Method for the in vitro production of a hepatocyte organoid

Abstract

The present invention relates to methods for the in vitro production of a hepatocyte organoid from primary human hepatocytes (PHH). The present invention further relates to a cell culture system for the in vitro expansion of functional foetal, neonatal, paediatric and adult PHH and the mass generation of organoids and use of the cell culture system for the in vitro mass generation and expansion of functional adult PHH and / or formation of hepatocyte organoid.

Description

[0001] METHOD FOR THE IN VITRO PRODUCTION OF A HEPATOCYTE ORGANOID

[0002] Description

[0003] The present invention relates to methods for the in vitro production of a hepatocyte organoid from primary human hepatocytes (PHH). The present invention further relates to a cell culture system for the in vitro expansion of functional foetal, neonatal, paediatric and adult primary human hepatocytes (PHH) and the mass generation of organoids and use thereof.

[0004] With the increasing research on tissue engineering and regenerative medicine, rapid production and large amounts of cells are demanded. A more recent and emerging technique is the mass expansion of cells through the generation of organoids. Organoids are miniature organs, produced in vitro in 3D, that resemble their organ of origin with respect to cell types and function, and can be established from a variety of organs and species. Organoids are derived from primary (stem) cells, embryonic stem cells or induced pluripotent stem cells, which self-organize in a three- dimensional culture. The production of organoids using cell culture technology, started with a shift from culturing cells in 3D culture conditions to allow for the development of the complex 3D structures of organs. As such, organoids are promising, not only for regenerative medicine applications, but also as in vitro models to study organ development, regeneration and disease. Organoids are also used to study (personalized) treatments in a laboratory, i.e. as an in vitro model which closely resembles an in vivo organ / cell of the patient, for example to test the efficacy or toxicity of future therapies.

[0005] The liver is an essential organ, performing a plethora of essential processes, such as blood detoxification and the production of serum proteins. Hepatocytes are the epithelial cells responsible for performing most of these crucial functions, making up 70-80% of the liver. In vitro, adult primary human hepatocytes (PHH) are the gold standard in toxicological studies and have the potential to treat patients with liver disease. However, PHH have short-term functionality and limited expansion potential in vitro. While notable advances have been made in the long-term maintenance of adult PHH, the efficient expansion of these cells in a functional state has yet to be reported. This is a major obstacle and limits the use of PHH.

[0006] Current cell culture system for hepatocyte organoid generation from adult primary human hepatocytes (PHH) has a plating efficiency of less than 1%, meaning that 99% of plated hepatocytes do not participate in organoid generation. This system has major reproducibility issues, provides no immunohistochemical data, and does not measure proliferation for several passages. Finally, due to the low plating efficiency and poor expansion potential of the present system, in vitro studies are difficult to perform and regenerative medicine applications are not practical. Considering the above, there is a need in the art for a method for the expansion of functional primary human hepatocytes. The method should provide for reproducible expansion of functional adult primary human hepatocytes, preferably as organoids, enabling long-term in vitro studies on (cycling) adult PHH, as well as regenerative medicine applications. In addition, there is a need in the art for a cell culture system suitable for the indicated method.

[0007] It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

[0008] Specifically, the above object, amongst other objects, is met, according to a first aspect, by the present invention by a method for the in vitro production of functional and proliferative hepatocyte organoids from primary human hepatocytes (PHH), comprising the steps of; a) aggregating primary hepatocyte cells in cell culture in a hepatocyte spheroid medium (HSM) to generate functional and proliferative hepatocyte spheroids wherein HSM is a hepatocyte culture media comprised of;

[0009] 0.5 to 100 ng / ml, preferably 1 to 75 ng / ml, more preferably 2 to 50 ng / ml, even more preferably 5 to 40 ng / mL, most preferably 10 to 25 ng / mL of at least one epidermal growth factor receptor (EGFR) agonist,

[0010] 1 to 50 ng / mL, preferably 5 to 40 ng / mL, more preferably 10 to 25 ng / mL of hepatocyte growth factor (HGF) or isoform thereof, and

[0011] 0.1 ng / mL to 2 pg / mL of at least one canonical Wnt activator, such as a Wnt protein or Wnt surrogate molecule, b) collecting the hepatocyte spheroids and transfer of the hepatocyte spheroids to a scaffold matrix, c) culturing of the hepatocyte spheroids transferred in the scaffold matrix in a hepatocyte organoid medium (HOM) to provide a hepatocyte organoid comprised of expanding functional PHH, wherein HOM is a hepatocyte culture media comprised of;

[0012] 0.5 to 100 ng / ml, preferably 1 to 75 ng / ml, more preferably 2 to 50 ng / ml, even more preferably 5 to 40 ng / mL, most preferably 10 to 25 ng / mL of at least one epidermal growth factor receptor (EGFR) agonist,

