Method for in vitro production of hepatocellular organoids

Abstract

The present invention relates to methods for the in vitro production of hepatocellular organoids from primary human hepatocytes (PHHs). The invention also relates to a cell culture system for the in vitro expansion of functional fetal, neonatal, pediatric and adult PHHs and the mass production of organoids, and to the use of said cell culture system for the in vitro mass production and expansion of functional adult PHHs and / or the formation of hepatocellular organoids.

Description

[0001] describe

[0002] The present invention relates to methods for generating hepatocyte organoids from primary human hepatocytes (PHH) in vitro. The present invention also relates to cell culture systems and uses thereof for in vitro expansion of functional fetal, neonatal, pediatric, and adult primary human hepatocytes (PHH) and for the mass production of organoids.

[0003] As research into tissue engineering and regenerative medicine increases, the rapid generation of cells and a large amount of cells are needed. The latest and emerging technology is to amplify cells in large quantities by generating organoids. Organoids are miniature organs produced in vitro 3D, which are similar to their source organs in terms of cell type and function, and can be established by a variety of organs and species. Organoids are derived from primary (stem) cells, embryonic stem cells or induced pluripotent stem cells, which are self-organized in three-dimensional culture. The use of cell culture technology to produce organoids begins with the transition to culturing cells under 3D culture conditions to allow the development of complex organ 3D structures. Therefore, organoids are promising: not only for regenerative medicine applications, but also as an in vitro model for studying organ development, regeneration and disease. Organoids are also used for research (personalized) treatment in the laboratory, i.e., as an in vitro model very similar to organs / cells in patients, for example, for testing the efficacy or toxicity of future treatments.

[0004] The liver is an important organ that performs many important processes, such as blood detoxification and serum protein production. Hepatocytes are epithelial cells responsible for performing most of these key functions, constituting 70% to 80% of the liver. In vitro, adult primary human hepatocytes (PHH) are the gold standard for toxicology studies and have the potential to treat patients with liver disease. However, PHH have short-term function and limited expansion potential in vitro. Although significant progress has been made in the long-term maintenance of adult PHH, the effective expansion of these cells in a functional state has not yet been reported. This is a major obstacle and limits the use of PHH.

[0005] The current cell culture system used to generate hepatocyte organoids from adult primary human hepatocytes (PHH) has a plating efficiency of less than 1%, meaning that 99% of the plated hepatocytes do not participate in organoid generation. This system has major reproducibility issues, does not provide immunohistochemistry data, and does not measure proliferation over multiple passages. Finally, the low plating efficiency and poor expansion potential of existing systems make in vitro studies difficult and regenerative medicine applications impractical.

[0006] In view of the above, there is a need in the art for methods for expanding functional primary human hepatocytes. Such methods should provide for the reproducible expansion of functional adult primary human hepatocytes, preferably into organoids, thereby enabling long-term in vitro studies of (circulating) adult PHH and applications in regenerative medicine. Furthermore, there is a need in the art for cell culture systems suitable for such methods.

[0007] It is an object of the present invention, among others, to address the above-mentioned needs in the art.The objects of the present invention, among others, are met by the invention as outlined in the accompanying claims.

[0008] In particular, the above objects, among others, are met according to a first aspect of the present invention by a method for generating functional and proliferative hepatocyte organoids in vitro from primary human hepatocytes (PHH), said method comprising the following steps:

[0009] a) Aggregating primary hepatocytes in cell culture in hepatocyte spheroid medium (HSM) to generate functional and proliferative hepatocyte spheroids, wherein HSM is a hepatocyte culture medium composed of:

[0010] 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,

[0011] 1 to 50 ng / mL, preferably 5 to 40 ng / mL, more preferably 10 to 25 ng / mL hepatocyte growth factor (HGF) or its isoform, and

[0012] 0.1 ng / mL to 2 μg / mL of at least one canonical Wnt activator, such as a Wnt protein or a Wnt surrogate molecule,

[0013] b) collecting the hepatocyte spheroids and transferring the hepatocyte spheroids to a scaffold matrix,

