WO2019185017A1

WO2019185017A1 - Medium for hepatocyte culture and preparation of liver organs

Info

Publication number: WO2019185017A1
Application number: PCT/CN2019/080422
Authority: WO
Inventors: 高栋; 刘文明; 何娟
Original assignee: 中国科学院上海生命科学研究院
Priority date: 2018-03-30
Filing date: 2019-03-29
Publication date: 2019-10-03
Also published as: CN110317775B; CN110317775A

Abstract

The present invention provides a proliferation culture medium and a differentiation culture medium for hepatocyte culture and preparation of liver organs. The proliferation culture medium and the differentiation culture medium are based on a culture medium for growth of mammalian cells, added with a supplemental L-Glutamine reagent, a pH regulator that maintains the pH stability of the culture medium, a primary cell culture antibiotic, a serum replacement, N-acetylcysteine, and an optional nicotinamide, plus one or more of a BMP inhibitor, a Wnt agonist, a growth factor, a ROCK signaling pathway inhibitor, a P38 signaling pathway inhibitor, a Notch signaling pathway inhibitor, dexamethasone, a BMP7 activator, and a cAMP activator. A functional liver organ can be obtained by culturing liver cells using the culture medium of the present invention.

Description

Medium for hepatocyte culture and preparation of liver organs

Technical field

The present invention relates to the cultivation of liver cells and the establishment of organoids. Specifically, the present invention relates to a culture medium for hepatocyte culture and preparation of liver organs.

Background technique

The liver is the body's largest detoxification organ, the largest digestive gland in the human body, and the center of energy metabolism in the body. The liver is mainly composed of two types of epithelial cells, namely hepatocytes and biliary cells. The main functions of the liver include: storage of liver glycogen, synthesis of basic secretory proteins, removal of foreign compounds, regulation of body metabolism, transport of drugs and excretion of bile. It is precisely because the liver has important physiological functions that many liver-related diseases, such as hepatitis, liver cancer, liver fibrosis, liver cirrhosis, acute and chronic liver failure, etc., have high morbidity and mortality. China is a high-risk country for hepatitis infection, so the incidence of severe liver disease in China is also very high. Liver transplantation remains the most effective treatment for severe liver disease. Due to the lack of liver donors, many patients with severe liver disease eventually die in conservative treatment, so there is an urgent need to develop new treatments to improve the survival rate of liver disease treatment. If a liver donor cell can be isolated and transplanted to multiple patients, the liver source can be greatly saved and more patients can be treated. Transplantation of hepatocytes has been studied in animal models for decades, and the results show that hepatocyte transplantation can significantly improve liver function and individual survival in liver-damaged animals. Human embryonic hepatocytes or adult hepatocytes transplantation can significantly improve the survival rate of patients with acute liver failure and have been used to prolong the lives of patients with acute liver failure. Therefore, liver cell transplantation therapy is the future trend of treating severe liver diseases. Although hepatocytes have strong regenerative capacity in vivo, long-term culture of mature hepatocytes in vitro remains a worldwide problem.

In vitro hepatocyte culture mainly adopts three strategies: one is direct culture of liver cells in vitro, the other is by in vitro differentiation of stem cells by hepatocytes, and the third is to induce differentiation of fibroblasts into hepatocyte-like cells. The cultivation of mature hepatocytes has always been a worldwide problem. Recent studies have shown that mature hepatocytes can only be maintained in vitro for about one week. Although cholangiocarcinoma cells can be cultured in vitro for a long time, they can only selectively restore liver function in liver-injured animals after induction of transdifferentiation into hepatocytes. Using human embryonic stem cells (hES) and in vitro differentiation techniques that induce pluripotent stem cells (hiPS), stem cells can be induced to differentiate into "hepatocyte-like cells" that have hepatocyte markers and partial hepatocyte functions. By in vitro chemical induction or transfection of hepatocyte-specific transcription factors, fibroblasts can be induced to transdifferentiate into hepatocytes with partial hepatocyte function. However, recent studies have shown that this in vitro reprogramming will lead to genetic and epigenetic changes, induce genomic instability and genetic mutations, and transplant GM biomaterials into the human body will face great ethical problems. The transplantation of "hepatocyte-like" cells that result in hES and hiPS sources poses a great risk. At present, it is urgent to establish a controllable new technology system for liver cell culture, and to culture normal mature liver cells with liver tissue and organ function in vitro.

Organoid research is a very important frontier hotspot, and it is a new in vitro culture technique, which is a three-dimensional in vitro tissue culture technique formed by stem cell-induced differentiation of cells, normal tissue cells or cell culture of patients. . The organoid model can mimic the microenvironment of cells in vivo and has unique advantages for constructing physiologically functional research models in vitro. At present, organ-like organs such as colon, stomach, prostate and pancreas have been successfully constructed in vitro using organ-like culture. Although organ-like organ culture research is still in its infancy, it has been widely used as an important research technique in stem cell and tumor research. Organoids have the following major advantages: (1) maintaining the high complexity of tissue and organ cells; (2) maintaining the polarity of contact between functional cells and microenvironmental matrices, better mimicking the in vivo microenvironment; (3) The clinical organization of organ-like organs is highly efficient and time-consuming, and can carry out long-term cultivation of cells; (4) It has both the advantages of genetic manipulation and the three-dimensional complex characteristics of the model. The latest research shows that organoids can quickly and accurately predict the efficacy of anticancer drugs. Organ-like technology was evaluated by Nature Methods as the most promising annual technology in the life sciences in 2017. The organoid model will play a huge application value in the fields of disease model construction, organ transplantation, drug screening, drug safety testing and drug efficacy evaluation.

Summary of the invention

The invention marks the mature liver cells of the mouse by the cell lineage tracing technique, combines the flow cell sorting and the organ-like organ culture technology of the hepatocytes, and finds that the mature mouse liver cells can rapidly proliferate and can be cultured in vitro for a long time. It also has liver cell-specific gene expression, such as Albumin, Hnf4α, Krt8, Cdh1 and the like. In addition, there is differentiation of biliary cells in liver organs, and a bile duct-like structure is formed in the organs. At the same time, the present invention also establishes a liver cell-like organ culture system derived from human embryonic liver and normal liver samples, and obtains a long-term cultured human liver-like organ. On the basis of this, by transplanting liver-like organ cells into a mouse model of liver injury, the inventors found that liver-like organs can significantly restore liver function in mice with liver injury and significantly improve the survival rate of animals.

Therefore, the first aspect of the present invention provides a serum-free cell culture medium which is based on a medium for growth of mammalian cells, is supplemented with an agent for supplementing L-glutamine, and maintains a pH of the culture medium. pH regulator, primary cell culture antibiotic, serum replacement, N-acetylcysteine and optionally nicotinamide, plus BMP inhibitor, Wnt agonist, growth factor, Rock signaling pathway inhibitor, P38 One or more of a signaling pathway inhibitor, a Notch signaling pathway inhibitor, dexamethasone, BMP7, and a cAMP activator.

In one or more embodiments, the serum-free cell culture medium is based on a medium for growth of mammalian cells, supplemented with an agent that supplements L-glutamine, and maintains a pH at which the pH of the medium is stable. Value modifier, primary cell culture antibiotic, serum replacement, N-acetylcysteine, and optionally nicotinamide, plus growth factor and Rock signaling pathway inhibitor, optionally with BMP inhibitor, Wnt One or more of an agonist, a P38 signaling pathway inhibitor, a Notch signaling pathway inhibitor, dexamethasone, BMP7, and a cAMP activator.

In one or more embodiments, the basal medium is an improved DMEM/F12 or modified RPMI medium.

In one or more embodiments, the BMP inhibitor is selected from one or more of Noggin, A-83-01, DAN, and DAN-like proteins; preferably, the BMP inhibitor is in the culture medium The final concentration is in the range of 0.5-800 ng/ml of medium.