[0013] 1 to 50 ng / mL, preferably 5 to 40 ng / mL, more preferably 10 to 25 ng / mL of at least one hepatocyte growth factor (HGF) or isoform thereof,

[0014] 0.1 ng / mL to 2 pg / mL of at least one canonical Wnt activator, such as a Wnt protein or Wnt surrogate molecule,

[0015] 50 to 500 ng / mL, preferably 100 to 300 ng / mL, more preferably 150 to 250 ng / mL of at least one R-spondin protein, 0.1 to 5 pM, preferably 0.5 to 3 pM, more preferably 1 to 2.5 pM of at least one transforming growth factor P (TGF- ) inhibitor,

[0016] 1 to 20 pM, preferably 2.5 to 15 pM, more preferably 5 to 10 pM of at least one ROCK inhibitor, and

[0017] 1 to 20 mM, preferably 2.5 to 15 mM, more preferably 5 to 10 mM of Nicotinamide (NAM).

[0018] The cell aggregation is preferably preformed in a cell culture system such as microwell plate, preferably an ultra-low attachment microwell plate. Aggregated PHH ‘spheroids’ as referred to herein are 3D hepatocyte cell aggregates that form in the absence of a predefined culture substrate to adhere to. As a result, the hepatocytes do not attach to, or adhere to the surface of the culture substrate or system. Techniques to generate spheroids include hanging-drop and ultra-low attachment cultures, which encourage cell-cell interaction while discouraging cell-substrate / matrix interaction. As a result, cells interact to form a compact sphere, although other shapes are possible. Fresh or cryopreserved adult (or foetal, neonatal or paediatric) primary human hepatocytes (PHH) are aggregated, for example, in ultralow attachment (ULA) microwells, and allowed to form spheroids in a defined hepatocyte spheroid medium (HSM).

[0019] The method of present invention preferably provides hepatocyte organoids that comprise 0.1 to 20%, preferably 2 to 17.5%, more preferably 5 to 15 %, most preferably 10 to 12.5% of non- parenchymal liver cells (NPCs), such as stellate cells, liver endothelial cells, and / or Kupfer cells. The presence of these NPCs add to the function of the liver organoid and provide further stimulation for the cells and their functioning in the overall organoid. The NPCs produce various growth factors important in the development and function of the liver organoid.

[0020] As used herein, ‘functional’ hepatocytes in cell culture refers to hepatocytes which retain key features and functions of hepatocytes in vivo. These include polarized expression of key drug transporters, such as MRP2 and / or BSEP; production and secretion of albumin; production and transport of bile; uptake of low-density lipoproteins; synthesis and storage of glycogen; biotransformation of xenobiotics through phase I and / or phase II drug metabolism. Furthermore, functional hepatocytes should remain susceptible to infection by hepatocyte specific pathogens, such as malaria and HBV. Importantly, transcriptomic data is not sufficient to denote functional hepatocytes in vitro. Polarized protein expression and activity must be demonstrated through various techniques which are well known in the art. Protein expression and localization may be demonstrated, for example, through immunolocalization or immunohistochemistry. Protein function may be assessed using various commercially available kits, for example, albumin and CYP ELISA kits, which measure secretion of albumin and biotransformation by CYP enzymes, respectively. As used herein, ‘proliferative’ or ‘expanding’ hepatocytes refers to hepatocytes which incorporate nucleoside analogues such as BrdU (5-bromo-2'-deoxyuridine) or EdU (5-ethynyl-2’- deoxyuridine) in newly synthesized DNA; express nuclear proteins specific to proliferative cells, such as Ki67 and PCNA; express cell cycle proteins, such as cyclin DI and Cdk2. Methods to assess hepatocyte proliferation are well known in the art, including but not limited to, immunofluorescent detection of proliferation proteins (i.e., Ki67, PCNA, cyclin DI), detection of incorporated nucleoside analogues (i.e., BrdU, EdU) and / or the generation of a growth curve through cell counts or alternatives to cell counts, such as the CellTiter-Glo® Luminescent Cell Viability Assay.

[0021] Following the formation of stable spheroids, PHH are transferred to a suitable scaffold matrix, for example Matrigel, and cultured in a defined hepatocyte organoid medium (HOM). Once transferred to the scaffold matrix (such as Matrigel) PHH spheroids begin to reorganize, proliferate, and invade the surrounding extracellular matrix. The method of present invention allows for reproducible and scalable expansion of functional adult primary human hepatocytes as organoids, enabling in vitro studies on (cycling) adult PHHs, as well as regenerative medicine applications. The method employs aggregation of primary hepatocytes into spheroids and allows for the mass-generation of primary human hepatocyte organoids from the first week of culture. The invention may employ ULA microwell plates or equivalent, allowing for the reproducible and controlled generation of thousands of spheroids. The expansion potential of adult PHH is far superior using the present solution than that of any known method and allows for the subculturing of expanded hepatocytes. The method of present invention has broad commercial applications, from regenerative medicine to in vitro toxicology studies, i.e. the pharmaceutical industry.