[0014] c) culturing the hepatocyte spheroids transferred into the scaffold matrix in hepatocyte organoid medium (HOM) to provide hepatocyte organoids composed of expanded functional PHHs, wherein HOM is a hepatocyte culture medium composed of:

[0015] 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,

[0016] 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 an isoform thereof,

[0017] 0.1 ng / mL to 2 μg / mL of at least one canonical Wnt activator, such as a Wnt protein or a Wnt surrogate molecule,

[0018] 50 to 500 ng / mL, preferably 100 to 300 ng / mL, more preferably 150 to 250 ng / mL of at least one R-spondin,

[0019] 0.1 to 5 μM, preferably 0.5 to 3 μM, more preferably 1 to 2.5 μM of at least one transforming growth factor β (TGF-β) inhibitor,

[0020] 1 to 20 μM, preferably 2.5 to 15 μM, more preferably 5 to 10 μM of at least one ROCK inhibitor, and

[0021] 1 to 20 mM, preferably 2.5 to 15 mM, more preferably 5 to 10 mM nicotinamide (NAM).

[0022] Cell aggregation is preferably carried out in a cell culture system such as a microplate, preferably an ultra-low attachment microplate. As mentioned herein, the PHH "spheroids" of aggregation are 3D hepatocyte cell aggregates formed in the absence of a predefined culture substrate to adhere to. Therefore, hepatocytes do not adhere or adhere to the surface of the culture substrate or system. The technology for generating spheroids includes hanging drops and ultra-low attachment culture, which promote cell-cell interactions while preventing cell-substrate / matrix interactions. Therefore, cells interact to form tight spheroids, but other shapes are also possible. Fresh or frozen adult (or fetus, neonate or child) primary human hepatocytes (PHH) are aggregated, for example, in ultra-low attachment (ULA) microwells, and are formed into spheroids in a defined hepatocyte spheroid culture medium (HSM).

[0023] The methods of the present invention preferably provide hepatocyte organoids comprising 0.1% to 20%, preferably 2% to 17.5%, more preferably 5% to 15%, and most preferably 10% to 12.5% ​​non-parenchymal liver cells (NPCs), such as stellate cells, hepatic endothelial cells, and / or Kupfer cells. The presence of these NPCs increases the function of the hepatic organoids and provides further stimulation for the cells and their function in the entire organoid. NPCs produce a variety of growth factors that are important in the development and function of hepatic organoids.

[0024] As used herein, "functional" hepatocytes in cell culture refer to hepatocytes that retain the key features and functions of hepatocytes in vivo. These include polarized expression of key drug transporters (e.g., MRP2 and / or BSEP), production and secretion of albumin, production and transport of bile, uptake of low-density lipoprotein, synthesis and storage of glycogen, biotransformation of xenobiotics by phase I and / or phase II drug metabolism. In addition, functional hepatocytes should remain sensitive to infection by hepatocyte-specific pathogens (e.g., malaria and HBV). Importantly, transcriptomic data are not sufficient to indicate functional hepatocytes in vitro. Polarized protein expression and activity must be shown by a variety of techniques well known in the art. Protein expression and localization can be shown, for example, by immunolocalization or immunohistochemistry. Protein function can be assessed using a variety of commercially available kits, such as albumin and CYP ELISA kits that measure the secretion of albumin and the biotransformation by CYP enzymes, respectively.

[0025] As used herein, "proliferating" or "expanding" hepatocytes refers to hepatocytes that: incorporate nucleoside analogs such as BrdU (5-bromo-2'-deoxyuridine) or EdU (5-ethynyl-2'-deoxyuridine) into newly synthesized DNA; express nuclear proteins specific for proliferating cells, such as Ki67 and PCNA; and express cell cycle proteins, such as cyclin D1 and Cdk2. Methods for assessing hepatocyte proliferation are well known in the art and include, but are not limited to, immunofluorescence detection of proliferation proteins (i.e., Ki67, PCNA, cyclin D1), detection of incorporated nucleoside analogs (i.e., BrdU, EdU), and / or by cell counting or alternatives to cell counting (e.g., CellTiter- Luminescent cell viability assay) to generate growth curves.

