Methods for in vitro production of hepatocyte organoids

Abstract

The present invention relates to a method for the in vitro production of hepatocyte organoids from primary human hepatocytes (PHHs).The invention further relates to a cell culture system for the in vitro expansion of functional fetal, neonatal, pediatric and adult PHHs and for the generation of populations of organoids, and to the use of the cell culture system for the in vitro generation and expansion of populations of functional adult PHHs and / or for the formation of hepatocyte organoids.

Description

[Technical Field]

[0001] The present invention relates to a method for the in vitro production of hepatocyte organoids from primary human hepatocytes (PHH).The present invention further relates to a cell culture system and its use for the in vitro expansion of functional fetal, neonatal, pediatric and adult primary human hepatocytes (PHH) and the mass generation of organoids. [Background technology]

[0002] Advances in research into tissue engineering and regenerative medicine require rapid cell production and large quantities of cells. A more recent emerging technology involves expanding cell populations through the generation of organoids. Organoids are three-dimensional in vitro mini-organs that resemble their parent organs in terms of 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 and self-organize in three-dimensional culture. The production of organoids using cell culture techniques began with a shift from culturing cells in three-dimensional culture conditions, allowing the complex three-dimensional structure of the organ to develop. Thus, organoids are promising not only for regenerative medicine applications, but also as in vitro models for studying organ development, regeneration, and disease. Organoids can also be used to study (individualized) treatments in the laboratory, i.e., as in vitro models closely resembling in vivo organs / cells in patients, for example, to test the efficacy or toxicity of future therapies.

[0003] The liver is a vital organ that performs many essential processes, such as blood detoxification and serum protein production. Hepatocytes, the epithelial cells responsible for most of these vital functions, comprise 70–80% of the liver. In vitro, adult primary human hepatocytes (PHHs) are the gold standard for toxicity studies and have the potential to treat patients with liver disease. However, PHHs only have short-term functionality in vitro and have limited expansion potential. While significant progress has been made in the long-term maintenance of adult PHHs, efficient expansion of these cells in a functional state has yet to be reported. This represents a major obstacle, limiting the use of PHHs.

[0004] Current cell culture systems for generating hepatic organoids from adult primary human hepatocytes (PHH) have a plating efficiency of less than 1%, meaning that 99% of plated hepatocytes do not participate in organoid formation. This system has significant problems with reproducibility, does not provide immunohistochemistry data, and does not allow for measuring proliferation over several passages. Ultimately, the low plating efficiency and insufficient expansion of the current system make in vitro studies difficult to perform and impractical for regenerative medicine applications.

[0005] In view of the above, what is needed in the art is a method for expanding functional primary human hepatocytes.This method can provide the reproducible expansion of functional adult primary human hepatocytes, preferably as organoid, and can be used for the long-term in vitro study of (circulating) adult PHH and for regenerative medicine applications.Furthermore, what is needed in the art is a cell culture system suitable for this method. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Hu et al., 2018, Cell 175, pp. 1591-1606 Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention, inter alia, to address the above-mentioned needs in the art. The object of the present invention is achieved, inter alia, by the invention as outlined in the appended claims. [Means for solving the problem]