[0022] The HSM and HOM are comprised of hepatocyte growth factor (HGF) or related isoforms (NK1, NK2) which is a c-Met agonist. c-MET activation by HGF controls a variety of processes, including development, proliferation, and branching morphogenesis.

[0023] According to a preferred embodiment, the present invention relates to the method, wherein the EGFR agonist is one or more selected from the group consisting of Amphiregulin (AREG), epidermal growth factor (EGF), Heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-alpha (TGFa), or related EGFR ligand, preferably AREG and / or EGF. Activation of EGFR induces a variety of intracellular processes, including proliferation and differentiation.

[0024] According to a preferred embodiment, the present invention relates to the method, wherein the at least one canonical Wnt activator is selected from the group consisting of Wnt surrogate molecule or Wnt protein, such as Wnt3a, Wnt2, Wnt9b, WntlOb, CHIR99021, Wnt ligands, glycogen synthase kinase 3b inhibitors, b-catenin and activators of b-catenin, preferably Wnt surrogate molecule or Wnt3a. According to another preferred embodiment, the present invention relates to the method, wherein the at least one canonical Wnt activator comprises 0.1 to 10 pg / mL, preferably 0.1 to 2 pg / mL, more preferably 0.2 to 1.5 pg / mL, more preferably 0.5 to 1 pg / mL of Wnt protein.

[0025] According to yet another preferred embodiment, the present invention relates to the method, wherein the at least one canonical Wnt activator comprises 0.1 to 100 ng / mL, preferably 10 to 80 ng / mL, more preferably 20 to 60 ng / mL of Wnt surrogate molecule.

[0026] According to a preferred embodiment, the present invention relates to the method, wherein the TGF-P inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947, or related ALK4-, ALK5-, ALK7- inhibitor, preferably A83- 01. A TGF inhibitor is a compound that reduces, blocks or inhibits TGF through the TGF RI and TGF RII receptors.

[0027] According to another preferred embodiment, the present invention relates to the method, wherein the hepatocytes are primary human hepatocytes selected from the group consisting of adult, foetal, neonatal or paediatric primary human hepatocytes, preferably adult. Hepatocytes may be primary cells isolated from a sample of immature or mature liver tissue or tumorous liver tissue. Liver tissue may be obtained from individuals of foetal age or of any age greater than 0 months.

[0028] According to another preferred embodiment, the present invention relates to the method, wherein the scaffolding matrix is a laminin-rich extracellular matrix hydrogel, preferably Matrigel, Laminin-Entactin Complex, Ultimatrix, Cultrex, or other basement membrane extract and / or synthetic (hydro)gels allowing organoid culture. The hepatocyte spheroids are transferred and cultured in 3D culture wherein the culture media and cells are mixed with a scaffold matrix, preferably the hydrogel which supports the growth and proliferation of cells in 3D and allow the cells to reorganize and expand as organoids. Preferably a hydrogel is used as suitable scaffold matrices, but other known natural and synthetic matrices such as fibrin, collagen, collagen / laminin, decellularized tissue-derived matrices, compressed collagen alginate, agarose, polyethylene glycol or basement membrane extracts may be used. The scaffold matrix may be chemically defined, for example a collagen or densified collagen hydrogel, or non-chemically defined, for example a complex protein hydrogel. Preferably, the scaffold matrix in the expansion medium is a complex protein hydrogel. Suitable complex protein hydrogels may comprise extracellular matrix components, such as laminin, collagen IV, entactin and heparin sulphate proteoglycans. Complex protein hydrogels may also include hydrogels of extracellular matrix proteins from Engelbreth- Holm-Swarm (EHS) mouse sarcoma cells. Suitable complex protein hydrogels are available from commercial sources and include Matrigel™ (Corning Life Sciences) or Cultrex™ BME 2 RGF (Amsbio™ Inc).

[0029] According to another preferred embodiment, the present invention relates to the method, wherein the hepatocyte spheroids are transferred to the scaffold matrix at a density of 1 to 1000 hepatocyte spheroids, preferably 100 to 500 hepatocyte spheroids, per 25 to 100 pL scaffold matrix. If too many spheroids are plated per droplet of scaffold matrix, i.e. more than 1,000 per 100 pL, they will fuse together, which is unwanted for expansion.