[0026] After forming stable spheroids, the PHHs are transferred to a suitable scaffold matrix, such as Matrigel, and cultured in a defined hepatocyte organoid medium (HOM). Once transferred to the scaffold matrix (e.g., Matrigel), the PHH spheroids begin to reorganize, proliferate, and invade the surrounding extracellular matrix. The method of the present invention allows for the reproducible and scalable expansion of functional adult primary human hepatocytes into organoids, enabling in vitro studies and regenerative medicine applications of (circulating) adult PHHs. The method uses primary hepatocytes to aggregate into spheroids and allows for the mass generation of primary human hepatocyte organoids starting from the first week of culture. The present invention can use ULA microplates or equivalents, which allow for the reproducible and controlled production of thousands of spheroids. The expansion potential of adult PHH using the solution of the present invention is far superior to that of any known method and allows for the passage and culture of the expanded hepatocytes. The method of the present invention has a wide range of commercial applications from regenerative medicine to in vitro toxicology studies, i.e., the pharmaceutical industry.

[0027] HSM and HOM are composed of hepatocyte growth factor (HGF) or related isoforms (NK1, NK2), which act as c-Met agonists. Activation of c-Met by HGF controls a variety of processes, including development, proliferation, and branching morphogenesis.

[0028] According to a preferred embodiment, the present invention relates to a 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 (TGFα), or related EGFR ligands, preferably AREG and / or EGF. Activation of EGFR induces a variety of intracellular processes, including proliferation and differentiation.

[0029] According to a preferred embodiment, the present invention relates to a method wherein the at least one classical Wnt activator is selected from Wnt replacement molecules or Wnt proteins, such as Wnt3a, Wnt2, Wnt9b, Wnt10b, CHIR99021, Wnt ligands, glycogen synthase kinase 3b inhibitors, b-catenin and activators of b-catenin, preferably Wnt replacement molecules or Wnt3a.

[0030] According to another preferred embodiment, the present invention relates to a method wherein the at least one canonical Wnt activator comprises 0.1 to 10 μg / mL, preferably 0.1 to 2 μg / mL, more preferably 0.2 to 1.5 μg / mL, more preferably 0.5 to 1 μg / mL Wnt protein.

[0031] According to yet another preferred embodiment, the present invention relates to a 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 the Wnt surrogate molecule.

[0032] According to a preferred embodiment, the present invention relates to a method wherein the TGF-β inhibitor is selected from A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947 or related ALK4 inhibitors, ALK5 inhibitors, ALK7 inhibitors, preferably A83-01. TGF inhibitors are compounds that reduce, block or inhibit TGF through TGF RI and TGF RII receptors.

[0033] According to another preferred embodiment, the present invention relates to a method wherein the hepatocytes are primary human hepatocytes selected from the group consisting of adult, fetal, neonatal, or pediatric primary human hepatocytes, preferably adult. The hepatocytes may be primary cells isolated from a sample of immature or mature liver tissue or tumor liver tissue. The liver tissue may be obtained from individuals of gestational age or any age greater than 0 months.

[0034] According to another preferred embodiment, the present invention relates to such a method: wherein the scaffold matrix is ​​an extracellular matrix hydrogel rich in laminin, preferably matrigel, laminin-nidogen complex, Ultimatrix, Cultrex, or other basement membrane extracts and / or synthetic (hydro) gels that allow organoid culture. The hepatocyte spheroids are transferred to a 3D culture and cultured in a 3D culture, wherein the culture medium and cells are mixed with a scaffold matrix, the scaffold matrix is ​​preferably a hydrogel, which supports the growth and proliferation of cells in 3D and allows cells to reorganize and expand into organoids. Preferably, hydrogel is used as a suitable scaffold matrix, but other known natural and synthetic matrices can be used, such as fibrin, collagen, collagen / laminin, a matrix derived from decellularized tissue, compressed collagen alginate, agarose, polyethylene glycol or basement membrane extract. The scaffold matrix can be chemically defined, such as collagen or dense collagen hydrogel, or non-chemically defined, such as a composite protein hydrogel. Preferably, the scaffold matrix in the expansion culture medium is a composite protein hydrogel. Suitable composite protein hydrogels can include extracellular matrix components such as laminin, collagen IV, entactin, and heparin sulfate proteoglycans. Composite protein hydrogels can also include hydrogels of extracellular matrix proteins from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Suitable composite protein hydrogels are available from commercial sources and include Matrigel TM (Corning Life Sciences) or Cultrex TM BME 2RGF (Amsbio TM Inc).

