParland et al., 2018 Nat Commun 9, 4383 (2018). https: / / doi.org:10.1038 / s41467-018-06318-7. The heatmap shows the expression of DEG between DM and EM2 that intersect with the gene list of pericentrally or periportally zonated genes from MacParland et al., 2018. b. Human liver tissue and Human hepatocyte organoids (h-HepOrg) expanded in EM1 or EM2 medium, or differentiated in DM medium or liver tissue were analysed for the expression of zonated genes. Human liver tissue (top) and h-HepOrg in DM medium (middle and bottom) stained for pericentrally (GS, CYP2E1, magenta) and periportally (ECAD and Albumin, grey) zonated liver markers. Top, representative images of a human liver tissue stained for the pericentral marker Glutamine synthetase (GS, magenta) and periportal marker (Albumin, grey). Middle and bottom, h-HepOrg stained for the pericentral markers GS (magenta, middle) and CYP2E1 (magenta, bottom), and periportal marker (E- CAD, E-cadherin, grey). Nuclei were stained with DAPI (cyan). Right panels, fluorescence intensities are indicated in Fire LUT to better distinguish regional differences. Central vein (CV) and portal vein (PV) are indicated with a yellow dashed-line in human liver tissue (top). Scale bars, 100 μm (tissue) and 50 μm (organoid). Representative images from n= 3 independent experiments from the same donor are shown. Figure 10. Human hepatocyte organoids retain liver function in vitro and retore liver function upon transplantation in vivo in Fah- / - / Rag2- / - / Il2rg- / - deficient mice. a. Human hepatocyte organoids expanded in EM2 and differentiated in DM were tested for functional cytochrome activity (left panels, CYP2C9 and CYP3A4) and albumin secretion (right panel) and compared to human liver cholangiocyte organoids (h-CholOrg) and fresh isolated primary human hepatocytes (PHH) cultured in as monolayer cultures. Graphs represent mean + / - SEM for n=4-7 donors from n= 3 independent experiments. Results are expressed as RLU (left panels, CYP2C9 and CYP3A4) or ng / ml (Albumin) normalized by the total cell count. Two-way ANOVA with Tukey test for multiple comparisons was used. b. Fah- / - / Rag2- / - / Il2rg- / - (FRG) mice were injected with 500.000 cells intrasplenically and cycled on NTBC treatment post transplantation as described in methods. Animal health was monitored every other day. Kaplan Meir survival curve shows that both undifferentiated as well as differentiated h-HepOrg rescue the survival of Fah mutant mice after NTBC withdrawal. Log-rank test p = 0.0112. The examples illustrate the invention. Example 1 – Expansion of liver cells Methods for culturing epithelial stem cells from a variety of tissues have previously been described in the abovementioned different patents. The present inventors have surprisingly found that combining WNT signalling activation with WNT ligand and YAP activation to induce hippo signalling inhibition allows freshly isolated, as well as cryopreserved, human hepatocytes to be cultured for an increased number of passages compared to when this combination is absent from the medium. Below details on the exemplification of the present invention are provided: At present, no cell culture methods allow the maintenance and in vitro expansion of human adult hepatocytes (freshly isolated or from cryopreserved sources) that retain apical polarity, metabolic and detoxifying functions while can be at the same time co-cultured with stromal populations to recapitulate liver tissue architecture. The existing methods allow the expansion only of liver cells from mouse or embryonic human tissue, but not the long-term expansion of adult human hepatocytes that retain the architecture and function to do functional cell-cell connections with the reminder of the liver cells in the tissue. To identify methods to expand human adult hepatocytes in culture first the isolation procedure was optimized. Classical enzymatic tissue digestion by mincing the liver tissue and plating was first compared with a 2-step collagenase perfusion. Viable hepatocyte organoid structures were only obtained following tissue digestion by collagenase perfusion when viability was > 50%. All other tissue dissociation procedures yield lower hepatocyte viability (<50%) and failed to generate any hepatocyte organoid structures. These results were independent