[0030] According to another preferred embodiment, the present invention relates to the method, wherein the method further comprises the step of d) subculturing of the hepatocyte organoids expanded in scaffold matrix with HOM to a fresh scaffold matrix every 1 to 60 days, preferably every 5 to 20 days, more preferably every 10 to 15 days. Hepatocyte organoids are expanded in the scaffold matrix for 1-30 days and are collected and transferred to fresh scaffold matrix at a 1:2 - 1:3 subculture ratio and provided with HOM for another 1-60 days. This process can be repeated as long as the cells continue to expand.

[0031] According to another preferred embodiment, the present invention relates to the method, wherein the HSM further comprises 50 to 500 ng / mL of at least one R-spondin protein.

[0032] According to another preferred embodiment, the present invention relates to the method, wherein the R-spondin protein is selected from the group consisting of R-spondin-3, R-spondin-2, R-spondin- 1, or other protein family member of the R-spondin protein family, preferably R- spondin-3. R-spondin amplifies the effect of Wnt protein and stimulates expansion. R-spondin 3 is more effective than the others and is also the R-spondin produced during liver regeneration. R- spondin is a secreted activator protein that positively regulates canonical and non-canonical Wnt signalling pathways. Preferably, R-spondin is human R-spondin. During canonical Wnt activation, R-spondin synergizes with Wnt molecules to amplify Wnt signals.

[0033] According to another preferred embodiment, the present invention relates to the method, wherein the HSM further comprises 1 to 20 pM of a ROCK inhibitor. ROCK inhibition blocks or inhibits Rho kinase (ROCK) activity. A ROCK inhibitor prevents cells going into apoptosis when cells undergo stress due to lacking cell-cell contact, for instance in stem cell cultures.

[0034] According to yet another preferred embodiment, the present invention relates to the method, wherein the ROCK inhibitor is selected from the group consisting of Y-27632 (trans-4-[(l R)-l-Aminoethyl]-N-4-pyridinylcyclohexanecarboxamide), fasudil, Y39983 (4-[(lR)-l- Aminoethy 1] - A / - 1 / - / -pyrrolo [2,3 -b]pyridin-4-y Ibenzamide dihydrochloride) , and azabenzimidazole-aminofurazans, preferably Y-27632.

[0035] According to yet another preferred embodiment, the present invention relates to the method, wherein the HSM and / or HOM further comprise 0.1 to 50 pM of a glucocorticoid. Glucocorticoid effect a wide range of processes, including survival, proliferation, and differentiation. HSM preferably comprises either 1-50 pM Hydrocortisone, or 0.1-1 pM Dexamethasone.

[0036] According to a preferred embodiment, the present invention relates to the method, wherein the glucocorticoid is selected from the group consisting of hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone, preferably hydrocortisone or dexamethasone, preferably 1 to 50 pM hydrocortisone or 0.1 to 1 pM dexamethasone.

[0037] According to another preferred embodiment, the present invention relates to the method, wherein the aggregation of functional primary hepatocyte cells in HSM is for about 1 to 30 days, preferably for about 3 to 10 days, more preferably for about 4 to 6 days. 4 to 6 days was found to be ideal for the generation of functional primary hepatocyte spheroids. Shorter (less than 2 days) or longer (over 30 days) times result in suboptimal results once the aggregates are transferred to the 3D matrix.

[0038] According to another preferred embodiment, the present invention relates to the method, wherein the culturing of the hepatocyte spheroids transferred in the scaffold matrix in HOM is for about 1 to 30 days, preferably about 5 to 20 days, more preferably about 8 to 15 days. Because the 3D matrix eventually degrades, most optimal every 5 to 15 days the cells are transferred to new 3D matrix and cultured for another round, as indicated above.

[0039] According to a preferred embodiment, the present invention relates to the method, wherein the hepatocytes into a microwell cell culture system are provided at a density of 5 to 1500 cells / microwell, preferably 25 to 250 cell / microwell, more preferably 50 to 100 cell / microwell.

[0040] According to yet another preferred embodiment, the present invention relates to the method, wherein the collected hepatocyte spheroids of step c) are on average 10 to 1000 pm in diameter, preferably 50 to 500 pm in diameter, more preferably 100 to 250 pm in diameter, as determined by bright field microscopy (i.e., by using a scale bar).

[0041] According to a preferred embodiment, the present invention relates to the method, wherein the HOM further comprises 1 to 1000 nM Gastrin. Gastrin is added within the specified concentration range to promote the expansion and viability of the cells in cell culture.