[0008] In particular, the above objects, among others, are achieved according to a first aspect of the present invention, which provides a method for the in vitro production of functional and proliferative hepatocyte organoids from primary human hepatocytes (PHH), comprising: a) aggregating primary hepatocytes in hepatocyte spheroid medium (HSM) in cell culture to generate functional and proliferative hepatocyte spheroids, wherein HSM comprises: 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, and most preferably 10 to 25 ng / ml of at least one epidermal growth factor receptor (EGFR) agonist; 1 to 50 ng / mL, preferably 5 to 40 ng / mL, more preferably 10 to 25 ng / mL of hepatocyte growth factor (HGF) or an isoform thereof, and 0.1 ng / mL to 2 μg / mL of at least one canonical Wnt activator, e.g., a Wnt protein or a Wnt surrogate molecule a hepatocyte culture medium comprising: b) harvesting the hepatocyte spheroids and transferring the hepatocyte spheroids to a scaffold matrix; c) Culturing the hepatocyte spheroids transferred to the scaffold matrix in hepatocyte organoid medium (HOM) to provide hepatocyte organoids containing expanding functional PHHs, wherein HOM is 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, and most preferably 10 to 25 ng / ml of at least one epidermal growth factor receptor (EGFR) agonist; 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 its isoform; 0.1 ng / mL to 2 μg / mL of at least one standard Wnt activator, e.g., a Wnt protein or a Wnt surrogate molecule; 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 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; 1 to 20 μM, preferably 2.5 to 15 μM, more preferably 5 to 10 μM, of at least one ROCK inhibitor; and 1 to 20 mM, preferably 2.5 to 15 mM, more preferably 5 to 10 mM nicotinamide (NAM) a hepatocyte culture medium comprising: It has been discovered by the present invention by a method comprising:

[0009] Cell aggregation is preferably performed in a cell culture system, such as a microwell plate, preferably an ultra-low attachment microwell plate. As referred to herein, aggregated PHH "spheroids" are three-dimensional hepatocyte aggregates formed in the absence of a specific culture substrate for attachment. As a result, hepatocytes do not attach or adhere to the surface of the culture substrate or culture system. Techniques for generating spheroids include hanging drop culture and ultra-low attachment culture, which promote cell-cell interactions while suppressing cell-substrate / matrix interactions. As a result, cells interact to form compact spheres, although other shapes are also possible. Fresh or cryopreserved adult (or fetal, neonatal, or pediatric) primary human hepatocytes (PHH) are aggregated, for example, in ultra-low attachment (ULA) microwells and allowed to form spheroids in a defined hepatocyte spheroid medium (HSM).

[0010] The methods of the present invention provide hepatic organoids containing preferably 0.1-20%, more preferably 2-17.5%, even more preferably 5-15%, and most preferably 10-12.5% ​​nonparenchymal hepatocytes (NPCs), such as stellate cells, hepatic endothelial cells, and / or Kupffer cells. The presence of these NPCs enhances the function of liver organoids and provides additional stimulation to the cells and their function in the organoid as a whole. NPCs produce various growth factors that are important for the development and function of liver organoids.

[0011] As used herein, "functional" hepatocytes in cell culture refer to hepatocytes that retain the major characteristics and functions of in vivo hepatocytes. These include polarized expression of major drug transporters, such as MRP2 and / or BSEP, albumin production and secretion, bile production and transport, low-density lipoprotein uptake, glycogen synthesis and storage, and xenobiotic biotransformation via phase I and / or phase II drug metabolism. Furthermore, functional hepatocytes should maintain susceptibility to infection by hepatocyte-specific pathogens, such as malaria and HBV. Importantly, transcriptome data are not sufficient to demonstrate functional hepatocytes in vitro. Polarity protein expression and activity must be demonstrated through various techniques well known in the art. Protein expression and localization can be demonstrated, for example, through immunolocalization or immunohistochemistry. Protein function can be assessed using various commercially available kits, such as albumin ELISA kits and CYP ELISA kits, which measure albumin secretion and biotransformation by CYP enzymes, respectively.

[0012] As used herein, "proliferative" or "expanding" hepatocytes refer to hepatocytes that incorporate nucleoside analogs, such as BrdU (5-bromo-2'-deoxyuridine) or EdU (5-ethynyl-2'-deoxyuridine), into newly synthesized DNA; hepatocytes that express nuclear proteins specific to proliferating cells, such as Ki67 and PCNA; and hepatocytes that 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 generation of proliferation curves by cell count or surrogate for cell count, e.g., the CellTiter-Glo® Luminescent Cell Viability Assay.