In one or more embodiments, the Wnt agonist is selected from one or more of R spondin, a GSK inhibitor, and Wnt3 (eg, Wnt 3a); preferably, each Wnt agonist has a final concentration of 1 -1500 ng/ml medium.

In one or more embodiments, the growth factor is selected from the group consisting of epidermal growth factor, transforming growth factor beta, basic fibroblast growth factor, hepatocyte growth factor, brain-derived neurotrophic factor, and keratinocyte growth factor. One or more; preferably, the final concentration of the growth factor is 1-1000 ng/ml of medium.

In one or more embodiments, the Rock signaling pathway inhibitor is selected from one or more of Y 27632, HA 1077, and H1152; preferably, the final concentration of the Rock inhibitor is in the range of 0.5-50 μM .

In one or more embodiments, the Notch signaling pathway inhibitor is selected from the group consisting of DAPT (GSI-IX), MK-0752, RO4929097, Semagacestat (LY450139), LY411575, Dibenzazepine (YO-01027), Avagacestat, Crenigacestat, NGP One or more of 555; preferably, the final concentration of the Notch signaling pathway inhibitor is from 0.1 to 50 [mu]M.

In one or more embodiments, the P38 signaling pathway inhibitor is selected from the group consisting of SB203580, Doramapimod, SB202190, LY2228820, VX-702, PH-797804, VX-745, TAK-715, BMS-582949, Losmapimod, Pexmetinib and One or more of Skepinoe-L; preferably, the final concentration of the P38 signaling pathway inhibitor is 1-20 μM.

In one or more embodiments, the cAMP agonist is Forskolin; preferably, the cAMP agonist has a final concentration of 1-200 [mu]M.

In one or more embodiments, the cell culture medium is a cell proliferation medium, a medium based on a medium for mammalian cell growth, a reagent supplemented with L-glutamine, and a pH of the maintenance medium. Stable pH regulators, primary cell culture antibiotics, serum replacements, N-acetylcysteine and nicotinamide, plus BMP inhibitors, Wnt agonists, growth factors, Rock signaling pathway inhibitors, P38 One or more of a signaling pathway inhibitor and a cAMP activator.

In one or more embodiments, the cell proliferation medium is supplemented with a BMP inhibitor, a Wnt agonist, a growth factor, and a Rock signaling pathway inhibitor, and is optionally added with a P38 signaling pathway inhibitor and a cAMP activator One or two of them.

In one or more embodiments, the cell culture medium is a cell proliferation medium comprising Noggin and A-83-01 as BMP inhibitors, EGF, FGF10 and FGF2 as mitotic growth factors, as Wnt agonists R spondin, and Y 27632 as a Rock signaling pathway inhibitor, supplemented with GlutaMAX-I, pH regulators that maintain medium pH stability, primary cell antibiotics, B27 serum replacement, nicotinamide, and N-acetyl half Cystine; preferably, the cell culture medium further contains a GSK inhibitor such as CHIR99021, and/or Wnt3a as a Wnt agonist.

In one or more embodiments, the proliferation medium further comprises any one, any two or all three of a P38 inhibitor such as SB202190, a cAMP activator such as Forskolin, and BMP7.

In one or more embodiments, the proliferation medium, when included: final concentration of Noggin is 5-15 ng/ml; final concentration of A-83-01 is 300-800 ng/ml; final concentration of EGF 20-80 ng / ml; FGF10 final concentration of 5-15ng / ml; FGF2 final concentration of 0.1-2ng / ml; R spondin final concentration of 50-150ng / ml; Y27632 final concentration of 5-15μM; The final concentration of SB202190 is 5-15μM; the final concentration of cAMP activator is 5-15μM; the final concentration of GSK inhibitor is 1-5μM; the final concentration of BMP7 is 10-40ng/ml; the final concentration of Wnt3a is 300-600ng /ml.

In one or more embodiments, the cell culture medium is a cell differentiation medium, a medium based on a medium for mammalian cell growth, a reagent supplemented with L-glutamine, and a pH of the maintenance medium. Stable pH regulators, primary cell culture antibiotics, serum replacements, and N-acetylcysteine, supplemented with BMP7, growth factors, Rock signaling pathway inhibitors, Notch signaling pathway inhibitors, and dexamethasone One or any of a variety.

In one or more embodiments, the differentiation medium is supplemented with BMP7, a growth factor, a Notch signaling pathway inhibitor, a dexamethasone, and a Rock signaling pathway inhibitor.

In one or more embodiments, the cell culture medium is a cell differentiation medium comprising BMP7, a growth factor, a Notch signaling pathway inhibitor, a Rock signaling pathway inhibitor, and dexamethasone, supplemented by GlutaMAX-I, maintained a pH-stabilized pH adjuster, a primary cellular antibiotic, a B27 serum replacement, and N-acetylcysteine; preferably, the growth factor comprises FGF10, FGF2 and HGF, and optionally FGF19, The Notch signaling pathway inhibitor is DAPT and the Rock signaling pathway inhibitor is Y27632.

In one or more embodiments, the BMP7 has a final concentration of 10-40 ng/ml in the differentiation medium, and the final concentration of the growth factor is 50-200 ng/ml, the Notch signaling pathway inhibitor The final concentration is 1-15 μM, the final concentration of the Rock signaling pathway inhibitor is 5-15 μM, and the final concentration of the dexamethasone is 0.01-30 μM.

In one or more embodiments, the final concentration of FGF10 in the differentiation medium is 5-15 ng/ml; the final concentration of FGF2 is 0.1-2 ng/ml; the final concentration of HGF is 10-40 ng/ml; When present, the final concentration of FGF19 is 10-100 ng/ml; the final concentration of Y 27632 is 5-15 μM; the final concentration of dexamethasone is 1-10 μM; and the final concentration of DAPT is 5-15 μM.

The present invention also provides a kit comprising a medium for growth of mammalian cells as a basal medium, a reagent for supplementing L-glutamine, a pH adjuster for maintaining a pH of the culture medium, and a primary cell. Culture antibiotics, serum replacements, N-acetylcysteine and optionally nicotinamide, as well as BMP inhibitors, Wnt agonists, growth factors, Rock signaling pathway inhibitors, P38 signaling pathway inhibitors, Notch signaling pathway inhibitors One or more of dexamethasone, BMP7 and cAMP activators.

In one or more embodiments, the kit further contains an extracellular matrix.

In one or more embodiments, the kit comprises the medium of any of the embodiments herein; preferably, the kit contains the cell proliferation medium and cell differentiation medium.

The invention also provides a cell culture comprising the medium of any of the embodiments herein and liver cells.

In one or more embodiments, the cell culture is a cell culture containing the culture medium and an organoid.

The present invention also provides a method for culturing a hepatocyte, the method comprising the steps of: preparing a hepatocyte suspension using the cell proliferation medium according to any one of the above, mixing the suspension with an extracellular matrix, and then performing culturing, and / or the step of differentiating cells using the cell differentiation medium described in any of the embodiments herein.

The invention also provides the use of the organoids described herein or the organoids prepared by the methods described for drug development, drug screening, and toxicity determination of food supplements.

DRAWINGS

Figure 1: 29 cytokine combinations were used to culture equal cells, and on day 1 of the organoids.

Figure 2: 29 cytokine combinations were used to culture equal cells, and on day 6 were organoids.

Figure 3: 29 cytokine combinations were used to culture equal cells, and on day 12, organoids.

Figure 4: 29 cytokine combinations were used to culture equal cells, and on the 14th day, organoids.

Figure 5: 29 cytokine combinations were used to culture equal cells, and on day 20, organoids.