of the gender or age of the donors (Figure 1). Wnt signalling is essential to activate the proliferative state of hepatocytes (Belenguer et al., 2022, Nat Commun, 13:334 and Hu et al., 2007, Gastroenterology, 133:1579–1591) and ductal cells (Huch et al., 2013, Nature, 494:247-250) in mouse models of liver regeneration, while mutations in core components of the pathway result in liver cancer (Monga, 2011, Int J Biochem Cell Biol, 43:1021-1029). Similarly, Hippo signalling has a crucial role in maintaining adult hepatic quiescence by limiting cell proliferation, as overexpression of YAP in the liver is sufficient to induce dramatic hepatomegaly in mice and zebrafish (Camargo et al., 2007, Curr Biol, 17:2054–2060 and Cox et al., 2016, Nat Cell Biol, 18:886–896. Notably, differential expression and Ingenuity Pathway Analysis (IPA) between expanding human liver cancer organoids and human healthy tissue, as well as between expanding mouse adult hepatocyte organoids and mouse tissue revealed that both type of expanded cultures presented high Wnt and low Hippo signalling activities. The analysis of the upstream regulators of the pathways revealed that Wnt signalling activators such as the pathway effector CTNNB1 or the cell surface and Rspondin receptor LGR5 were upregulated, while LATS1 and MST1, the kinases responsible for phosphorylating YAP / TAZ and inducing its proteasomal degradation (Russell and Camargo. Nat. Rev. Gastroenterol and Hepatol. 2022), were found amongst the most downregulated upstream regulators, indicating that YAP / TAZ were activated and canonical Hippo signalling pathway was off (Figure 4 A and B). Therefore, to identify a culture medium that would allow the long-term expansion of human adult hepatocytes as hepatocyte organoids that would retain hepatic specific features of hepatocyte cell polarity, metabolic and detoxifying activity, it was opted to activate Wnt and suppress Hippo signalling pathways, respectively. To activate Wnt signalling, the culture medium was supplemented with a Wnt ligand agonist, namely Wnt surrogate, a water-soluble FZD–LRP5 / LRP6 heterodimerizer, with broad FZD reactive binding domains, which elicits canonical Wnt signalling activation through β-catenin / TCF4 signalling, and, when injected in vivo, promotes hepatocyte proliferation (Janda et al., 2017, Nature, 545:234-237). To inhibit Hippo signalling pathway and activate YAP / TAZ, it was opted to inhibit the kinase LATS1 / 2 with the specific inhibitors TRULI or TDI-011536. Both, TRULI and TDI-011536 are LATS1 / 2 specific inhibitors, which have been shown to increase YAP / TAZ activity by reducing phosphorylated YAP and increasing its nuclear translocation and, in vivo, promote retinal epithelial and cardiomyocyte proliferation, respectively (Kastan et al., 2021, Nat Commun, 12:3100). The addition of Wnt-S alone or TRULI or TDI-011536 alone allowed the expansion of the cultures up to 1 month and 2-3 passages, respectively. However, after this time, the cultures deteriorated and could not be further expanded. Notably, a synergistic effect was observed when combining the activation of WNT and YAP by LATS1 / 2 inhibition (Figure 2-3). The cultured cells and resulting organoids expanded for longer and increased the passage number. As expected, in the presence of WNT-surrogate and LATS1 / 2 inhibition, the expanded PHH organoids expressed WNT targets and YAP targets and also presented YAP nuclear localization (Figure 3). The expanded PHH organoids expressed markers of hepatoblast (e.g AFP) and hepatocyte markers such as HNF4A and MRP2, indicative of presence of bile canaliculi. However, the presence of cholangiocyte marker expression (e.g. EpCAM or KRT19) suggested that the obtained hepatocytes lacked maturation. Also, their bile canaliculi were too thick compared to published tissue control. Therefore, it was hypothesized that the expanding PHH organoids presented an immature hepatocyte state, which could prevent their functionality (Figure 3). It is well known that YAP activation by means of doxycycline- inducible expression of YAP- S127A or acute deletion of Nf2 in adult mouse hepatocytes triggers their dedifferentiation towards a cholangiocyte fate (Yimlamai et al., 2014, Cell, 157:1324-1338). However, cessation of YAP- S127A expression allows cells to re-differentiate