[0042] According to a preferred embodiment, the present invention relates to the method, wherein the HOM further comprises 1 to 100 ng / mL, preferably 10 to 75 ng / mL, more preferably 25 to 50 ng / mL of tumour necrosis factor alpha (TNFa). TNFa addition in the HOM promotes activation of NF-KB signalling and induces further proliferation of hepatocytes.

[0043] According to a preferred embodiment, the present invention relates to the method, wherein the HOM further comprises 1 to 100 ng / mL of fibroblast growth factor 19 (FGF19). FGF19 activates fibroblast growth factor receptor 4 (FGFR4) signalling, modulating metabolic capabilities, such as bile acid synthesis.

[0044] According to another preferred embodiment, the present invention relates to the method, wherein the HSM and / or HOM further comprises 1 to 5mM of N-acetylcysteine (NAC). According to yet another preferred embodiment, the present invention relates to the method, wherein the HSM and / or HOM further comprises 0.1 to 10% v / v foetal calf serum (FCS) or foetal bovine serum (FBS).

[0045] According to another preferred embodiment, the present invention relates to the method, wherein at least 50%, more preferably at least 65%, even more preferably at least 85%, most preferably at least 90% of the hepatocytes present at the start of cell culturing contribute to the organoid formation. In contrast to the method of present invention, with the known and currently applied methods for producing organoids about 1% of the cells that have been provided at the start of culture survive and contribute to the organoid formation and production. The method of present invention is far superior in this respect, wherein at least 50% of the viable cells that have been used at the start of cell culture contribute to the provision or generation of the organoid.

[0046] The method of present invention makes use of HSM and HOM media. Both HSM and HOM may be comprised of a basal medium further supplemented with a media supplement, such as B27 supplement, N21 supplement, N2 supplement or ITS(+) supplement, to constitute a nutrient medium. The nutrient medium may further comprise L-glutamine or substitutes, such as L-alanyl- L- glutamine (e.g. Glutamax™), N-acetylcysteine (NAC), MEM Non-Essential Amino Acids Solution, sodium pyruvate, 2-Phospho-L-ascorbic acid, and buffers, such as HEPES, and antibiotics such as penicillin and streptomycin. Suitable basal media include Iscove’s Modified Dulbecco’s Medium (IMDM), Ham’s F12, (Advanced) Dulbecco’s modified eagle medium (DMEM) or DMEM / F12, Williams E medium, or RPMI1640. For example, the basal medium may be supplemented with 1-4% (v / v) GlutaMax, 1-4% (v / v) Non-Essential Amino Acids, 1% (v / v) Penicillin-Streptomycin, 10-20 mM HEPES, 1-10 mM Sodium Pyruvate, 100-1,000 pM 2- Phospho-L-ascorbic acid trisodium salt, 1-5% (v / v) B27, 1-5 mM N-acetylcysteine. Preferably HSM is comprised of Advanced DMEM / F12, supplemented with 1-4% (v / v) GlutaMax, 1-4% (v / v) Non-Essential Amino Acids, 1% (v / v) Penicillin-Streptomycin, 10-20 mM HEPES, 1-10 mM Sodium Pyruvate, 100-1,000 pM 2-Phospho-L-ascorbic acid trisodium salt, 1-5% (v / v) B27, 1-5 mM N-acetylcysteine, 1-100 ng / mL EGF, 1-100 ng / mL HGF, 100-500 ng / mL R-spondin 3, 1-20 pM Y-27632, 1-50 pM Hydrocortisone, and 100-1,000 ng / mL Wnt3a or 0.1-10 nM Wnt surrogate molecule. Preferably HOM is comprised of Advanced DMEM / F12, supplemented with 1-4% (v / v) GlutaMax, 1-4% (v / v) Non-Essential Amino Acids, 1% (v / v) Penicillin-Streptomycin, 10-20 mM HEPES, 1-10 mM Sodium Pyruvate, 100-1,000 pM 2-Phospho-L-ascorbic acid trisodium salt, 1- 5% (v / v) B27, 1-20 mM Nicotinamide, 1-5 mM N-acetylcysteine, 1-1,000 nM gastrin, 1-100 ng / mL EGF, 1-100 ng / mL HGF, 100-500 ng / mL RPSO3, 1-100 ng / mL Amphiregulin, 1-50 pM Y- 27632, 1-5 pM A-83-01 and a canonical Wnt activator such as 100-1,000 ng / mL Wnt3a or 0.1-10 nM Wnt surrogate.

[0047] The present invention, according to a second aspect, relates to a cell culture system for the in vitro mass generation and expansion of functional adult primary human hepatocytes (PHH) and / or formation of hepatocyte organoid, wherein the system is comprised of a microwell cell culture system comprising PHH, a hepatocyte spheroid medium (HSM), and / or a hepatocyte organoid medium (HOM) as defined above. The cell culture system is suitable for mass generation of hepatocyte spheroids and the system is preferably comprised of ultralow attachment (ULA) micro wells.