[0013] After stable spheroid formation, PHHs are transferred to an appropriate 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 as organoids, enabling in vitro studies of (circulating) adult PHHs as well as applications in regenerative medicine. This method aggregates primary hepatocytes into spheroids, enabling the generation of a population of primary human hepatocyte organoids from the first week of culture. The present invention can use ULA microwell plates or equivalent, allowing for the reproducible and controlled generation of thousands of spheroids. The expansion capacity of adult PHHs is far superior to any known method using this solution, enabling the passage of expanded hepatocytes. The method of the present invention has a wide range of commercial applications, from regenerative medicine to in vitro toxicology studies, including the pharmaceutical industry.

[0014] HSMs and HOMs contain the c-Met agonist hepatocyte growth factor (HGF) or related isoforms (NK1, NK2). c-MET activation by HGF regulates a variety of processes, including development, proliferation, and branching morphogenesis.

[0015] 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 various intracellular processes, including proliferation and differentiation.

[0016] According to a preferred embodiment, the present invention relates to a method, wherein the at least one canonical Wnt activator is selected from the group consisting of a Wnt surrogate molecule or a Wnt protein, such as Wnt3a, Wnt2, Wnt9b, Wnt10b, CHIR99021, a Wnt ligand, a glycogen synthase kinase 3b inhibitor, b-catenin and an activator of b-catenin, preferably a Wnt surrogate molecule or Wnt3a.

[0017] According to another preferred embodiment, the present invention relates to a method, wherein the at least one standard 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, even more preferably 0.5 to 1 μg / mL of Wnt protein.

[0018] According to yet another preferred embodiment, the present invention relates to a method, wherein the at least one canonical Wnt activator comprises 0.1-100 ng / mL, preferably 10-80 ng / mL, more preferably 20-60 ng / mL of a Wnt surrogate molecule.

[0019] According to a preferred embodiment, the present invention relates to a method, wherein the TGF-β inhibitor is selected from the group consisting of A83-01, SB431542, RepSox, SB505124, galunisertib, LY364947 or related ALK4-, ALK5-, ALK7-inhibitors, preferably A83-01. TGF inhibitors are compounds that reduce, block or inhibit TGF via the TGF RI and TGF RII receptors.

[0020] In 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 primary human hepatocytes. The hepatocytes can be primary cells isolated from a sample of immature or mature liver tissue, or tumorous liver tissue. The liver tissue can be obtained from an individual of fetal age or any age above 0 months.

[0021] In another preferred embodiment, the present invention relates to a method in which the scaffold matrix is ​​a laminin-rich extracellular matrix hydrogel, preferably Matrigel, laminin-entactin complex, Ultimatrix, Cultrex, or other basement membrane extract and / or synthetic (hydro)gel that allows organoid culture. Hepatocyte spheroids are transferred to a three-dimensional culture medium and cultured. The culture medium and cells are mixed with a scaffold matrix, preferably a hydrogel that supports cell growth and proliferation in three dimensions and allows the cells to reorganize and grow as organoids. Preferably, hydrogels are used as suitable scaffold matrices, but other known natural and synthetic matrices, such as fibrin, collagen, collagen / laminin, decellularized tissue-derived matrix, compressed collagen alginate, agarose, polyethylene glycol, or basement membrane extracts, can also be used. The scaffold matrix can be chemically defined, such as collagen or densified collagen hydrogel, or chemically undefined, such as a complex protein hydrogel. Preferably, the scaffold matrix in the expansion medium is a complex protein hydrogel. Suitable complex protein hydrogels may include extracellular matrix components such as laminin, collagen IV, entactin, and heparin sulfate proteoglycans. Complex protein hydrogels may also include hydrogels of extracellular matrix proteins derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. Suitable complex protein hydrogels are commercially available, including Matrigel™ (Corning Life Sciences) and Cultrex™ BME 2 RGF (Amsbio™ Inc.).