into hepatocytes, highlighting the plasticity of liver cell fate. Therefore, it was hypothesized that for the cells to acquire mature hepatocyte fate, removal of YAP activation would be required. This brought us to define a novel hepatocyte differentiation medium by removing YAP activation while keeping WNT activation. For that, PHH organoids cultured in expansion medium were split and maintained in YAP activation for 2-3 days. Thereafter, the medium was replaced with a hepatocyte differentiation medium in which YAP activation was removed while maintaining WNT activation and PHH organoids were cultured for 7-9 days. Following treatment with differentiation medium, the cells reduced the expression of the embryonic and cholangiocyte markers AFP and KRT19, respectively. Mature markers, markers for xenobiotic metabolism, lipid metabolism and detoxifying enzymes were highly upregulated, almost to the same levels as fresh isolated human hepatocytes (Figure 5). Immunofluorescence analysis revealed that the cells have acquired the right apical polarity, with expression of bile canaliculi marker at the apical side of two adjacent hepatocytes, while E-cadherin, was present basolaterally (Figure 5). Hepatic differentiated PHH organoids also presented mature hepatic functions including cytochrome p450 activities and albumin secretion (Figure 5). RNAseq analysis revealed that the differentiated organoids clustered together with the fresh isolated hepatocytes and far away from cholangiocyte organoids published in Huch et al (Figure 5D). RNAseq analysis indicates that bile acid metabolism as well as fatty acid metabolism are all similar to primary human liver tissue (Figure 5C). It was noted that after several passages TRULI treated cultures presented better morphology (no cysts / cholangiocyte-like morphology), compared to TDI-011536 treated cultures (Figure 8b). This can be explained because TRULI is also an inhibitor of PKC and, to lesser extent, of PKA. PKC is known to be activated upon bile accumulation in hepatocytes, which triggers its trans-differentiation to cholangiocytes. It was noted that upon TRULI addition, many PKC genes are down-regulated, confirming the effect of TRULI through this pathway and suggesting that also PKC inhibition is essential for hepatocyte expansion. Therefore, it was opted to continue with the combination of MM + WntS and TRULI, named h-HepOrg-EM1 from hereon. Importantly, the combined use of Wnt-surrogate and YAP activation with TRULI allowed the long-term expansion of hepatocellular carcinomas derived from liver cancer patients with well-differentiated tumours, which was previously not possible (see Broutier et al., 2017, Nat Med). The expanded cultures expressed tumour markers such as TERT and GPC3 amongst others (Figure 4). Figure Combining Wnt activation and LATS1 / 2 inhibition in MM medium allowed the cultures to be serially expanded for several passages. However, MM is composed of many factors targeting critical pathways such as FGFR, EGF or HGF. Therefore, the requirement of all these components to grow h- HepOrg was tested by removing them one by one. Notably, removal of nicotinamide improved organoid formation efficiency by nearly 10-fold and sustained long-term expansion for at least 3 months in a serial passage (1:2 each week, >10 passages) (Figure 7b-e and Figure 8c). Under these conditions, the cultures expanded as solid structures with no lumina (FigureFigure 8a). All the other components were essential (Figure 8c). These results were consistent with the IPA analysis, where we found that the NAD signalling pathway was inactive in all the datasets analysed (Figure 7a), and were in agreement with published studies suggesting that excess nicotinamide leads to hepatotoxicity in rats and humans. Using these conditions (MM supplemented with Wnt and TRULI and without nicotinamide, hereafter called h- HepOrg-EM2), long-term expandable human hepatocyte organoids (h-HepOrg) were successfully generated from patients ranging between 11-85 years old from both sexes (n=12, in h-HepOrg-EM2 medium) (Table 3). Table 3: List of patients / donors under EM1 or EM2 culture conditions. Organoid formation and expansion potential are indicated. Note that for some donors used for the growth expansion graph we also indicate the passage number reached.