[0035] According to another preferred embodiment, the present invention relates to a 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 μL of scaffold matrix. If too many spheroids are plated per scaffold matrix droplet, i.e., more than 1,000 per 100 μL, they will fuse together, which is undesirable for expansion.

[0036] According to another preferred embodiment, the present invention relates to a method wherein the method further comprises the step of: d) subculturing the hepatocyte organoids expanded in the scaffold matrix with HOM to fresh scaffold matrix every 1 to 60 days, preferably every 5 to 20 days, and more preferably every 10 to 15 days. The hepatocyte organoids are expanded in the scaffold matrix for 1 to 30 days, harvested, and transferred to fresh scaffold matrix at a subculturing ratio of 1:2 to 1:3, and HOM is provided for an additional 1 to 60 days. This process can be repeated as long as the cells continue to expand.

[0037] According to another preferred embodiment, the present invention relates to a method wherein said HSM further comprises 50 to 500 ng / mL of at least one R-spondin.

[0038] According to another preferred embodiment, the present invention relates to a method wherein the R-spondin is selected from R-spondin-3, R-spondin-2, R-spondin-1, or another member of the R-spondin family, preferably R-spondin-3. R-spondin proteins amplify the effects of Wnt proteins and stimulate their expansion. R-spondin-3 is more potent than other proteins and is also produced during liver regeneration. R-spondin proteins are secreted activator proteins that positively regulate both canonical and non-canonical Wnt signaling pathways. Preferably, the R-spondin proteins are human R-spondin proteins. During canonical Wnt activation, R-spondin proteins cooperate with Wnt molecules to amplify Wnt signals.

[0039] According to another preferred embodiment, the present invention relates to a method wherein the HSM further comprises 1 to 20 μM ROCK inhibitor. ROCK inhibition blocks or inhibits Rho kinase (ROCK) activity. ROCK inhibitors prevent cells from entering apoptosis when they are stressed due to a lack of cell-cell contact (e.g., in stem cell cultures).

[0040] According to another preferred embodiment, the present invention relates to a method wherein the ROCK inhibitor is selected from Y-27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridylcyclohexanecarboxamide), fasudil, Y39983 (4-[(1R)-1-aminoethyl]-A / -1 / - / -pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride) and azabenzimidazole-aminofurazan, preferably Y-27632.

[0041] According to another preferred embodiment, the present invention relates to a method wherein the HSM and / or HOM further comprises 0.1 to 50 μM glucocorticoids. Glucocorticoids influence a wide range of processes, including survival, proliferation, and differentiation. The HSM preferably comprises 1 to 50 μM hydrocortisone or 0.1 to 1 μM dexamethasone.

[0042] According to a preferred embodiment, the present invention relates to a 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 μM hydrocortisone or 0.1 to 1 μM dexamethasone.

[0043] According to another preferred embodiment, the present invention relates to a method wherein the aggregation of functional primary hepatocytes in HSM is continued for about 1 to 30 days, preferably about 3 to 10 days, more preferably about 4 to 6 days. 4 to 6 days were found to be ideal for generating functional primary hepatocyte spheroids. Once the aggregates are transferred to a 3D matrix, shorter (less than 2 days) or longer (more than 30 days) times lead to suboptimal results.

[0044] According to another preferred embodiment, the present invention relates to a method wherein the hepatocyte spheroids transferred into the scaffold matrix are cultured in the HOM for about 1 to 30 days, preferably about 5 to 20 days, and more preferably about 8 to 15 days. Since the 3D matrix eventually degrades, the cells are optimally transferred to a new 3D matrix every 5 to 15 days and cultured for another round, as described above.