Figure 6: A liver-like organ culture system was established by human fetal liver cell culture medium. The isolated tissue blocks were mechanically sheared and collagenase digested into single cells, and then subjected to 3D culture on Matrigel to form organoids. The figure shows the formation of organoids in different liver samples under bright field after 5-7 days.

Figure 7: A long-term culture system of liver-like organs was established using human fetal liver cell culture medium.

Figure 8: Embryo liver organ formation efficiency.

Figure 9: Statistics of embryonic liver organ formation efficiency.

Figure 10: Embryonic liver organs maintain the expression of hepatocyte genes.

Figure 11: Low expression of AFP in embryonic liver organs.

Figure 12: Embryonic liver organs maintain the histological features of the liver.

Figure 13: Markers of mature liver cells expressing embryonic liver organs.

Figure 14: Markers associated with high expression of hepatocyte function in embryonic liver organs.

Figure 15: Genes of the cytochrome P450 enzyme family that are highly expressed in embryonic liver organs.

Figure 16: Embryonic liver organs have the function of a mature liver.

Figure 17: Embryonic liver organs can prolong the lifespan of mice with liver injury.

Figure 18: Embryonic liver organs can be integrated into the liver of liver-injured mice.

Figure 19: Embryonic liver-like organs have the function of repairing the liver of FRG model mice.

Figure 20: Embryonic liver-like organ transplantation into the FRG liver injury model has the function of secreting ALB.

Figure 21: Long-term culture of embryonic liver organs in vitro has a tendency to transdifferentiate into biliary cells.

Figure 22: Establishment of an adult hepatocyte-like organ culture system.

Figure 23: Detection of organohepatocyte-like organ formation efficiency.

Figure 24: Adult hepatocyte organogenesis efficiency.

Figure 25: Statistics of the efficiency of adult hepatocyte organogenesis.

Figure 26: Adult hepatocyte-like organs maintain hepatocyte gene expression.

Figure 27: Markers of mature hepatocyte-like organs expressing mature hepatocytes.

Figure 28: Genes of the cytochrome P450 enzyme family that are highly expressed in adult hepatocyte-like organs.

Figure 29: Adult hepatocyte-like organs are mainly composed of hepatocytes.

Figure 30: Adult hepatocyte-like organs have the function of a mature liver.

Figure 31: Adult liver-like organs have the potential to differentiate into the bile duct.

Figure 32: Four days of differentiation medium induced differentiation for 14 days of cell growth.

Figure 33: Expression of hepatocyte-associated markers at 14 days of differentiation induced by four differentiation media.

Figure 34: Hepatic organ-biliary cell-like organ structure formed by differentiated liver organs.

Figure 35: Differentiated mature hepatocytes - biliary cell-like organs have a gene expression profile similar to liver tissue.

Figure 36: Differentiated mature hepatocytes - biliary cell-like organs have mature hepatocyte function.

Figure 37: Differentiated mature hepatocytes - biliary cell-like organs have cytochrome P450 enzyme family 3A4 activity.

Figure 38: Differentiated mature hepatocytes - biliary cell-like organs have the function of mature biliary cells.

Figure 39: Differentiated mature hepatocytes - biliary cell-like organs have the function of repairing the liver of mice with acute liver injury.

Figure 40: Differentiated mature hepatocytes - biliary cell-like organs can be integrated into the liver of mice with acute liver injury.

Figure 41: Differentiation of mature hepatocytes - biliary cell-like organs can prolong the lifespan of mice with chronic liver injury.

Figure 42: Differentiated mature hepatocytes - biliary cell-like organs transplanted into the FRG liver injury model have the function of secreting ALB.

Figure 43: Differentiated mature hepatocytes - Cholangioblastic organ can be integrated into the liver of chronic liver injury mice.

Figure 44: Differentiated mature hepatocytes - biliary cell-like organs have the function of repairing damaged liver.

Figure 45: Differentiated mature hepatocytes - biliary cell-like organs form a bile duct structure in vivo.

detailed description

It should be understood that within the scope of the present invention, the above various technical features of the present invention and the technical features specifically described in the following (as in the embodiments) may be combined with each other to constitute a preferred technical solution.

It is well known in the art that mature hepatocytes typically survive under in vitro culture conditions for no more than one week and are unable to proliferate under normal in vitro culture conditions. To solve this problem, the present invention provides a method for culturing liver cells, which comprises the step of contacting a cell culture fluid containing isolated liver cells with an extracellular matrix (ECM). The present inventors have found that culturing hepatocytes by the method of the present invention can improve the survival time of hepatocytes, maintain the differentiation characteristics of mature hepatocytes, and achieve the persistence of differentiated hepatocytes. In the absence of ECM, hepatocyte cultures could not be cultured for a long time, and no differentiated hepatocytes were observed to persist. In addition, three-dimensional tissue-like organs can be cultured in the presence of ECM. In particular, in certain embodiments, hepatocytes cultured using the methods described herein can develop into organoids of hepatocyte-cholangiocarcinoma cells that contain lumens lined with bile duct-like epithelium to form lumens The cells maintain the function of the bile duct. The resulting organoids have both the characteristics of hepatocytes and the function of the bile duct. More surprisingly, the inventors have discovered that in the absence of stem cell nevi, the single isolated primary hepatocytes can be grown into hepatocyte-choline tubular organoids using the methods described herein. The open upper portion of the unit is closed and the chamber is filled with apoptotic cells. The newly formed liver-like organs exert liver function and have the function of repairing the damaged liver. In the absence of ECM, three-dimensional tissue-like organs can not be cultured.

As used herein, "liver cell" refers to any cell isolated from the liver including, but not limited to, primary hepatocytes, embryonic liver stem/progenitor cells, and liver cancer cells. Methods for isolating liver cells from liver tissue are known in the art, and major methods of isolation include non-perfusion methods (Spotorno et al., 2006) and perfusion methods (Kuniyoshi et al., 2004). The non-perfusion method is further divided into: mechanical separation method (Kravchenko et al., 2002): by using surgical instruments, the liver tissue is separated into small pieces, and the hepatocytes are separated by blow-off extrusion; trypsin digestion method (Wang Et al., 2017): A method of using pancreatic enzymes to disrupt bridges between hepatocytes and separating hepatocytes; collagenase digestion (Ehrhardt and Schmicke, 2016): using collagenase to disrupt fibers between cells Ingredients, the tissue is now cut into small pieces and washed once with calcium-free and magnesium-free PBS, and then digested with collagenase, and then the reaction is stopped with serum-containing DMEM, and the liver cell suspension is obtained by centrifugation. The perfusion method refers to a method of sequentially injecting a plurality of separation solutions into the liver through the inferior vena cava to obtain hepatocytes. In certain embodiments, the invention employs an ex vivo collagenase perfusion method to obtain mouse hepatocytes. In vitro collagenase perfusion is a method of combining ex vivo perfusion with collagenase digestion. When hepatocytes are isolated by this method, the hepatic portal vein and inferior vena cava of the mouse are dissected and a tube is built in the inferior vena cava to establish a perfusion channel. . Firstly, 80-150 mL of perhydrated calcium-free and magnesium-free EBSS solution was perfused, and EBSS solution containing calcium and magnesium ions was used to continuously perfuse 50-80 mL, and then collagenase was used for continuous perfusion to separate hepatocytes from interstitial and release. Hepatocyte. Then, the hepatocyte stop solution is used to terminate the digestion of collagenase, and the hepatocyte suspension is obtained by filtering, centrifuging, discarding the supernatant, and resuspending the pellet with the hepatocyte culture solution. In certain embodiments, the invention employs mechanical separation in combination with collagenase digestion to obtain human hepatocytes. First remove the fibrous components such as the liver tissue capsule and blood vessels, cut the liver tissue into small pieces of about 1 mm ³ with scissors, transfer the small pieces of liver tissue to a centrifuge tube containing the hepatocyte separation medium, and gently blow with a straw. After the sediment is set, the supernatant is discarded. After washing several times, an appropriate amount of 4-8 mg/ml type IV collagenase is added and shaken in a 37 ° C incubator for a period of time (for example, 40-80 min, such as about 60 min). After the lumps were not visible to the naked eye, the reaction was stopped with high glucose DMEM containing 10% fetal bovine serum, centrifuged at 600-1000 rpm for 3-6 min, and the supernatant was discarded. The pellet contains the desired hepatocytes.