[0003] *In all instances where there is a diagnosis (not cryo-preserved PHH) the healthy adjacent part of the tissue was collected and used for hepatocyte isolation and organoid generation. The expanded cultures retained chromosome stability after long-term passaging (P20 and P27) (Figure 8d). Interestingly, h-HepOrg cultured in EM2 can be easily frozen and thawed while maintaining expansion potential upon recovery, allowing us to generate organoids from up to 12 different donors (Figure 8e and Table 3). In addition, the expanded cultures could be freeze down, kept in liquid N2 for months and later on thawed while retaining the expansion potential and features of the cells, suggesting that the method described here is suitable for biobanking approaches (Figure 8e). In conclusion, this invention relates to the ability to expand both healthy primary human hepatocytes (PHH) as well as hepatocellular carcinoma tissue (HCC) long-term while retaining the features of the corresponding tissues. This is advantageously as it allows more cells to be obtained from a single starting cell or from a collection of starting cells compared to previous methods. This enables a large number of applications, for example, drug screening / testing for personalized medicine approaches, in which a large amount of material is required to test various different drugs for the same patient. Similarly, the ability to expand healthy hepatocyte cells is advantageous for toxicology applications, as well as for applications where it is necessary to compare results between experiments. Hence, the ability to generate the cells from a single starting source is advantageous for applications where it is necessary to compare results between experiments. Example 2 – Properties of expanded liver cells The liver lobule is characterized for presenting zonal expression of some genes. Around the portal area hepatocytes have an oxidative metabolism (lipid β- oxidation) and amino acid catabolism while near the central vein area, hepatocytes mostly present the opposite metabolism, with glycolytic metabolism and lipid and amino acid biosynthesis (Campana et al., (2021), Nat Rev Mol Cell Bio.; 22(9):608-624). We investigated whether our HepOrg expanded in EM2 and differentiated in DM as described in methods would present features of liver zonation. We found that some pericentrally zonated genes, such as CYP2E1 and GLUL (Glutamine Synthase, GS), as well as some periportally zonated genes, such as ALB, or ASGR2, were highly upregulated (Figure 9a). Immunofluorescence analysis confirmed the heterogeneity of expression for some of these markers, with some of the cells presenting higher levels of pericentral markers CYP2E1 or GS while others expressing higher levels of the periportal marker ECAD. These results indicated that some cells present a gradient of expression of zonated genes, at least for those genes tested (Figure 9b). To evaluate the extent of maturation of the hepatocytes grown as hepatocyte organoids in EM2 (MM+WntS+TRULI w / o Nic) medium and differentiated in Differentiation medium (DM) we performed functional assays in vitro and compared them to the gold-standard used in pharmaceutical industry: hepatocytes grown as monolayer culture, in 2D. We observed that differentiated h-HepOrg secreted high levels of albumin in the medium, even higher than hepatocytes grown in 2D culture, and, to a lesser extent, cytochrome p450 activity (Figure 10a). Human liver monogenic diseases are caused by single-gene mutations. Although their prevalence is low, and are defined as rare diseases, all together, account for 10 in every 1000 births according to the World Health Organisation (Fagiuoli, et al. (2013), Journal of Hepatology, 59(3):595-612). Amongst these ones, Alpha1 antitrypsin deficiency, tyrosinemia or urea cycle disorders like citrin deficiency are amongst the most common. In the majority of cases, there is no therapeutic intervention that rescues the patients to undergo liver failure, and present, organ transplantation remains the standard of care for the majority of these diseases, offering either a curative approach or an improvement in the quality of life (Fagiuoli, et al. (2013), Journal of Hepatology, 59(3):595-612). Notably, a recovery of liver function of ~20-40% would suffice to rescue the patients from undergoing liver failure. Given the shortage of available donor organs for transplantation, alternative therapies have been extensively explored. Cell therapy transplantation, by repopulating the damaged livers with wild-type, healthy mature hepatocytes, proofs very effective in rodent models, but still suffers from the caveat that at present hepatocytes could only be obtained from another deceased donor. Given that our protocol allows the long-term expansion of human hepatocytes that retained their genetic stability and function in vitro, we next, we investigated whether the expanded hepatocyte organoids could be utilized as cellular source for liver cell therapy transplantation. For that, we expanded hepatocytes as hepatocyte organoids and engrafted both expanded and differentiated organoids in vivo in a mouse model of tyrosinemia type I (Fah- / -Rag2- / -Il2rg- / mouse) (Azuma et al. (2007), Nat Biotechnol., 25:903-10). Remarkably, after