[0022] According to another preferred embodiment, the present invention relates to a method in which hepatocyte spheroids are transferred to a scaffold matrix at a density of 1-1000 hepatocyte spheroids, preferably 100-500 hepatocyte spheroids per 25-100 μL of scaffold matrix. If too many spheroids are seeded per droplet of scaffold matrix, i.e., more than 1000 per 100 μL, the spheroids will fuse together, which is undesirable for expansion.

[0023] According to another preferred embodiment, the present invention relates to a method further comprising the step of: d) subculturing hepatocyte organoids expanded in a scaffold matrix with HOM into a fresh scaffold matrix every 1-60 days, preferably every 5-20 days, more preferably every 10-15 days. The hepatocyte organoids are grown in the scaffold matrix for 1-30 days, harvested, transferred to a fresh scaffold matrix at a subculture ratio of 1:2-1:3, and provided with HOM for another 1-60 days. This process can be repeated as long as the cells continue to proliferate.

[0024] According to another preferred embodiment, the present invention relates to a method, wherein the HSM further comprises 50 to 500 ng / mL of at least one R-spondin protein.

[0025] According to another preferred embodiment, the present invention relates to a 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 members of the R-spondin protein family, and preferably R-spondin-3. R-spondin amplifies the effect of Wnt protein and stimulates its expansion. R-spondin-3 is more effective than others, and is also the R-spondin produced during liver regeneration. R-spondin is a secreted activator protein that positively regulates the canonical Wnt signaling pathway and non-canonical Wnt signaling pathway. Preferably, R-spondin is human R-spondin. During canonical Wnt activation, R-spondin synergizes with Wnt molecules to amplify Wnt signaling.

[0026] In another preferred embodiment, the present invention relates to a method in which the HSM further comprises 1-20 μM of a ROCK inhibitor, which blocks or inhibits the activity of Rho kinase (ROCK). ROCK inhibitors prevent cells from undergoing apoptosis when stressed by a loss of cell-cell contact, for example in stem cell culture.

[0027] According to yet another preferred embodiment, the present invention relates to a method, wherein the ROCK inhibitor is selected from the group consisting of Y-27632 (trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide), fasudil, Y39983 (4-[(1R)-1-aminoethyl]-A / -1 / - / -pyrrolo[2,3-b]pyridin-4-ylbenzamide dihydrochloride), and azabenzimidazole-aminofurazan, preferably Y-27632.

[0028] According to yet another preferred embodiment, the present invention relates to a method in which the HSM and / or HOM further comprises 0.1-50 μM of a glucocorticoid. Glucocorticoids affect a wide range of processes, including survival, proliferation, and differentiation. The HSM preferably contains either 1-50 μM of hydrocortisone or 0.1-1 μM of dexamethasone.

[0029] 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 1-50 μM hydrocortisone or 0.1-1 μM dexamethasone.

[0030] According to another preferred embodiment, the present invention relates to a method for forming functional primary hepatocyte spheroids in HSM for about 1-30 days, preferably about 3-10 days, and more preferably about 4-6 days. It has been found that 4-6 days is ideal for generating functional primary hepatocyte spheroids. Shorter times (less than 2 days) or longer times (more than 30 days) result in suboptimal results when the aggregates are transferred to a three-dimensional matrix.

[0031] In another preferred embodiment, the present invention relates to a method in which hepatocyte spheroids transferred to a scaffold matrix in HOM are cultured for about 1 to 30 days, preferably about 5 to 20 days, and more preferably about 8 to 15 days. Because the three-dimensional matrix eventually degrades, optimally, as described above, the cells are transferred to a new three-dimensional matrix every 5 to 15 days and cultured again.

[0032] According to a preferred embodiment, the present invention relates to a method in which hepatocytes are provided to a microwell 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.