As used herein, an "extracellular matrix" is secreted by connective tissue cells and comprises a plurality of polysaccharides, water, elastin and glycoproteins, wherein the glycoproteins include collagen, fibronectin, nestin and laminin. Different types of ECM are known which include different compositions, such as different combinations containing different types of glycoproteins or glycoproteins. Examples of cells producing extracellular matrix are chondrocytes mainly producing collagen and proteoglycan, fibroblasts mainly producing type IV collagen, laminin, interstitial procollagen and fibronectin, and mainly producing collagen. Colonic myofibroblasts of proteins (types I, III and V), chondroitin proteoglycan, hyaluronic acid, fibronectin and muscle glycoprotein C. Herein, the polysaccharide, elastin, glycoprotein, collagen, fibronectin, nestin and laminin contained in the ECM are various polysaccharides, elastins, glycoproteins, collagens contained in ECMs well known in the art. Protein, fibronectin, nestin and laminin. For example, collagen may be collagens of type I, III, IV and V contained in natural ECM as is well known in the art.

ECMs suitable for use herein are commercially available. Examples of commercially available extracellular matrix cells include extracellular matrix proteins (Invitrogen, R & D systems) and Matrigel (Matrixgel ^TM, BD Biosciences) and the like. In certain embodiments, the ECM used in the culture methods herein comprises at least two different glycoproteins, such as two different types of collagen or one collagen and one laminin. The ECM can be a synthetic hydrogel extracellular matrix or a naturally occurring ECM. The most preferred ECM is made of Matrigel (

^{3-D Culture Matrix TM) (} R & D systems) provided, comprising laminin.

After isolation of the hepatocytes to be cultured, the cell suspension can be prepared from the proliferation medium described herein, mixed with ECM, and subjected to 3D culture. Cell culture media (also referred to as basal media) suitable for use in the methods described herein can be any cell culture media, particularly cell culture media for human cell culture. A preferred cell culture medium is a defined synthetic medium buffered with a carbonate-based buffer to a pH of 7.2-7.6, preferably 7.4. For example, a suitable medium is serum-free (eg, free of fetal bovine serum or calf serum), and insulin-containing modified DMEM/F12 or modified RPMI medium. Generally, in place of fetal bovine serum, eliminating the effects of unknown growth factors, an appropriate amount of serum replacement such as B27 supplement (Gibco) can be added to the cell culture medium of the present invention. If necessary, other nutrients needed to maintain cell growth may be added to the culture medium, including but not limited to L-glutamine-added reagents (such as GlutaMAX-I) and pH adjusters (such as maintaining medium pH stability). Reagents such as HEPES, primary cell culture antibiotics (such as primocin), and penicillin-streptomycin. The amount of these additives added can be determined by a conventional method as the case may be. For example, the final concentration of primary cell culture antibiotics can range from 50 to 200 ug/ml. In certain embodiments, nicotinamide and N-acetylcysteine are also added to the basal medium to provide the desired environment for cell growth. The amount of nicotinamide added is usually from 1 to 50 mM, for example from 10 to 20 mM. The amount of N-acetylcysteine added is usually from 0.5 to 20 mM, for example, from 1 to 5 mM.

In certain embodiments, one or more of a BMP inhibitor, a Wnt agonist, a growth factor, a Rock inhibitor, a P38 inhibitor, and a cAMP activator are also added to the cell proliferation medium of the present invention. In certain embodiments, at least a BMP inhibitor, a Wnt agonist, a growth factor, and a Rock inhibitor are added to the cell proliferation medium of the invention. In certain embodiments, the cell culture medium of the invention is supplemented with a BMP inhibitor, a Wnt agonist, a growth factor, and a Rock inhibitor, and optionally with one or both of a P38 inhibitor and a cAMP activator.

BMP binds to the receptor site of the TGF-β superfamily, thereby recruiting and activating SMAD family transcription factors and regulating gene expression. A BMP inhibitor is an agent that binds to a BMP molecule to form a complex that neutralizes BMP activity by preventing or inhibiting binding of a BMP molecule to a BMP receptor. Herein, a BMP inhibitor refers to inhibiting BMP-dependent activity in a cell to at most 90%, more preferably at most 80%, more preferably at most 70%, more preferably, relative to the level of BMP activity in the absence of the inhibitor. Formulations of up to 50%, more preferably up to 30%, more preferably up to 10%, more preferably 0%. As is known to those skilled in the art, BMP activity can be determined by measuring the transcriptional activity of BMP (Franceschi et al., 2000). The BMP inhibitor can be an agent that acts as an antagonist or inverse agonist, such as an antibody, which binds to the BMP receptor and prevents binding of the BMP to the receptor.

Several classes of natural BMP-binding proteins are known, including Noggin (Peprotech), notochordin and choline protein-like proteins (R&D sytems), follistatin, and follistatin-related proteins containing the follistatin domain. (R&D sytems), DAN and DAN-like proteins (R&D sytems), sclerostin (SOST, R&D sytems), core proteoglycans (R&D sytems) and α2 macroglobulin (R&D systems) containing the DAN cysteine knot domain . In certain embodiments, the BMP inhibitor also includes a TGF-beta inhibitor, such as A-83-01.

Several BMP inhibitors can be added simultaneously in the medium of the present invention. In certain embodiments, the BMP inhibitors used herein may be selected from Noggin, A-83-01, DAN, and DAN-like proteins (R&D sytems) including Cerberus and Gremlin. These diffusible proteins bind to BMP ligands with different affinities and inhibit these BMP ligands from approaching signal transduction receptors. Addition of any of these BMP inhibitors to the basal medium prevents hepatocyte loss, which would otherwise occur after about one week of culture.

The amount and frequency of BMP inhibitors added to the basal medium can be determined based on the inhibitory activity of the different inhibitors, typically the final concentration of the BMP inhibitor in the medium is in the range of 0.5-800 ng/ml of medium. For example, in certain embodiments, noggin is used herein. Noggin is an important signaling molecule that plays an important role in embryonic development of the animal's body. The release of the notochord regulates the expression of BMP during growth and development. As a secreted polypeptide, noggin binds to a TGF-β superfamily member such as bone morphogenetic protein-4 (BMP-4), rendering it inactive. Usually, the final concentration of noggin in the medium is 0.5-500 ng/ml. In certain embodiments, the final concentration of noggin is 1-200 ng/ml, such as 1-150 ng/ml, 1-100 ng/ml, 1-50 ng/ml, 5-15 ng/ml, or 20-100 ng/ml. In certain embodiments, a TGF-β inhibitor, such as A-83-01, is used herein as a BMP inhibitor; typically, it has a final concentration in the culture medium of 100-800 nM, such as 300-800 nM, 300-600 nM or 400-600nM. In certain embodiments, noggin and A-83-01 are added to the culture medium; typically, the final concentration of noggin is 1-200 ng/ml, such as 50-150 ng/ml or 5-15 ng/ml, A- The final concentration of 83-01 is 300-600 nM, such as 400-600 nM. In the course of culturing the liver cells, it is preferred to add a BMP inhibitor to the medium every two days, preferably every three days.