[0045] According to a preferred embodiment, the present invention relates to a method wherein hepatocytes are provided to the microporous cell culture system at a density of 5 to 1500 cells / microwell, preferably 25 to 250 cells / microwell, more preferably 50 to 100 cells / microwell.

[0046] According to yet another preferred embodiment, the present invention relates to a method wherein the collected hepatocyte spheroids of step c) have an average diameter of 10 to 1000 μm, preferably a diameter of 50 to 500 μm, more preferably a diameter of 100 to 250 μm, as determined by bright field microscopy (i.e. by using a scale bar).

[0047] According to a preferred embodiment, the present invention relates to a method wherein the HOM further comprises 1 to 1000 nM gastrin.Gastrin is added within a specified concentration range to promote the expansion and viability of cells in cell culture.

[0048] According to a preferred embodiment, the present invention relates to a method wherein the HOM further comprises 1 to 100 ng / mL, preferably 10 to 75 ng / mL, more preferably 25 to 50 ng / mL tumor necrosis factor alpha (TNFα). The addition of TNFα to HOM promotes the activation of NF-κB signaling and induces further proliferation of hepatocytes.

[0049] According to a preferred embodiment, the present invention relates to a method wherein the HOM further comprises 1 to 100 ng / mL fibroblast growth factor 19 (FGF19). FGF19 activates fibroblast growth factor receptor 4 (FGFR4) signaling, which regulates metabolic capacity, such as bile acid synthesis.

[0050] According to another preferred embodiment, the present invention relates to a method wherein said HSM and / or HOM further comprises 1 to 5 mM N-acetylcysteine ​​(NAC). According to yet another preferred embodiment, the present invention relates to a method wherein said HSM and / or HOM further comprises 0.1 to 10% v / v fetal calf serum (FCS) or fetal bovine serum (FBS).

[0051] According to another preferred embodiment, the present invention relates to a method in which at least 50%, more preferably at least 65%, even more preferably at least 85%, and most preferably at least 90% of the hepatocytes present at the start of the cell culture contribute to organoid formation. Compared to the method of the present invention, using known and currently used methods for generating organoids, approximately 1% of the cells provided at the start of the culture survive and contribute to organoid formation and generation. The method of the present invention is much superior in this respect, in which at least 50% of the viable cells used at the start of the cell culture contribute to the provision or generation of organoids.

[0052] The methods of the present invention utilize HSM and HOM medium. Both HSM and HOM can be composed of a basal medium that is also supplemented with a medium supplement (e.g., B27 supplement, N21 supplement, N2 supplement, or ITS(+) supplement) to form a nutrient medium. The nutrient medium may also contain L-glutamine or a substitute, such as L-alanyl-L-glutamine (e.g., Glutamax TM), N-acetylcysteine ​​(NAC), MEM non-essential amino acid solution, sodium pyruvate, 2-phospho-L-ascorbic acid and buffer (such as HEPES) and antibiotics (such as penicillin and streptomycin). Suitable basal culture 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 can be supplemented with 1% to 4% (v / v) GlutaMax, 1% to 4% (v / v) non-essential amino acids, 1% (v / v) penicillin-streptomycin, 10 to 20 mM HEPES, 1 to 10 mM sodium pyruvate, 100 to 1,000 μM 2-phospho-L-ascorbic acid trisodium salt, 1% to 5% (v / v) B27, 1 to 5 mM N-acetylcysteine. Preferably, HSM is composed of Advanced DMEM / F12 supplemented with 1% to 4% (v / v) GlutaMax, 1% to 4% (v / v) non-essential amino acids, 1% (v / v) penicillin-streptomycin, 10 to 20 mM HEPES, 1 to 10 mM sodium pyruvate, 100 to 1,000 μM 2-phospho-L-ascorbic acid trisodium salt, 1% to 5% (v / v) B27, 1 to 5 mM N-acetylcysteine, 1 to 100 ng / mL EGF, 1 to 100 ng / mL HGF, 100 to 500 ng / mL R-spondin 3, 1 to 20 μM Y-27632, 1 to 50 μM hydrocortisone, and 100 to 1,000 ng / mL Wnt3a or 0.1 to 10 nM Wnt surrogate molecules.Preferably, HOM consists of Advanced DMEM / F12 supplemented with 1% to 4% (v / v) GlutaMax, 1% to 4% (v / v) non-essential amino acids, 1% (v / v) penicillin-streptomycin, 10 to 20 mM HEPES, 1 to 10 mM sodium pyruvate, 100 to 1,000 μM 2-phospho-L-ascorbic acid trisodium salt, 1% to 5% (v / v) B27, 1 to 20 mM nicotinamide, 1 to 5 mM N-acetylcysteine, 1 to 1,000 nM gastrin, 1 to 100 ng / mL EGF, 1 to 100 ng / mL HGF, 100 to 500 ng / mL RPSO3, 1 to 100 ng / mL amphiregulin, 1 to 50 μM Y-27632, 1 to 5 μM A-83-01, and canonical Wnt activators such as 100 to 1,000 ng / mL Wnt3a or 0.1 to 10 nM Wnt surrogate.