transplantation in vivo, both expanded and differentiated hepatocyte organoids retained their hepatic function and were able to rescue the lethal phenotype of the mice upon transplantation (Figure 10b). These results indicate that our method could facilitate the production of hepatocytes as a cellular source for liver cell therapy avoiding the need of liver transplant. Therefore, it means that many cells are available for use in potential cell therapy transplants and that multiple patients may be transplanted with cells obtained from a useful donor, in other words, from one donor from which cryopreserved hepatocytes could be obtained, several patients could be transplanted at once. Example 3 – Material and Methods Primary human hepatocyte organoid culture Primary human hepatocytes were isolated by two-step collagenase perfusion as follows: tissue was perfused with perfusion solution A (8 g / L NaCl + 0.4 g / L KCl + 0.35 g / L NaHCO3 + 0.06 g / L NaH2PO4·2H2O + 0.048 g / L Na2HPO4·12H2O + 1 g / L glucose + 10 mM HEPES + 2.5 mM EGTA in distilled water) at 37°C for 15-30 min at ratio of 15 mL / 20 sec. Then, perfusion solution was change to perfusion solution B (8 g / L NaCl + 0.4 g / L KCl + 0.35 g / L NaHCO3 + 0.06 g / L NaH2PO4·2H2O + 0.048 g / L Na2HPO4·12H2O + 1 g / L glucose + 100 mM HEPES + 4.8 mM CaCl2 + 1 g / L collagenase P) and perfused at 37°C for 5-15 min at ratio of 15 mL / 20 sec. The digestion was stopped by adding cold Williams E medium supplemented with 1% HEPES + 1% GlutaMax + 1% Penicillin / Streptomycin and tissue was minced with blade. The primary human hepatocytes were detached from the tissue by shaking using forceps filtered through a 100 µm Nylon cell strainer and spun 5 min at 50-100g. The pellet was resuspended in Williams E medium supplemented with 1% HEPES + 1% GlutaMax + 1% Penicillin / Streptomycin and kept cold and spun at 50-100g for 5 min. The cell pellet was mixed with Matrigel (BD bioscience) and 25000-50000 cells were seeded per well in a 48 well / plate. After Matrigel had solidified, culture medium was added. Culture media was based on AdDMEM / F12 (Invitrogen) supplemented with B27 without retinoic acid (Gibco), 1.25 mM N-Acetylcysteine (Sigma), 10 nM gastrin (Sigma) and the growth factors: 50 ng / ml EGF (Peprotech), 15% RSPO1 conditioned media (home- made), 100 ng / ml FGF10 (Peprotech), 100 ng / ml FGF7 (Peprotech), 50 ng / ml HGF, 10 mM Nicotinamide (Sigma), 2 µM A83-01 (Tocris Bioscience), 3 µM CHIR99021 (Tocris Bioscience), 10 µM Y-27632 (Tocris), 0.5 nM Wnt Surrogate FC Fusion Protein (Life Science Incubator) and 10 µM TRULI (Axon). One week-10 days later organoids were removed from the Matrigel, mechanically dissociated into small fragments, and transferred to fresh Matrigel. Passage was performed at 1:2-1:3 split ratio once per week for at least 3 months. To prepare frozen stocks, organoid cultures were dissociated and mixed with Recovery cell culture freezing medium (Gibco) and froze following standard procedures. Hepatocyte differentiation To enhance hepatocyte cell fate, primary human hepatocyte organoids were seeded and kept 2-4 days under the liver expansion conditions explained above. Then, medium was changed to AdDMEM / F12 medium supplemented with 1% B27 without retinoic acid (Gibco) and containing 1.25 mM N- Acetylcysteine (Sigma), EGF (50 ng / ml), HGF (50 ng / ml, Peprotech), FGF19 (100 ng / ml), A83-01 (2 µM, Tocris Bioscience), CHIR99021 (3 µM, Tocris Bioscience), 10 µM Y-27632 (Tocris), 0.5 nM Wnt Surrogate FC Fusion Protein (Life Science Incubator), 15% RSPO1 conditioned media (home-made) and Dexamethasone (1.6-3 µM). Medium was changed every other day for a period of 7-9 days. Freezing and thawing of PHH-organoids PHH-Organoids were removed from Matrigel with same process of passaging and were gently resuspended in cold-Recovery Cell Culture Freezing Medium (Thermo Fischer Scientific (one well per one vial). PHH-Organoids were moved to the cryopreserved tube and tubes were placed in the precooled cell freezing container. PHH-Organoids were frozen in -80 °C for 24 hours and transferred in liquid nitrogen. Thawing was performed as standard by placing the cells at a water bath at 37 °C for 5 min and immediately transferred to a tube with pre-warmed medium, spun at 100G for 5 min, resuspended in Matrigel, seeded in non-attaching plates and, once Matrigel was solidified, overlayed with EM medium described above. Hepatocyte functional studies To determine albumin secretion, primary human hepatocyte organoids were differentiated as described. Culture medium was changed every other day and culture supernatant was collected d 24h after the last medium change.2D primary human hepatocytes and human cholangiocyte organoids were cultured for 24h in the same medium without growth factors and were used as positive and negative control respectively. The amount of albumin in culture supernatant was determined using a human specific Albumin ELISA kit (both from Assay Pro). To measure Cytochrome P450 activity the cultures were differentiated as described and the day of the experiment the cells were removed from the matrigel and cultured with the Luciferin-H substrate (100 µM) or Luciferin-IPA (3 µM) in Williams’ E medium supplemented with 1% HEPES + 1% GlutaMax + 1% Penicillin / Streptomycin. As controls, 2D primary human hepatocytes and human cholangiocyte organoids were cultured for 24h and the day of the experiment transferred to Williams’ E medium supplemented with 1% HEPES + 1% GlutaMax + 1% Penicillin / Streptomycin and Luciferin-H substrate (100 µM) or Luciferin-IPA (3 µM). Cytochrome P450 activity was measured 8h later using the P450-Glo Assay Kit (Promega) according to manufacturer’s instructions. Immunofluorescence Organoids were fixed for 30 minutes with 4% paraformaldehyde (PFA) at 4°C, washed with PBS and transferred to a µ-Slide 8 Well Chamber Slide (Ibidi). Blocking was done using 2% BSA 0.1% Triton-X in PBS for 1 hour on the rocker at room temperature. After blocking, organoids were washed with PBS and incubated with primary antibodies for 24 hours on the rocker at 4°C, followed by washes with PBS. Next, they were incubated with secondary antibodies for 12 hours on the rocker at 4°C, and then washed with PBS. All antibodies were diluted in the blocking solution. The antibodies and dilutions used are listed in Table 2. Table 2: List of antibodies Nuclei were stained with Hoechst33342 (Molecular Probes) or DAPI. Stained organoids were cleared with clearing solution (16.5 mL of glycerol, 3.5 mL of dH2O and 14.86 g of fructose) before imaging. Pictures were taken with a Zeiss LSM 880 Airy inverted microscope and a Zeiss LSM 780 NLO microscope (Zeiss). RT-PCR and qPCR analysis RNA was extracted from organoid cultures or freshly isolated tissue using the RNeasy Mini RNA Extraction Kit (Qiagen), and reverse-transcribed using reverse-transcribed using Moloney Murine Leukemia Virus reverse transcriptase (Promega). All targets were amplified (40 cycles) using gene- specific primers and MiIQ syber green (Bio-Rad). Data were analyzed using BioRad CFX manager. cDNA was amplified in a thermal cycler (GeneAmp PCR System 9700; Applied Biosystems, London, UK) as previously described (Huch et al, 2009). Functional assays For functional assays, hepatocyte organoids were cultured in expansion and differentiation medium as described above. As negative controls we used cholangiocyte organoids grown as described above. As positive controls we used fresh isolated human primary hepatocytes cultured in standard 2D-hepatocyte sandwich culture79. Briefly, fresh isolated PHH were plated onto collagen (1.8 mg / mL, RatColTMcollagen, Advanced Biomatrix) coated 24-well plates at 500,000 or 250,000 cells / well in Williams E medium (PAN Biotech), substituted with 10% FBS, penicillin / streptomycin and 100 nM Dexamethasone for 3 hours for attachment and then cultured for 24 hours in organoid medium. To determine albumin secretion, supernatant from 24 hours was collected and the amount of albumin was determined using a human specific Albumin ELISA kit (both from Assay Pro) following manufacturer’s instructions on an ELISA plate reader. To measure Cytochrome P450 activity, on the day of the experiment 2D-hepatocytes cultures or cholangiocyte and hepatocyte organoids were removed from Matrigel using Cell Recovery solution (Corning) and cells were cultured with Williams’ E medium supplemented with 1% HEPES + 1% GlutaMax + 1% Penicillin / Streptomycin and cultured with the Luciferin-H substrate (100 µM) or Luciferin-IPA (3 µM) for 6 hours. Cytochrome activity was measured using the P450-Glo Assay Kit (Promega) according to manufacturer’s instructions on a PerkinElmer Envision Plate reader. Xenotransplantation in Fah- / - / Rag2- / - / Il2rg- / - (FRG) Male and female Fah- / - / Rag2- / - / Il2rg- / - (FRG) mice were obtained from Jackson Laboratory. Mice were housed and maintained under specific pathogen-free conditions in accordance with the Principles of Laboratory Animal Care and the Guide set by the HYU Industry-University Cooperation Foundation. For their maintenance, mice were administered ad libitum NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3- cyclohexanedione) in drinking water. Mice aged 8-16 weeks old from both sexes were kept on NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)- 1,3-cyclohexanedione) in drinking water until 3 days prior to the experiment, when NTBC was withdrawn. Human hepatocyte organoids expanded in h-HepOrg-EM2 and differentiated in h-HepOrg-DM medium were dissociated into single cells and prepared for injection. For transplantation experiments commercially available frozen PHHs were used (F-PHH2, Supplementary Table 2). Organoids cultured under h-HepOrg-EM2 medium as well as isolated hepatocytes (PHHs) from the same donors were used as controls. Following dissociation, 500,000 dissociated organoid cells or 800,000 primary human hepatocytes (PHHs) were resuspended in 100 μl of AdDMEM / F-12 medium and injected into the spleen. The non-injected negative control group received 100 μl of PBS instead of cells. Mice were cycled in and out of NTBC treatment for 3 days every time their body weight dropped below 80% of the initial weight. Data analysis All values are represented as mean ± standard error of the mean (S.E.M.). Unless differently stated in the legend, Man-Whitney non-parametric test was used. p<0.05 was considered statistically significant. In all cases data from at least 3 independent experiments was used.