The Wnt signaling pathway functions when Wnt proteins bind to cell surface receptors of seven transmembrane receptors (Frizzled family members). This results in activation of the Dishevelled family of proteins, which results in inhibition of the disruption complex of the protein comprising axin, GSK3 and protein APC to inhibit the degradation of βcatenin. The resulting nuclear-enriched βcatenin enhances transcription through the TCF/LEF family of transcription factors.

The inventors have found that the addition of at least one Wnt agonist to the basal medium is essential for hepatocyte proliferation. Herein, a Wnt agonist is defined as an agent that activates TCF/LEF-mediated transcription in a cell. Thus, Wnt agonists are selected from true Wnt agonists that bind to and activate members of the Frizzled receptor family (including any and all Wnt family proteins), inhibitors of intracellular beta-catenin degradation, and activators of TCF/LEF. The Wnt agonist stimulates Wnt activity in the cell by at least 10%, more preferably at least 20%, more preferably at least 30%, more preferably at least 50%, more preferably relative to the level of Wnt activity in the absence of the Wnt agonist. At least 85%, more preferably at least 100%. Wnt activity can be determined by measuring the transcriptional activity of Wnt as known to those skilled in the art, for example by pTOP FLASH and pTOP FLASH Tcf luciferase reporter constructs (Ma et al., 2015).

Wnt agonists include secreted glycoproteins including Wnt-1/Int-1, Wnt-2/Irp (Int-1 related protein), Wnt-2b/13, Wnt-3/Int-4, Wnt-3a, Wnt-4, Wnt-5a, Wnt-5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a/8d, Wnt-8b, Wnt-9a/14, Wnt-9b/14b/ 15. Wnt-10a, Wnt-10b/12, Wnt-11, Wnt-12, Wnt-13, Wnt-14, Wnt-14b, Wnt-15, Wnt-16. An overview of human Wnt proteins is provided in "THE WNT FAMILY OF SECRETED PROTEINS" (https://www.rndsystems.com/cn/resources/articles/wnt-family-secreted-proteins), R&D Systems Catalog, 2004. Other Wnt agonists include the R spondin family of secreted proteins involved in the activation and regulation of the Wnt signaling pathway and are composed of four members R spondin 1, R spondin 2, R spondin 3 and R spondin 4 ; Wnt agonists Also included is Norrin (Norrie or NDP), which functions similarly to the secretory regulatory protein of the Wnt protein, binds to the Frizzled 4 receptor with high affinity and induces activation of the Wnt signaling pathway (Zhang et al., 2017). A small molecule agonist of the Wnt signaling pathway, an aminopyrimidine derivative (Lin et al., 2016), has recently been identified and is also explicitly included in Wnt agonists.

In certain embodiments, the Wnt agonist is a GSK inhibitor. Known GSK inhibitors include small interfering RNA (siRNA; Cell Signaling), lithium (Sigma), 1-Azakenpaullone (selleck), TWS119 (selleck), SB216763 (selleck), CHIR-99021, CHIR-98014, and can block GSK3 FRAT family members interacting with axin and FRAT-derived peptides. Methods and assays for determining the level of inhibition of GSK3 are known to those skilled in the art and include, for example, the methods and assays described (Dandekar et al., 2017; Uwai et al., 2016).

Several Wnt agonists can be added simultaneously. The amount of each Wnt agonist added is also related to the agonistic activity of different Wnt agonists, typically in the range of 1-1500 ng/ml basal medium. For example, suitable addition amounts include 1-800 ng/ml, 1-500 ng/ml, 1-300 ng/ml, 1-200 ng/ml, 1-100 ng/ml, 20-300 ng/ml, 20-100 ng/ml, and the like. Inside.

In certain embodiments, Wnt agonists for use herein include one or more of R spondin 1-4, Norrin, and GSK inhibitors. In certain embodiments, the Wnt agonist is R spondin, a GSK inhibitor, and/or Wnt3 (eg, Wnt 3a). Preferably, when a protein of the R spondin family is used as a Wnt agonist, its final concentration in the medium may be in the range of 50-1500 ng/ml, such as 50-1000 ng/ml, 50-500 ng/ml, 50- 200 ng/ml or 50-150 ng/ml; when a GSK inhibitor such as CHIR-99021 or CHIR-98014 is used as a Wnt agonist, the final concentration is in the range of 0.1-20 uM, such as 1-5 μM, 5-15 μM or 3 -12 μM; when Wnt3 such as Wnt3a is used as a Wnt agonist, the final concentration may be in the range of 1-1000 ng/ml, such as 10-500 ng/ml, 300-600 ng/ml, 400-600 ng/ml or 50-250 ng. /ml. During the culture of hepatocytes, fresh medium is preferably replaced every three days.

The growth factor added to the cell proliferation medium is typically a purified growth factor, which may be a natural, semi-synthetic or synthetic growth factor. In certain embodiments, the growth factor added to the basal medium is a mitotic growth factor, the growth factor family comprising epidermal growth factor (EGF, Peprotech), transforming growth factor beta (TGFβ, Peprotech), basic fibroblasts Cell growth factor (bFGF, Peprotech), brain-derived neurotrophic factor (BDNF, R&D Systems), hepatocyte growth factor and keratinocyte growth factor (KGF, Peprotech). Such growth factors are commercially available, for example, Peprotech's epidermal growth factor, transforming growth factor beta, basic fibroblast growth factor, and keratinocyte growth factor can be used; brain-derived properties provided by R&D Systems can be used Neurotrophic factor. EGF is an effective mitogen for a variety of cultured ectoderm and mesoderm cells and has important implications for in vivo and in vitro differentiation of specific cells and differentiation of certain fibroblasts in cell culture. The EGF precursor is in the form of a membrane-bound molecule that is cleaved by proteolysis to produce a 53 amino acid peptide hormone that stimulates cells. Therefore, a preferred mitotic growth factor is EGF. EGF can be replaced with TGF-β, and KGF can be replaced with FGF2 or FGF10. One or several mitotic growth factors may be added, and the amount of each mitotic growth factor may be determined according to its biological activity, for example, the amount of each mitotic growth factor is usually 0.1-1000 ng/ml (final concentration), preferably 1 Within the range of 500 ng/ml. For example, EGF can be added to the basal medium at a concentration of 5-500 ng/ml. A preferred final concentration range is from 5 to 200 ng/ml, more preferably from 20 to 100 n/ml, still more preferably from 20 to 80 ng/ml. In certain embodiments, a preferred concentration is at least 10, 18, 28, 36, 45 or 50 ng/ml and no more than 500, 400, 300, 200, 150 or 100 ng/ml. The same concentration can be used for FGF, preferably for FGF10 or FGF2. If more than one FGF, such as FGF2 and FGF10, is used, the concentration of FGF is defined as described above and refers to the total concentration of FGF used. In certain embodiments, when present, the final concentration of FGF10 can range from 1 to 100 ng/ml, such as from 1 to 50 ng/ml, from 1 to 20 ng/ml, or from 5 to 15 ng/ml. In certain embodiments, when present, the final concentration of FGF2 can range from 0.1 to 10 ng/ml, such as from 0.1 to 5 ng/ml or from 0.1 to 2 ng/ml. During the culture of hepatocytes, the medium is preferably changed every three days. Any member of the bFGF family can be used. Preferably, FGF2 or FGF10 is used. In certain embodiments, the mitotic growth factor is selected from one or more of the group consisting of: EGF, TGF-β, KGF, FGF10, and FGF2. In certain embodiments, the mitotic growth factors are: EGF, TGF-β and KGF; EGF, TGF-β and FGF2; EGF, TGF-β and FGF10; EGF and KGF; EGF and FGF2; EGF and FGF10; TGF-β and KGF; TGF-β and FGF2; or EGF, FGF2 and FGF10.