[0053] According to a second aspect, the present invention relates to a cell culture system for the in vitro generation and expansion of functional adult primary human hepatocytes (PHH) and / or the formation of hepatocyte organoids, wherein the system is composed of a microporous cell culture system comprising PHH, hepatocyte spheroid medium (HSM) and / or hepatocyte organoid medium (HOM) as defined above. The cell culture system is suitable for the large-scale generation of hepatocyte spheroids and is preferably composed of ultra-low attachment (ULA) microwells.

[0054] According to another aspect, the present invention relates to the use of the cell culture system for mass production and expansion of functional adult primary human hepatocytes (PHHs) and / or formation of hepatocyte organoids. The present invention and the system for mass production and expansion of functional adult primary human hepatocytes can be used to engineer artificial liver models or artificial liver organs for in vitro assessment of hepatotoxicity, particularly hepatogenotoxicity, and / or the effects of drugs or compounds.

[0055] The present invention will be further described in detail with reference to the following examples and accompanying drawings, in which:

[0056] Figure 1 : Shows adult cryopreserved human hepatocytes (PHH) seeded as single cells in ULA microwells and cultured in HSM (day 0), Figure 1 A. After 4 days, PHHs aggregated to form stable hepatocyte spheroids with a generally uniform shape and a diameter of approximately 50 to 250 μm. Figure 1 B. Hepatocyte spheroids were then transferred to the scaffold matrix and cultured in HOM (day 0). Figure 1 C. Hepatocytes undergo expansion and morphogenesis over time (day 6). Figure 1 D.

[0057] Figure 2 : shows healthy functional hepatocyte organoids obtained according to the present invention, and shows the expression of mature functional hepatocyte markers by immunofluorescence (IF) staining. Figure 2 A shows IF staining in hepatocyte organoids, with green for CYP3A4 (a marker of mature hepatocytes involved in phase I drug metabolism), red for E-cadherin (a marker of epithelial hepatocytes), and blue for DAPI as nuclear staining. Figure 2 B shows IF staining in hepatocyte organoids, where green is HNF4α (a marker for hepatocytes), red is albumin (a marker for mature hepatocytes), and blue DAPI is nuclear staining.

[0058] Figure 3 : Shows the results on day 0 ( Figure 3 A) and on day 6 in HSM ( Figure 3 B), Day 6 in culture medium from Hu et al., 2018 ( Figure 3 C) and on day 6 in HOM ( Figure 3 D) Single adult human hepatocytes seeded directly in Matrigel without prior aggregation. Example

[0059] Isolation of primary adult hepatocytes

[0060] Primary human hepatocytes were isolated from liver surgical resections after informed consent.Hepatocytes were detached by two-step collagenase perfusion and filtered through a 70 μM cell strainer.

[0061] Generation of hepatocyte spheroids

[0062] Fresh or cryopreserved adult primary human hepatocytes are resuspended in Hepatocyte Spheroid Medium (HSM). HSM consisted 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 μM 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 μM Y-27632 (Selleckchem), 10 μM hydrocortisone (STEMCELL Tech.), and 1 μg / mL Wnt3a (R&D Systems).