Claims

CLAIMS 1. A method for expanding liver cells, wherein the method comprises (a) expanding the liver cells within an extracellular matrix in an expansion medium, wherein the expansion medium comprises a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway, and preferably with the proviso that the expansion medium does not comprise nicotinamide.

2. The method of claim 1 further comprising after step (a) (b) isolating the expanded liver cells, wherein the expanded liver cells are preferably in the form of organoids.

3. The method of claim 1 or 2 further comprising before step (a) (a’) adding the liver cells into the extracellular matrix.

4. The method of any one of claims 1 to 3 further comprising after step (a) and, if present, before step (b) (b’) differentiating the expanded liver cells in a differentiation medium that comprises a Wnt surrogate or activator and does not comprise an inhibitor of the Hippo signalling pathway.

5. The method of any one of claims 1 to 4, wherein the cells are expanded (i) for at least 5 passages, preferably at least 10 passages and most preferably at least 15 passages; and / or (ii) for at least 50 days, preferably at least 100 days and most preferably at least 200 days.

6. The method of any one of claims 1 to 5, wherein the Wnt surrogate or activator is SZN-1326, SZN-413 or a water-soluble and Wnt receptor frizzled (FZD)-specific surrogate Wnt agonist, preferably a water-soluble FZD-LRP5 / LRP6 heterodimerizer.

7. The method of any one of claims 1 to 6, wherein the inhibitor of the Hippo signalling pathway is an activator of the yes-associated protein (YAP) / WW-domain-containing transcriptional regulator (TAZ) and / or an inhibitor of the large tumor suppressor kinase 1 / 2 (LATS1 / 2).

8. The method of any one of claims 1 to 7, wherein the inhibitor of the Hippo signalling pathway is selected from one or more of TRULI, TDI-011214, TDI-011241, TDI-011536, DB-202, ION-537, VT-3989, GH-658, TM-25659, TY-0584, XMU-MP-1, I3MT-3 IHMT-MST1-58, and SBP-3264, PY-60, GA-017, TT-10, MA-5 and VT02956.