Rock (Rho kinase) inhibitors prevent anoikis, especially when culturing a single stem cell. Suitable Rock inhibitors are preferably selected from the group consisting of Y 27632 (selleck), HA 1077 (Cayman Chemical) and H1152 (Tocris Bioschience) and the like. The amount of the Rock inhibitor can be a conventional amount. For example, in certain embodiments, the final concentration of a Rock inhibitor such as Y 27632 is in the range of 0.5-50 μM, such as 5-20 μM or 5-15 μM. In culturing the hepatocytes, it is preferred to continuously add the Rho kinase inhibitor, such as Y 27632.

The component that can also be added to the basal medium is an inhibitor of the P38 signaling pathway. P38 protein kinase is a tyrosine phosphoprotein kinase isolated and purified by Han et al. using endotoxin to stimulate mammalian cells. P38 is the most important member of the MAPK family to control inflammation, which is activated by physiological stress, lipopolysaccharide, osmotic stress, and ultraviolet radiation. Key enzymes of the P38 pathway include TAK, ASK, and MLK of MKK3, MKK6, and MAPKKK of the MAPKK class. TAK is activated by TAK-binding protein (TAB) and mediates signal transduction of transforming growth factor (TGF-β). TAK can also activate MKK4, which in turn activates P38. Nuclear translocation occurs after P38 activation and phosphorylation and activation of many protein kinases and transcription factors. Inhibitors of the P38 signaling pathway include SB203580, Doramapimod (BIRB796), SB201190, LY2228820, VX-702 (inhibition of p38a MAPK), PH-797804, VX-745 (acting on p38a), TAK-715 (acting on p38a), BMS -582949 (inhibition of p38a MAPK), Losmapimod (GW856553X, R-1503/Ro4402257), Pexmetinib (ARRY-614), Skepinoe-L. The present inventors have found that the addition of one or more of the P38 signaling pathway inhibitors to the liver organ culture can improve the proliferation rate of the organoids. The amount of the P38 signaling pathway inhibitor such as SB202190 is usually in the range of 1-50 μM (final concentration). Preferably, SB202190 is used in an amount ranging from 1 to 20 μM, such as from 5 to 15 μM. The present inventors have found that the addition of a P38 signaling pathway inhibitor (such as SB202190) to the basal medium has little effect on the cell proliferation rate, but can delay the loss of hepatocyte characteristics. When cultured for 28 days, the RT- can be utilized. The expression of the biliary cell marker KRT19 was detected by PCR or immunohistochemistry.

Another component that can also be added to the basic culture is the eukaryotic adenylate cyclase (AC) activator (cAMP agonist), such as Forskolin. Forskolin is a ubiquitous cAMP agonist in a variety of cell types and is commonly used to improve cAMP levels in cell physiology studies. The present inventors have found that the addition of a cAMP agonist during the cultivation of liver organs has a certain effect on maintaining the proliferation of adult liver organs. The concentration of the cAMP agonist in the medium can generally be in the range of 1-200 [mu]M, such as 1-100 [mu]M, 5-50 [mu]M or 5-15 [mu]M.

Thus, in certain embodiments, it is practiced in an extracellular matrix-containing cell culture medium supplemented with at least a bone morphogenetic protein (BMP) inhibitor, a Wnt agonist, a mitotic growth factor, and a Rock inhibitor as described herein. Appropriate amounts of bone morphogenetic protein (BMP) inhibitors, Wnt agonists, mitotic growth factors and Rock inhibitors can be added to the cell culture medium at the same time or at the wrong time. For example, as described above, the medium is changed every three days, so bone morphogenetic protein (BMP) inhibitors, Wnt agonists, mitotic growth factors, and Rock inhibitors can be added simultaneously on the same day, and all cultures are changed every three days. base. The addition amount and timing of addition of a bone morphogenetic protein (BMP) inhibitor, a Wnt agonist, a mitotic growth factor, and a Rock inhibitor can be appropriately adjusted according to the current culture state. Any one or both of the inhibitors of the P38 signaling pathway and the cAMP agonist described above are optionally added to the medium.

In certain embodiments, the cell proliferation medium described herein comprises Noggin and A-83-01 as BMP inhibitors, EGF, FGF10 and FGF2 as mitotic growth factors, R spondin as a Wnt agonist, and as Rho inhibitor Y 27632, supplemented with GlutaMAX-I, medium pH stable reagent, primary cellular antibiotic, B27 serum replacement, nicotinamide and N-acetylcysteine. The amount of each component in the medium can be as described in any of the preceding embodiments. Preferably, in the medium, the final concentration of Noggin is 1-20 ng/ml, preferably 5-15 ng/ml, and the final concentration of EGF is 10-100 ng/ml, preferably 30-80 ng/ml, and the final concentration of FGF10 is 1. -20 ng/ml, preferably 5-15 ng/ml, the final concentration of FGF2 is 0.1-5 ng/ml, preferably 0.5-2 ng/ml, and the final concentration of R spondin is 10-200 ng/ml, preferably 50-150 ng/ml, Y The final concentration of 27632 is 1-20 μM, preferably 5-15 μM. In certain embodiments, the cell culture medium further comprises a GSK inhibitor, such as CHIR99021 and/or Wnt3a, as a Wnt agonist, which may have a final concentration of 1-5 [mu]M and 300-700 ng/ml, respectively. In certain embodiments, the medium described herein further comprises any one, any two or all three of a P38 inhibitor such as SB202190, a cAMP activator such as Forskolin, and BMP7, and when present, the final concentration can be They are 5-15 μM, 5-15 μM and 10-40 ng/ml, respectively.

The cell culture medium herein supports the isolation of isolated hepatocytes in a three-dimensional culture comprising Matrigel as an extracellular matrix. This medium promoted the cultured cells to survive for 25 days.

The cultivation can be carried out under conventional conditions, for example, in a 5-10% CO ₂ environment. The temperature can be a conventional hepatocyte culture temperature. In certain embodiments, the cultures described herein comprise preparing a liver cell suspension using the medium described herein (cell density can be from 1 x 10 ³ to 1 x 10 ⁸ per 200 μl suspension), with extracellular matrix After mixing, it is planted in a corresponding container, placed in an incubator, and after the droplets are solidified, the medium described herein is added to continue the cultivation. Typically, it is mixed with the extracellular matrix in an equal volume ratio.

The culture methods described herein can also be used for the culture of epithelial stem cells sorted from the pancreas and lungs.

When a single hepatocyte is cultured by the method and medium described herein, the biliary cell is transdifferentiated in the later stage, and the differentiation medium provided herein can be used to obtain the characteristics of the hepatocyte in the partially differentiated cholangiocarcinoma cell to form a complex hepatocyte. - Organoid structure of biliary cells. A preferred cell differentiation medium is a defined synthetic medium buffered with a carbonate-based buffer to a pH of 7.2-7.6, preferably 7.4. For example, a suitable medium is serum-free (eg, free of fetal bovine serum or calf serum), and insulin-containing modified DMEM/F12 or modified RPMI medium. Generally, in place of fetal bovine serum, eliminating the effects of unknown growth factors, an appropriate amount of serum replacement such as B27 supplement (Gibco) can be added to the cell culture medium of the present invention. If necessary, other nutrients needed to maintain cell growth may be added to the culture medium, including but not limited to L-glutamine-added reagents (such as GlutaMAX-I) and pH adjusters (such as maintaining medium pH stability). Reagents such as HEPES, primary cell culture antibiotics (such as primocin), and penicillin-streptomycin. The amount of these additives added can be determined by a conventional method as the case may be. For example, the final concentration of primary cell culture antibiotics can range from 50 to 200 ug/ml. In certain embodiments, N-acetylcysteine and BMP7 are also added to the basal medium to provide the desired environment for cell growth. The amount of N-acetylcysteine added is usually from 0.5 to 20 mM, for example, from 1 to 5 mM. The final concentration of BMP7 is 10-40 ng/ml, for example 20 ng/ml.