[0063] Cells were plated in HSM in ultra-low attachment microwell chambers at a density of approximately 20 cells / well and allowed to form spheroids in approximately 96 hours. To refresh the medium, 100% (v / v) fresh HSM was added to each well after 48 hours of culture.

[0064] Organoid Culture of Adult Primary Human Hepatocytes

[0065] The hepatocyte spheroids generated in HSM were collected and centrifuged at 100 g for 5 minutes. The supernatant was removed and the spheroids were resuspended in a matrigel solution obtained by mixing matrigel (Corning) with advanced DMEM / F12. The hepatocyte spheroids were plated at a density of approximately 200 spheroids / 50 μL matrigel droplets. After the matrigel polymerized into a stable hydrogel (20 to 30 minutes), HSM was added to each well.

[0066] HOM consisted 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 μM 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), 2 μM A83-01, 10 μM Y-27632 (Selleckchem), 1 μg / mL Wnt3a (R&D Systems), and 10 mM NAM.

[0067] During the entire culture period, the culture medium was refreshed every 2 to 3 days. Figure 1 A shows PHH seeded as single cells in ULA and cultured in HSM. Figure 1 B shows PHHs after stable spheroid formation on day 4 of culture in HSM. Figure 1 C shows PHH spheroids at day 0 after transfer to Matrigel. Figure 1 D shows the PHH spheroids at 6 days after being transferred to matrigel, which show that they are significantly expanded into hepatocyte organoids. Afterwards, hepatocyte organoids (HO) are passaged at a distribution ratio of 1:2 to 1:3 every 8 to 12 days. In order to make HO mature, 10 μM hydrocortisone and / or 50 ng / mL FGF19 are added to HOM.

[0068] Next, single adult human hepatocytes (as a control) were seeded in Matrigel without a prior aggregation step (in contrast to the method of the present invention) and supplemented with HSM as described above ( Figure 3 B)HOM( Figure 3 D), or supplemented with the hepatocyte organoid culture medium composition published by Hu et al., 2018, Cell 175, 1591–1606 ( Figure 3 D). On day 0 ( Figure 3 A, in the presence of HSM) and monitoring cultures for organoid formation on day 6. Figure 3As seen in Figure 2, direct transfer of hepatocytes into a scaffold matrix without prior aggregation formed very few organoids with limited expansion and morphogenesis. In contrast, in the method according to the present invention, when single hepatocytes were first aggregated in HSM and subsequently transferred to Matrigel and cultured in HOM, the hepatocytes formed organoids that proliferated and underwent morphogenesis while invading the surrounding matrix, as shown in Figure 2. Figure 1 As observed in .

Claims

1. A method for generating functional and proliferative hepatocyte organoids in vitro from primary human hepatocytes (PHH), comprising the following steps: a) aggregating primary hepatocytes in cell culture in hepatocyte spheroid medium (HSM) to generate functional and proliferative hepatocyte spheroids, wherein HSM is a hepatocyte culture medium composed 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 hepatocyte growth factor (HGF) or an isoform thereof, and 0.1 ng / mL to 2 μg / mL of at least one canonical Wnt activator, b) collecting the hepatocyte spheroids and transferring the hepatocyte spheroids to a scaffold matrix, c) culturing the hepatocyte spheroids transferred into the scaffold matrix in hepatocyte organoid medium (HOM) to generate hepatocyte organoids composed of expanded functional PHHs, wherein HOM is a hepatocyte culture medium composed 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 its isoform, 0.1 ng / mL to 2 μg / mL of at least one canonical Wnt activator, 50 to 500 ng / mL of at least one R-spondin, 0.1 to 5 μM of at least one transforming growth factor beta (TGF-β) inhibitor, 1 to 20 μM of at least one ROCK inhibitor, and 1 to 20 mM nicotinamide (NAM).

2. The 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 (TGFα) or related EGFR ligands, preferably AREG and / or EGF.

3. The method according to claim 1 or 2, wherein the at least one canonical Wnt activator is selected from Wnt replacement molecules or Wnt proteins Wnt3a, Wnt2, Wnt9b, Wnt10b, CHIR99021, preferably Wnt replacement molecules or Wnt3a. 4 . The method according to claim 1 , wherein the at least one canonical Wnt activator comprises 0.1 to 10 μg / mL, preferably 0.1 to 2 μg / mL, more preferably 0.2 to 1.5 μg / mL, more preferably 0.5 to 1 μg / mL Wnt protein. 5 . The method according to any one of claims 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 Wnt surrogate molecule.