[0048] The present invention, according to a further aspect, relates to a use of this cell culture system for mass generation and expansion of functional adult primary human hepatocytes (PHH) and / or formation of hepatocyte organoids. The present invention and the system for mass generation and expansion of functional adult PHH can be used for engineering an artificial liver model or an artificial liver organ, use for assessing in vitro the liver toxicity, in particular liver genotoxicity, and / or the effects of a drug or a compound.

[0049] The present invention will be further detailed in the following examples and figures wherein:

[0050] Figure 1: shows adult cryopreserved human hepatocytes (PHH) seeded as single cells in

[0051] ULA microwells and cultured in HSM (day 0), Figure 1A. After 4 days PHH have aggregated, forming stable hepatocyte spheroids, which are substantial uniform in shape and about 50 to 250 pm in diameter, Figure IB. Hepatocyte spheroids are then transferred to scaffolding matrix and cultured in HOM (day 0), Figure 1C. Hepatocytes undergo expansion and morphogenesis over time (day 6), Figure ID.

[0052] Figure 2: shows healthy functional hepatocyte organoids obtained according to present invention and shows the expression of mature functional hepatocyte markers by Immunofluorescence (IF) staining. Figure 2A shows IF staining in hepatocyte organoids, wherein in green CYP3A4 (a marker of mature hepatocytes involved in phase I drug metabolism), in red E-cadherin (marker of epithelial liver cells) and the blue DAPI as nuclear staining. Figure 2B shows IF staining in hepatocyte organoids, wherein in green HNF4a (a marker of hepatocytes), in red Albumin (a marker of mature hepatocytes) and the blue DAPI as nuclear staining.

[0053] Figure 3: shows single adult hepatocytes seeded directly in Matrigel without prior aggregation on day 0 (Figure 3A) and day 6 in HSM (Figure 3B), day 6 in media from Hu et al., 2018 (Figure 3C) and day 6 in HOM (Figure 3D). Examples

[0054] Isolation of primary adult human hepatocytes

[0055] Primary human hepatocytes were isolated from liver surgical resections after informed consent. Hepatocytes were isolated by two-step collagenase perfusion and filtered through a 70 pM cell strainer.

[0056] Generation of hepatocyte spheroids

[0057] Fresh or cryopreserved adult primary human hepatocytes were resuspended in hepatocyte spheroid medium (HSM). HSM is comprised of Advanced DMEM / F12, supplemented with 1% (v / v) GlutaMax, 1% (v / v) Non-Essential Amino Acids, 1% (v / v) Penicillin-Streptomycin, 10 mM HEPES (all Thermo), 4 mM Sodium Pyruvate (Sigma-Aldrich), 440 pM 2-Phospho-L-ascorbic acid trisodium salt (Sigma), 2% (v / v) B27 (Gibco), 1.5 mM N-acetylcysteine (Sigma- Aldrich), 4% (v / v) FBS (Sigma), 10 ng / mL EGF (Peprotech), 5 ng / mL HGF (Peprotech), 250 ng / mL R-spondin 3 (Peprotech), 10 pM Y-27632 (Selleckchem), 10 pM Hydrocortisone (STEMCELL Tech.), and 1 pg / mL Wnt3a (R&D Systems).

[0058] Cells were plated in ultra-low attachment microwell cavities at a density of ~20 cells per microwell in HSM and allowed to form spheroids over about 96 hours. To refresh medium, 100% (v / v) fresh HSM was added to each well after 48 hours of culture.

[0059] Organoid culture of adult primary human hepatocytes

[0060] Hepatocyte spheroids generated in HSM were collected and centrifuged at 100g for 5 minutes. The supernatant was removed, and spheroids were resuspended in Matrigel solution obtained by mixing Matrigel (Corning) with advanced DMEM / F12. Hepatocyte spheroids were plated at a density of about 200 spheroids per 50 pL Matrigel droplet. Following polymerization of Matrigel to stable hydrogel (20-30 minutes), HOM was added to each well.

[0061] HOM consists of Advanced DMEM / F12, supplemented with 1% (v / v) GlutaMax, 1% (v / v) Non-Essential Amino Acids, 1% (v / v) Penicillin-Streptomycin, 10 mM HEPES (all Thermo), 4 mM Sodium Pyruvate (Sigma-Aldrich), 440 pM 2-Phospho-L-ascorbic acid trisodium salt (Sigma), 10 nM gastrin (Sigma), 2% (v / v) B27 (Gibco), 1.5 mM N-acetylcysteine (Sigma- Aldrich), 50 ng / mL EGF (Peprotech), 25 ng / mL HGF (Peprotech), 250 ng / mL R-spondin 3 (Qkine), 2pM A83-01, 10 pM Y-27632 (Selleckchem), 1 pg / mL Wnt3a (R&D Systems), and 10 mM NAM.