In certain embodiments, the differentiation medium of the invention may further comprise one or any of a variety of growth factors, Rock signaling pathway inhibitors, Notch signaling pathway inhibitors, and dexamethasone as described herein.

The growth factor added in the cell differentiation medium preferably includes a fibroblast growth factor and a hepatocyte growth factor. Preferably, the fibroblast growth factor is FGF10 and FGF2; when included, the final concentration of FGF10 can range from 1 to 100 ng/ml, such as from 1 to 50 ng/ml, from 1 to 20 ng/ml or from 5 to 15 ng/ml. The final concentration of FGF2 can range from 0.1 to 10 ng/ml, such as from 0.1 to 5 ng/ml or from 0.1 to 2 ng/ml. FGF19 is also added in certain embodiments, and when present, the final concentration of FGF19 is 10-200 ng/ml, such as 50-150 ng/ml. Hepatocyte growth factor (HGF) is also included in certain embodiments, and when present, has a final concentration of 10-40 ng/ml.

The final concentration of the Rock signaling pathway inhibitor, such as Y 27632, added in the cell differentiation medium is in the range of 0.5-50 μM, such as 5-20 μM or 5-15 μM. The final concentration of dexamethasone added in the cell differentiation medium may be 0.01-30 μM, such as 1-10 μM or 1-5 μM.

Herein, the Notch signaling pathway inhibitor may be selected from one or more of DAPT (GSI-IX), MK-0752, RO4929097, Semagacestat (LY450139), LY411575, Dibenzazepine (YO-01027), Avagacestat, Crenigacestat, NGP 555. Kind. Typically, the final concentration of the Notch signaling pathway inhibitor is 0.1-50 [mu]M, such as 0.1-10 [mu]M or 5-30 [mu]M in the cell differentiation medium.

Therefore, the differentiation of liver cell-like organs is based on a medium based on a medium for growth of mammalian cells, a reagent supplemented with L-glutamine, a pH adjuster for maintaining the pH of the culture medium, and primary cell culture. Antibiotics, serum replacement and N-acetylcysteine, plus BMP7, growth factor, Rock signaling pathway inhibitor, Notch signaling pathway inhibitor, and one or any of a variety of extracellular matrix-containing cells of dexamethasone It was carried out in a differentiation medium and the medium was changed every three days. Preferably, the cell differentiation medium comprises BMP7, a growth factor, a Rock signaling pathway inhibitor, a Notch signaling pathway inhibitor, and dexamethasone.

In certain embodiments, the cell differentiation medium comprises growth factors FGF10, FGF2 and FGF19, Y27632, a Rock signaling pathway inhibitor, and a Notch signaling pathway inhibitor, DAPT, supplemented by GlutaMAX-I, maintaining a pH-stabilized medium pH regulator, primary cell antibiotic, B27 serum replacement, BMP7 and N-acetylcysteine. The medium was induced to differentiate, and after about 2 weeks, the transdifferentiated cholangio cells were partially restored to the characteristics of hepatocytes and functioned as hepatocytes.

In certain embodiments, the cell differentiation medium of the invention is serum-free and insulin-containing modified DMEM/F12 or modified RPMI medium is a basal medium containing GlutaMAX-I, medium pH stable reagent, primary cell Antibiotic, B27 serum substitute, BMP7, N-acetylcysteine, FGF10, FGF2, Y 27632, HGF, dexamethasone and DAPT, optionally containing FGF19; wherein, in the medium, the final concentration of BMP7 is 10 -40 ng/ml, the final concentration of FGF10 is 5-15 ng/ml, the final concentration of FGF2 is 0.1-5 ng/ml, the final concentration of Y 27632 is 5-30 μM, and the final concentration of HGF is 10-40 ng/ml. The final concentration of rice pine is 1-5 μM, the final concentration of DAPT is 5-30 μM, and the final concentration of the optional FGF 19 is 50-200 ng/ml.

After approximately 2 weeks of proliferation and 2 weeks of differentiation, a structure similar to the organoid structure of hepatocyte-cholangiocarcinoma obtained using intact liver was formed. Histological analysis of these organoids also indicated that it retained the basic hepatocyte-choline tubular cell structure, the presence of all differentiated liver epithelial cell types, and the absence of non-epithelial components.

Also provided herein is any of the cell culture media described in any of the preceding embodiments. In certain embodiments, the serum-free cell culture medium provided herein is a medium based on a medium for mammalian cell growth, supplemented with an agent that supplements L-glutamine, and maintains a pH at which the pH of the medium is stable. Modulators, primary cell culture antibiotics, serum replacements, N-acetylcysteine and optionally nicotinamide, as well as growth factor and Rock signaling pathway inhibitors, optionally with BMP inhibitors, Wnt activation One or more of a dose, a P38 signaling pathway inhibitor, a Notch signaling pathway inhibitor, dexamethasone, BMP7, and a cAMP activator. In these embodiments, the optional component may be optionally added to the medium, such as nicotinamide, a BMP inhibitor, a Wnt agonist, a P38 signaling pathway inhibitor, a Notch signaling pathway inhibitor, and a ground. A cell proliferation medium and a cell differentiation medium according to any of the preceding embodiments are prepared by using dexamethasone, BMP7, a cAMP activator or the like.

In certain embodiments, provided herein is a kit comprising the serum-free cell culture medium of any of the embodiments herein. Preferably, the kit comprises the proliferation medium and differentiation medium of any of the embodiments herein. Preferably, the kit further contains an ECM. In certain embodiments, the kit contains the individual components of the individually packaged medium for formulating the medium described herein, such as individually packaged basal medium, BMP7, bone morphogenetic protein (BMP) inhibitor, Wnt agonist. , growth factors and Rock inhibitors, optionally inhibitors of the P38 signaling pathway and/or cAMP agonists, Notch signaling pathway inhibitors, and dexamethasone; kits may optionally also be packaged individually or as a mixture One or more of GlutaMAX-I, a pH regulator that maintains a pH stability of the culture medium, a primary cellular antibiotic, a B27 serum replacement, nicotinamide, and N-acetylcysteine are provided. In certain embodiments, the kit may contain separately packaged L-glutamine-added reagents, pH-stabilizing pH regulators, primary cell culture antibiotics, serum replacements, N- Basal media for acetylcysteine, growth factor and Rock signaling pathway inhibitors, and individually packaged nicotinamide, BMP inhibitors, Wnt agonists, P38 signaling pathway inhibitors, Notch signaling pathway inhibitors, dexamethasone One or more of BMP7 and cAMP activators.

Also included herein is the use of the cell culture media described herein in liver cell culture, particularly in prolonging the survival of liver cells, maintaining the differentiation characteristics of mature hepatocytes, and/or achieving the persistence of differentiated hepatocytes. Also included herein is the use of the cell culture media described herein in the preparation of liver-like organs.

In certain embodiments, also provided herein is a cell culture comprising liver cells prepared using the methods described herein. Optionally, the cell culture further comprises a cell culture medium as described herein. In certain embodiments, the culture contains the medium, liver cells, and ECM described herein.

In certain embodiments, the cell culture is a histoid of hepatocyte-cholangiocarcinoma cells comprising a central lumen lined with bile duct-like epithelial cells, which are passed through a cell culture medium as described herein. Produced by the transformation of cultured liver cells. Preferably, the organoid-biliary cell-like organ is obtained using the methods described herein.

Also provided herein are assemblies of organoids of hepatocyte-cholangiocarcinoma cells, each aggregate comprising more than 10, preferably more than 20, more preferably more than 40 organoids. The organoid assembly of hepatocyte-cholangiocarcinoma preferably comprises at least 20% viable cells, more preferably at least 50% viable cells, more preferably at least 60% viable cells, more preferably at least 70% viable cells, more preferably At least 80% of living cells, more preferably at least 90% of viable cells. Cell viability can be assessed by Hoechst staining or propidium iodide staining in FACS.