6. The method according to any one of claims 1 to 5, wherein the TGF-β inhibitor is selected from A83-01, SB431542, RepSox, SB505124, Galunisertib, LY364947 or related ALK4 inhibitors, ALK5 inhibitors, ALK7 inhibitors, preferably A83-01.

7. The method according to any one of claims 1 to 6, wherein the hepatocytes are primary human hepatocytes selected from the group consisting of adult, fetal, neonatal or pediatric primary human hepatocytes, preferably adult.

8. The method according to any one of claims 1 to 7, wherein the scaffold matrix is ​​a laminin-rich extracellular matrix scaffold matrix, preferably Matrigel, laminin-nidogen complex, Ultimatrix, Cultrex or other basement membrane extracts.

9. The method according to any one of claims 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 μL of scaffold matrix.

10. The method according to any one of claims 1 to 9, wherein the method further comprises the following steps: d) subculturing the hepatocyte organoids expanded in the scaffold matrix to fresh scaffold matrix using HOM every 1 to 60 days, preferably every 5 to 20 days, more preferably every 10 to 15 days.

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

12. The method according to any one of claims 1 to 11, wherein the R-spondin is selected from R-spondin-3, R-spondin-2, R-spondin-1 or other protein family members of the R-spondin family, preferably R-spondin-3.

13. The method of any one of claims 1 to 12, wherein the HSM further comprises 1 to 20 μM ROCK inhibitor.

14. The method according to any one of claims 1 to 13, wherein the ROCK inhibitor is selected from Y-27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridylcyclohexanecarboxamide), fasudil, Y39983 (4-[(1R)-1-aminoethyl]-A / -1 / - / -pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), and azabenzimidazole-aminofurazan, preferably Y-27632.

15. according to the method described in any one of claim 1 to 14, wherein said HSM and / or HOM also comprise 0.1 to 50 μ M glucocorticoid.

16. method according to claim 15, wherein said glucocorticoid is selected from hydrocortisone, dexamethasone, cortisone, prednisone, prednisolone, methylprednisolone, betamethasone, triamcinolone, triamcinolone acetonide, fluocinonide, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone and beclomethasone, preferably hydrocortisone or dexamethasone, preferably 1 to 50 μM hydrocortisone or 0.1 to 10 μM dexamethasone.

17. The method according to any one of claims 1 to 16, wherein aggregation of functional primary hepatocytes in HSM lasts for about 1 to 30 days, preferably for about 3 to 10 days, more preferably for about 4 to 6 days.

18. The method according to any one of claims 1 to 17, wherein the hepatocyte spheroids transferred into the scaffold matrix are cultured in HOM for about 1 to 60 days, preferably about 5 to 20 days, more preferably about 10 to 15 days.

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

20. The method according to any one of claims 1 to 19, wherein the hepatocyte spheroids collected in step c) have an average diameter of 10 to 1000 μm, preferably 50 to 500 μm, more preferably 100 to 250 μm, as determined by bright field microscopy.

21. The method of any one of claims 1 to 20, wherein the HOM further comprises 1 to 1000 nM gastrin.

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

23. The method of any one of claims 1 to 22, wherein the HOM further comprises 1 to 100 ng / mL tumor necrosis factor alpha (TNFα).

24. The method of any one of claims 1 to 23, wherein the HSM and / or HOM further comprises 1 to 5 mM N-acetylcysteine ​​(NAC).

25. The method of any one of claims 1 to 24, wherein the HSM and / or HOM further comprises 0.1 to 10% v / v fetal calf serum (FCS) or fetal bovine serum (FBS).

26. The method according to any one of claims 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 culture contribute to the organoid formation.

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

28. Use of the cell culture system according to claim 27 for generating and expanding functional adult primary human hepatocytes (PHH) in large quantities and / or forming hepatocyte organoids.

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