[0062] Medium was refreshed every 2-3 days throughout the culture period. Figure 1A shows the PHH seeded as single cells in ULA and cultured in HSM. Figure IB shows PHH on day 4 of culture in HSM after stable formation of spheroids. Figure 1C shows the PHH spheroids 0 days after being transferred to Matrigel. Figure ID shows PHH spheroids 6 days after being transferred to Matrigel, showing significant expansion as hepatocyte organoids. After Hepatocyte organoids (HOs) were passaged every 8-12 days at a split ratio of 1:2- 1:3. For maturation of HOs 10 pM Hydrocortisone and / or 50 ng / mL FGF19 was added to HOM. Next, single adult hepatocytes (as a control) were seeded in Matrigel without a prior aggregation step (in contrast to the method of present invention) and supplemented with either HSM (Figure 3B), HOM (Figure 3D) as indicated above, or supplemented with the hepatocyte organoid media composition published by Hu et al., 2018, Cell 175, 1591-1606 (Figure 3D). Cultures were monitored at day 0 (Figure 3 A, with HSM) and 6 for organoid formation. As seen in Figure 3, hepatocytes transferred directly to the scaffolding matrix without prior aggregation form few organoids with limited expansion and morphogenesis. In contrast, when single hepatocytes are first aggregated in HSM, followed by transfer to Matrigel and culture in HOM in the method according to present invention, hepatocytes form organoids which proliferate and undergo morphogenesis as they invade the surrounding matrix, as observed in Figure 1.

Claims

Claims1. A method for the in vitro production of functional and proliferative hepatocyte organoids from primary human hepatocytes (PHH), comprising the steps of; a) aggregating primary hepatocyte cells in cell culture in a hepatocyte spheroid medium (HSM) to generate functional and proliferative hepatocyte spheroids, wherein HSM is a hepatocyte culture media comprised of 0.5 to 100 ng / ml, preferably 1 to 50 ng / mL of at least one epidermal growth factor receptor (EGFR) agonist, 1 to 50 ng / mL of hepatocyte growth factor (HGF) or isoform thereof, and 0.1 ng / mL to 2 pg / mL of at least one canonical Wnt activator, b) collecting the hepatocyte spheroids and transfer of the hepatocyte spheroids to a scaffold matrix, c) culturing of the hepatocyte spheroids transferred in the scaffold matrix in a hepatocyte organoid medium (HOM) to generate a hepatocyte organoid comprised of expanding functional PHH, wherein HOM is a hepatocyte culture media comprised of 0.5 to 100 ng / ml, preferably 1 to 50 ng / mL of at least one epidermal growth factor receptor (EGFR) agonist, 1 to 50 ng / mL of at least one hepatocyte growth factor (HGF) or isoform thereof, 0.1 ng / mL to 2 pg / mL of at least one canonical Wnt activator, 50 to 500 ng / mL of at least one R-spondin protein, 0.1 to 5 pM of at least one transforming growth factor (TGF- ) inhibitor, 1 to 20 pM of at least one ROCK inhibitor, and 1 to 20 mM of Nicotinamide (NAM).

2. Method according to claim 1, wherein the EGFR agonist is one or more selected from the group consisting of Amphiregulin (AREG), epidermal growth factor (EGF), Heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-alpha (TGFa), or related EGFR ligand, preferably AREG and / or EGF.

3. Method according to claim 1 or 2, wherein the at least one canonical Wnt activator is selected from the group consisting of Wnt surrogate molecule or Wnt protein, Wnt3a, Wnt2, Wnt9b, WntlOb, CHIR99021, preferably Wnt surrogate molecule or Wnt3a.

4. Method according to any one of claim 1 to 3, wherein the at least one canonical Wnt activator comprises 0.1 to 10 pg / mL, preferably 0.1 to 2 pg / mL, more preferably 0.2 to 1.5 pg / mL, more preferably 0.5 to 1 pg / mL of Wnt protein.

5. Method according to any one of claim 1 to 3, wherein the at least one canonical Wnt activator comprises 0.1 to 100 ng / mL, preferably 10 to 80 ng / mL, more preferably 20 to 60 ng / mL of Wnt surrogate molecule.

6. Method according to any one of claim 1 to 5, wherein the TGF-P inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947, or related ALK4-, ALK5-, ALK7- inhibitor, preferably A83-01.