9. The method of any one of claims 1 to 8, wherein the expansion medium further comprises one ormore of (i) a Wnt ligand, wherein the Wnt ligand is preferably Wnt, Norrin or a GSK-inhibitor, wherein the GSK inhibitor is preferably a GSK3b inhibitor and is most preferably CHIR99021 (6-[[2-[[4- (2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3- pyridinecarbonitrile), tideglusib, AL-001, dronabinol, elraglusib, AP-001, AP-1, IB-AD, MD-053; (ii) a FGFR2 / FGFR4 agonist, wherein the FGFR2 / FGFR4 agonist is preferably FGF and is most preferably FGF10 and / or FGF7; (iii) a Rho-kinase inhibitor, wherein the Rho-kinase inhibitor is preferably an inhibitor of ROCK1 or ROCK2 and is most preferably selected from Y-27632, RKI-1447, DJ4, H-1152, Chroman 1, Ripasudil, GSK269962A, Fausdil, belumosudil, OPL-0401, TDI-01, zelasudil, NRL- 1049, ANG-4201, CVT-100069, CVT-100077, KD-045, REDX-10616, RXC-008, TRX-101; (iv) a TGF-inhibitor, wherein the TGF-inhibitor is preferably an inhibitor of ALK4, ALK5 or AKL7 and is most preferably A83-01, SB-431542, SB-5225334 LY364947, SS-208, SNJ 2511, EW-7197, SB505124, RepSox, GW788388, SD-208, Galunisertib, EW-7197, or LY2109761; (v) B-27 Supplement, wherein the B-27 Supplement is preferably without retinoic acid; (vi) N-acetylcysteine; (vii) gastrin; (viii) a growth factor, preferably EGF and / or HGF; and (ix) R-Spondin (RSPO) conditioned media supplement (preferably prepared from cell transfectant stably secreting RSPO) or RSPO protein, wherein RSPO is preferably RSPO1, RSPO2, RSPO3 or RSPO4.

10. The method of any one of claims 3 to 9, wherein the differentiation medium, if present, further comprises one or more of (i) a Wnt ligand, wherein the Wnt ligand is preferably Wnt, Norrin or a GSK-inhibitor, wherein the GSK inhibitor is preferably a GSK3b inhibitor and is most preferably selected from CHIR99021, tideglusib, AL-001, dronabinol, elraglusib, AP-001, AP-1, and IB-AD, MD-053; (ii) a FGFR2 / FGFR4 agonist, wherein the FGFR2 / FGFR4 agonist is preferably a Pan-FGFR inhibitor and is most preferably FGF19; (iii) a Rho-kinase inhibitor, wherein the Rho-kinase inhibitor is preferably an inhibitor of ROCK1 or ROCK2 and is most preferably selected from Y-27632, RKI-1447, DJ4, H-1152, Chroman 1, Ripasudil, GSK269962A, Fausdil, belumosudil, OPL-0401, TDI-01, zelasudil, NRL- 1049, ANG-4201, CVT-100069, CVT-100077, KD-045, REDX-10616, RXC-008, and TRX-101; (iv) a TGF-inhibitor, wherein the TGF-inhibitor is preferably an inhibitor of ALK4, ALK5 or AKL7 and is most preferably A83-01, SB-431542, SB-5225334 LY 364947, SS-208 or SNJ 2511; (v) B-27 Supplement, wherein the B-27 Supplement is preferably without retinoic acid; (vi) N-acetylcysteine; (vii) a growth factor, preferably EGF and / or HGF; (viii) dexamethasone; and (ix) R-Spondin (RSPO) conditioned media supplement (preferably prepared from cell transfectant stably secreting RSPO) or RSPO protein, wherein RSPO is preferably RSPO1,RSPO2, RSPO3 or RSPO4.

11. The method of any one of claims 1 to 10, wherein the extracellular matrix (i) comprises one or more and preferably all of collagen (preferably collagen type IV), entactin, perlecan (preferably heparan sulfate proteoglycan), and laminin; and / or (ii) is a reconstituted basement membrane derived from extracts of mammalian cells, preferably mouse cells, and most preferably Engelbreth-Holm-Swarm mouse tumor cells.

12. A liver cell expansion medium that comprises a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway as defined in any one of the preceding claims.

13. A liver cells expansion kit that comprises (i) a liver cells expansion medium comprising a Wnt surrogate or activator and an inhibitor of the Hippo signalling pathway as defined in any one of the preceding claims, and (ii) a liver cells differentiation medium comprising a Wnt surrogate or activator and does not comprise an inhibitor of the Hippo signalling pathway as defined in any one of the preceding claims.

14. Use of the expansion medium of claim 12 or the kit of claim 13 for expanding liver cells.

15. The method, medium or kit of any one of the preceding claims, wherein the liver cells are (i) adult liver cells; (ii) mammalian liver cells, preferably human liver cells; (iii) liver tumor cells, preferably liver cancer cells; and / or (iv) liver epithelial or mesenchymal cells, preferably hepatocytes.

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