[0033] According to yet another preferred embodiment, the present invention relates to a method, wherein the recovered hepatocyte spheroids of step c) have an average diameter of 10-1000 μm, preferably 50-500 μm, more preferably 100-250 μm, as determined by bright field microscopy (i.e., by using a scale bar).

[0034] According to a preferred embodiment, the present invention relates to a method further comprising a gastrin having an HOM of 1-1000 nM, the gastrin being added within a concentration range to promote cell growth and viability in cell culture.

[0035] 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 of tumor necrosis factor alpha (TNFα). Addition of TNFα to the HOM promotes activation of NF-κB signaling and induces further proliferation of hepatocytes.

[0036] According to a preferred embodiment, the present invention relates to a method further comprising fibroblast growth factor 19 (FGF19) at a HOM of 1 to 100 ng / mL. FGF19 activates fibroblast growth factor receptor 4 (FGFR4) signaling and modulates metabolic capacity, such as bile acid synthesis.

[0037] According to another preferred embodiment, the present invention relates to a method, wherein the 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 the HSM and / or HOM further comprises 0.1 to 10% v / v fetal calf serum (FCS) or fetal bovine serum (FBS).

[0038] According to another preferred embodiment, the present invention relates to a method, wherein at least 50% of the hepatocytes present at the beginning of cell culture are contributed to organoid formation, more preferably at least 65%, even more preferably at least 85%, and most preferably at least 90%.In contrast to the method of the present invention, in the known and currently applied method for producing organoid, about 1% of the cells provided at the beginning of culture survive and contribute to the formation and production of organoid.The method of the present invention is extremely superior in this respect, and at least 50% of the living cells used at the beginning of cell culture are contributed to the provision or generation of organoid.

[0039] The methods of the present invention use HSM and HOM media. Both HSM and HOM may include a basal medium further supplemented with a medium supplement, such as B27 supplement, N21 supplement, N2 supplement, or ITS(+) supplement, to form a nutrient medium. The nutrient medium may further include L-glutamine or a substitute thereof, such as L-alanyl-L-glutamine (e.g., Glutamax™), N-acetylcysteine ​​(NAC), MEM non-essential amino acid solution, sodium pyruvate, 2-phospho-L-ascorbic acid, and a buffer such as HEPES, as well as antibiotics such as penicillin and streptomycin. Suitable basal media include Iscove's Modified Dulbecco's Medium (IMDM), Ham's F12 medium, (Advanced) Dulbecco's Modified Eagle's Medium (DMEM) or DMEM / F12, Williams' Medium, or RPMI 1640. 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 μM 2-phospho-L-ascorbic acid trisodium salt, 1-5% (v / v) B27, and 1-5 mM N-acetylcysteine. Preferably, the HSM contains 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 molecule.Preferably, the HOM contains 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, and 1% (v / v) of 2-phospho-L-ascorbic acid trisodium salt. The formulation includes Advanced DMEM / F12 supplemented with standard Wnt activators such as erythromycin, 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 μM Y-27632, 1-5 μM A-83-01, and 100-1,000 ng / mL Wnt3a or 0.1-10 nM Wnt surrogate.

[0040] According to a second aspect, the present invention relates to a cell culture system for in vitro population generation and proliferation of functional adult primary human hepatocytes (PHH) and / or formation of hepatocyte organoids, the cell culture system comprises a microwell cell culture system comprising PHH as defined above, hepatocyte spheroid medium (HSM), and / or hepatocyte organoid medium (HOM).The cell culture system is suitable for population generation of hepatocyte spheroids, and the system preferably comprises ultra-low attachment (ULA) microwells.

[0041] According to another aspect, the present invention relates to the use of this cell culture system for the generation and proliferation of functional adult primary human hepatocyte (PHH) population and / or for the formation of hepatocyte organoid.The present invention and this system for the generation and proliferation of functional adult PHH population can be used to evaluate the liver toxicity, particularly the liver genotoxicity, and / or the effect of drugs or compounds in vitro, for designing artificial liver model or artificial liver organ.