7. Method according to any one of claim 1 to 6, wherein the hepatocytes are primary human hepatocytes selected from the group consisting of adult, foetal, neonatal or paediatric primary human hepatocytes, preferably adult.

8. Method according to any one of claim 1 to 7, wherein the scaffold matrix is a laminin- rich extracellular matrix scaffold matrix, preferably Matrigel, Laminin-Entactin Complex, Ultimatrix, Cultrex, or other basement membrane extract.

9. Method according to any one of claim 1 to 8, wherein the hepatocyte spheroids are transferred to the scaffold matrix at a density of 1 to 1000 hepatocyte spheroids, preferably 100 to 500 hepatocyte spheroids, per 25 to 50 pL scaffold matrix.

10. Method according to any one of claim 1 to 9, wherein the method further comprises the step of d) subculturing of the hepatocyte organoid expanded in scaffold matrix with HOM to a fresh scaffold matrix every 1 to 60 days, preferably every 5 to 20 days, more preferably every 10 to 15 days.

11. Method according to any one of claim 1 to 10, wherein the HSM further comprises 50 to 500 ng / mL of at least one R-spondin protein.

12. Method according to any one of claim 1 to 11, wherein the R-spondin protein is selected from the group consisting of R-spondin-3, R-spondin-2, R-spondin-1, or other protein family member of the R-spondin protein family, preferably R-spondin-3.

13. Method according to any one of the claims 1 to 12, wherein the HSM further comprises 1 to 20 pM of a ROCK inhibitor.

14. Method according to any one of the claims 1 to 13, wherein the ROCK inhibitor is selected from the group consisting of Y-27632 (trans-4-[(l R)-l-Aminoethyl]-N-4- pyridinylcyclohexanecarboxamide), fasudil, Y39983 (4-[(lR)-l- Aminoethyl]-A / -l / - / -pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), and azabenzimidazole-aminofurazans, preferably Y- 27632.

15. Method according to any one of claim 1 to 14, wherein the HSM and / or HOM further comprise 0.1 to 50 pM of a glucocorticoid.

16. Method according to claim 15, wherein the glucocorticoid is selected from the group consisting of hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinolone acetonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, and beclomethasone, preferably hydrocortisone or dexamethasone, preferably 1 to 50 pM hydrocortisone or 0.1 to 10 pM dexamethasone.

17. Method according to any one of claim 1 to 16, wherein the aggregation of functional primary hepatocyte cells in HSM is for about 1 to 30 days, preferably for about 3 to 10 days, more preferably for about 4 to 6 days.

18. Method according to any one of claim 1 to 17, wherein the culturing of the hepatocyte spheroids transferred in the scaffold matrix in HOM is for about 1 to 60 days, preferably about 5 to 20 days, more preferably about 10 to 15 days.

19. Method according to any one of claim 1 to 18, wherein the hepatocytes into a microwell cell culture system are provided at a density of 5 to 1500 cells / microwell, preferably 50 to 100 cell / microwell.

20. Method according to any one of claim 1 to 19, wherein the collected hepatocyte spheroids of step c) are on average 10 to 1000 pm in diameter, preferably 50 to 500 pm in diameter, more preferably 100 to 250 pm in diameter, as determined by bright field microscopy.

21. Method according to any one of claim 1 to 20, wherein the HOM further comprises 1 to 1000 nM of Gastrin.

22. Method according to any one of claim 1 to 21, wherein the HOM further comprises 0.1 to 100 ng / mL of fibroblast growth factor 19 (FGF19).

23. Method according to any one of claim 1 to 22, wherein the HOM further comprises 1 to 100 ng / mL of tumor necrosis factor alpha (TNFa).

24. Method according to any one of claim 1 to 23, wherein the HSM and / or HOM further comprises 1 to 5 mM of N-acetylcysteine (NAC).

25. Method according to any one of claim 1 to 24, wherein the HSM and / or HOM further comprises 0.1 to 10% v / v foetal calf serum (FCS) or foetal bovine serum (FBS).

26. Method according to any one of claim 1 to 25, wherein at least 50%, more preferably at least 65%, even more preferably at least 85%, most preferably at least 90% of the hepatocytes present at the start of cell culturing contribute to the organoid formation.

27. Cell culture system for the in vitro mass generation and expansion of functional adult primary human hepatocytes (PHH) and / or formation of hepatocyte organoid, wherein the system is comprised of a microwell cell culture system comprising PHH, a hepatocyte spheroid medium (HSM), and / or a hepatocyte organoid medium (HOM) as defined in any of the claims 1 to 26.

28. Use of a cell culture system according to claim 27 for mass generation and expansion of functional adult primary human hepatocytes (PHH) and / or formation of hepatocyte organoids.

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