[0042] The invention is explained in more detail in the following examples and figures. [Brief explanation of the drawings]

[0043] [Figure 1] Adult cryopreserved human hepatocytes (PHH) are seeded as single cells into ULA microwells and cultured in HSM (day 0, Figure 1A). After 4 days, PHH aggregated to form stable hepatocyte spheroids, which were substantially uniform in shape and approximately 50–250 µm in diameter (Figure 1B). The hepatocyte spheroids were then transferred to a scaffold matrix and cultured in HOM (day 0, Figure 1C). Hepatocytes proliferated over time (day 6) and morphogenetically formed (Figure 1D). [Figure 2] Figure 2 shows healthy functional hepatocyte organoids obtained by the present invention, and shows the expression of mature functional hepatocyte markers by immunofluorescence (IF) staining. Figure 2A shows IF staining of hepatocyte organoids, showing CYP3A4 (a marker for mature hepatocytes involved in phase I drug metabolism) in green, E-cadherin (a marker for epithelial hepatocytes) in red, and DAPI as a nuclear stain in blue. Figure 2B shows IF staining of hepatocyte organoids, showing HNF4α (a marker for hepatocytes) in green, albumin (a marker for mature hepatocytes) in red, and DAPI as a nuclear stain in blue. [Figure 3] Figure 3 shows single adult hepatocytes seeded directly onto Matrigel without prior aggregation on days 0 (Figure 3A) and 6 (Figure 3B) in HSM, day 6 in Hu et al., 2018 medium (Figure 3C), and day 6 in HOM (Figure 3D). DETAILED DESCRIPTION OF THE INVENTION [Example]

[0044] Isolation of adult primary human hepatocytes Primary human hepatocytes were isolated from surgical liver resections after informed consent. Hepatocytes were isolated by two-step collagenase perfusion and filtered through a 70 μM cell strainer.

[0045] Generation of hepatocyte spheroids Fresh or cryopreserved adult primary human hepatocytes were resuspended in hepatocyte spheroid medium (HSM). HSM contained 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), and 10 μM Hydrocortisone (STEMCELL). Tech.), and 1 μg / mL Wnt3a (R&D Systems).

[0046] Cells were seeded into ultra-low attachment microwells in HSM at a density of approximately 20 cells per microwell and allowed to form spheroids for approximately 96 hours. To refresh the medium, 100% (v / v) fresh HSM was added to each well after 48 hours of culture.

[0047] Organoid culture of adult primary human hepatocytes Hepatocyte spheroids formed in HSM were collected and centrifuged at 100 g for 5 minutes. The supernatant was removed, and the spheroids were resuspended in Matrigel solution prepared by mixing Matrigel (Corning) with advanced DMEM / F12. Hepatocyte spheroids were seeded at a density of approximately 200 spheroids per 50 μL Matrigel droplet. After allowing Matrigel to polymerize into a stable hydrogel (20–30 minutes), HOM was added to each well.

[0048] HOM contained 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.

[0049] The medium was refreshed every 2–3 days during the culture period. Figure 1A shows PHHs seeded as single cells on ULA and cultured in HSM. Figure 1B shows PHHs on day 4 of culture in HSM after stable spheroid formation. Figure 1C shows PHH spheroids on day 0 after transfer to Matrigel. Figure 1D shows PHH spheroids on day 6 after transfer to Matrigel, demonstrating significant expansion as hepatocyte organoids. Hepatocyte organoids (HO) were subcultured every 8–12 days at a split ratio of 1:2–1:3. To mature HOs, 10 μM hydrocortisone and / or 50 ng / mL FGF19 were added to the HOM.

[0050] Next, single adult hepatocytes (as a control) were seeded onto Matrigel without a prior aggregation step (as opposed to the method of the present invention) and supplemented with either HSM (Figure 3B), HOM (Figure 3D), as described above, or the hepatocyte organoid medium composition published by Hu et al., 2018, Cell 175, pp. 1591-1606 (Figure 3D). Cultures were monitored for organoid formation on days 0 (Figure 3A, with HSM) and 6. As evident in Figure 3, hepatocytes transferred directly to the scaffold matrix without prior aggregation formed few organoids, and showed limited expansion and morphogenesis. In contrast, when single hepatocytes were first aggregated in HSM, then transferred to Matrigel and cultured in HOM in the method of the present invention, the hepatocytes formed organoids, invading the surrounding matrix while proliferating and morphogenetically forming, as observed in Figure 1.

Claims

1. A method for in vitro production of functional and proliferative hepatocyte organoids from primary human hepatocytes (PHH), comprising: 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 containing 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 an isoform thereof, and 0.1 ng / mL to 2 μg / mL of at least one standard Wnt activator; b) harvesting the hepatocyte spheroids and transferring the hepatocyte spheroids to a scaffold matrix; c) culturing the hepatocyte spheroids transferred to the scaffold matrix in a hepatocyte organoid medium (HOM) to generate hepatocyte organoids containing expanding functional PHHs, wherein HOM is a hepatocyte culture medium containing 0.5 to 100 ng / ml, preferably 1 to 50 ng / mL, at least one epidermal growth factor receptor (EGFR) agonist, 1 to 50 ng / mL, at least one hepatocyte growth factor (HGF) or its isoform, 0.1 ng / mL to 2 μg / mL, at least one standard Wnt activator, 50 to 500 ng / mL, at least one R-spondin protein, 0.1 to 5 μM, at least one transforming growth factor β (TGF-β) inhibitor, 1 to 20 μM, at least one ROCK inhibitor, and 1 to 20 mM, nicotinamide (NAM); A method comprising:

2. 2. The method of 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, and preferably AREG and / or EGF.

3. 3. The method of claim 1 or 2, wherein the at least one canonical Wnt activator is selected from the group consisting of a Wnt surrogate molecule or Wnt protein, Wnt3a, Wnt2, Wnt9b, Wnt10b, CHIR99021, preferably a Wnt surrogate molecule or Wnt3a.

4. 4. The method of any one of claims 1 to 3, wherein the at least one standard 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, even more preferably 0.5 to 1 μg / mL of Wnt protein.

5. 4. The method of 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 of a Wnt surrogate molecule.

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

7. 7. The method of 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 primary human hepatocytes.

8. 8. The method of claim 1, 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. 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. d) the method according to any one of claims 1 to 9, further comprising the step of subculturing the hepatocyte organoid that is grown in scaffold matrix using HOM into fresh scaffold matrix every 1 to 60 days, preferably every 5 to 20 days, more preferably every 10 to 15 days.

11. 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. 12. The method according to any one of claims 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 members of the R-spondin protein family, preferably R-spondin-3.

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

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

15. 15. The method of any one of claims 1 to 14, wherein the HSM and / or HOM further comprises 0.1 to 50 μM of a glucocorticoid.

16. 16. The method of 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 μM hydrocortisone or 0.1 to 10 μM dexamethasone.

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

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

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

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

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

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

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

24. 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. 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 of fetal calf serum (FCS) or fetal bovine serum (FBS).

26. 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%, and most preferably at least 90% of the hepatocytes present at the start of cell culture contribute to organoid formation.

27. 27. A cell culture system for in vitro population generation and proliferation of functional adult primary human hepatocytes (PHHs) and / or formation of hepatocyte organoids, comprising a microwell cell culture system comprising PHHs according to any one of claims 1 to 26, hepatocyte spheroid medium (HSM), and / or hepatocyte organoid medium (HOM).

28. 28. Use of the cell culture system of claim 27 for the generation and proliferation of a population of functional adult primary human hepatocytes (PHH) and / or for the formation of hepatocyte organoids.

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