The hepatocyte-cholangiocarcinoid organ prepared using the methods described herein or in the cell culture medium comprises a lumen lining the bile duct-like epithelium. The chamber is open at successive time intervals to release the contents into the medium. The organoids can be passaged and can be cultured for at least 6 months without losing important properties.

The cell cultures described herein, particularly hepatocyte-cholangiocarcinoma cells, can be substituted for commercial primary hepatocyte cell lines for drug development, drug screening, and toxicity determination of food supplements. Accordingly, provided herein are cell cultures described herein, particularly the use of such organoids in drug development, drug screening, toxicity assays, or regenerative medicine. In addition, the cell cultures described herein, especially the organoids, can also be used for the detection of cytochrome P450 enzyme activity, liver detoxification function research and the like. In detecting the enzymatic activity of the organ-like organ, the change of the enzyme activity of the CYP450 family can be directly detected by high performance liquid chromatography after adding the corresponding substrate, and the corresponding inducer can also be added to observe the change of the enzyme activity. In the detection of liver metabolic activity, phenacetin, coumarin, dextromethorphan, etc. can be added exogenously to observe liver detoxification function. In addition, transplantation of an organoid described herein into a liver-injured animal can also repair a damaged liver.

A large number of liver cells can be provided using the methods and media described herein. China is a big country of hepatitis B. Although early antiviral treatment can relieve the development of liver cirrhosis and liver failure to a certain extent, many patients still develop liver failure. The poor quality of life caused by liver failure in patients seriously affects the quality of life of patients, and in order to maintain the survival of patients, heavy medical expenses often impose a serious burden on families. Unlike dialysis for kidney failure, there is no mature artificial liver that can mimic the function of the liver. Due to the complexity of the structure of the liver and the diversity of functions, the establishment of artificial liver has not achieved a good breakthrough. Mechanically simulating liver function is not as feasible as using hepatocytes cultured in vitro to help exercise liver function. The use of the method described herein can obtain a large number of cells capable of stably performing liver function, which will make the research of artificial liver a big step.

The invention will be elucidated below in the context of specific embodiments. It is understood that the examples are merely illustrative and are not intended to limit the scope of the invention. The methods and reagents obtained in the examples, unless otherwise stated, are routine methods and reagents in the art.

I. Materials and methods

1. Tissue block separation hepatocyte method

Primary hepatocytes are isolated from adjacent tissues of liver cancer patients. Fetal liver cells are derived from the liver tissue of a hospital aborted fetus. Liver cancer tissue adjacent or aborted fetus liver tissue was isolated tissue culture medium (Advance DMEM / F12 was added GlutaMAX ^TM, Hepes, penicillin / streptomycin, primocin, Rock signaling pathway inhibitor), washed three times in order to remove blood , impurities, bacteria, cut the tissue into small pieces (<0.1mm ³ ) by surgical scissors, transfer to the centrifuge tube, wait for the liver tissue block to deposit on the bottom of the tube, discard the supernatant, and use the appropriate amount of 6mg/ml IV The collagenase solution (containing the Rock signaling pathway inhibitor) was resuspended and placed in a 37 ° C incubator for 1 h. Be no visible block structure, with high glucose DMEM (added GlutaMAX ^TM, penicillin / streptomycin, 10% FBS) to terminate the reaction, 300g centrifugation 5min, discarded the supernatant. Washed with hepatocyte culture medium (high glucose DMEM was added GlutaMAX ^TM, penicillin / streptomycin, 10% FBS; percol solution; calcium ion, the magnesium ion solution is EBSS) cells were resuspended using differential centrifugation to remove dead cells and Cell debris, lysing red blood cells with red blood cell lysate. Finally, it was resuspended in hepatocyte culture medium and filtered through a 70 μm filter to obtain individual hepatocytes. In order to further obtain hepatocytes with higher purity and better vigor, we used the flow sorting method to treat the hepatocytes obtained above. The filtered hepatocytes were resuspended at 1*10 ⁷ /mL, and the human EPCAM antibody (Miltenyi Biotec) was stained at a ratio of 1:250, and the dead cells were labeled by DAPI, and the viable cell was sorted by flow cytometry. Hepatocyte.

2. Hepatocyte culture method

The following 29 kinds of proliferation medium were used to prepare hepatocyte suspension (cell density: 1×10 ⁶ per 200 μl suspension), mixed with the same volume of matrigel, planted in a six-well plate, and placed in an incubator for gel drop. After solidification, the culture was continued by adding 3 ml of the corresponding medium, and the culture was carried out at 37 ° C in a 5% CO ₂ atmosphere. These 29 media were prepared by modifying DMEM/F12 (Invitrogen)-based medium and adding the additives shown in Table 1 as shown in Table 2.

Table 1

Table 2

The proliferated liver-like organs were induced to differentiate using the following four differentiation media. When it is desired to prepare a functional hepatocyte-cholangiocarcinoid organ, the following four differentiation media are added to a well-proliferated liver organ. The cultivation was carried out at 37 ° C in a 5% CO ₂ atmosphere. These four media were prepared by modifying DMEM/F12 (Invitrogen)-based medium and adding the additives shown in Table 3 as shown in Table 4.

table 3

Table 4

分化培养基1Differentiation medium 1	AA	BB	CC	DD	EE	FF
分化培养基2Differentiation medium 2		BB	CC	DD	EE	FF
分化培养基3Differentiation medium 3	AA	BB	CC	DD	EE
分化培养基4Differentiation medium 4		BB	CC	DD	EE

3. Identification of hepatocyte-cholangiocarcinoma cell population

By single-cell sequencing analysis of hepatocyte-like organs cultured in different periods, as well as hepatocyte-cholangiocarcinoid organs, most of the cells in the pre-proliferation phase were found to be hepatocytes, and in the later stage, hepatocytes showed a tendency to differentiate into cholangiocarcinoma cells. After induction of differentiation, it can form a complex structure containing both hepatocytes, biliary cells, and stem cells.

4. Identification of related features and functions of hepatocyte organs and hepatocytes-cholangiocarcinoid organs

The obtained organoids were functionally identified in vitro and in vivo. In vitro identification methods mainly include morphology, gene expression and hepatocyte function identification.

Liver-like organs and hepatocyte-choline-like organ-like hepatocytes have a gene-wide expression profile similar to that of hepatocytes, which is expressed in mRNA levels and protein levels of hepatocytes, including: liver-related transcription factors. (HNF4A, HNF1A), secreted protein gene (ALB, SERPINA), cytoskeletal gene (KRT8), cell junction gene (CDH1), liver metabolism function related genes (CYP450 family, glucose metabolism related G6PC), etc., using QPCR to detect related genes mRNA expression level and identification of whole gene expression by RNA sequencing; detection of relative content of human albumin in culture medium by ELISA (Human Albumin ELISA Quantitation Set: E80-129); detection of liver-specific markers by immunofluorescence (goat-anti-ALB; rabbit-anti-HNF4A; rabbit-anti-FAH) expression of rabbit protein (rabbit-anti-KRT19) and connectin (mouse-anti-CDH1). The hepatocyte-cholangiocarcinoma cells also have biliary cell expression characteristics such as KRT19, and stem cell expression characteristics such as EpCAM.

In the functional identification, hepatocytes mainly include glycogen staining, oil red staining, indocyanine green and other liver-related specific staining, and high performance liquid chromatography to detect the cytochrome P450 family enzyme activity. The in vivo identification method comprises the above-mentioned cultured liver-like organs obtained by using the above-mentioned subculture method to obtain a small organ-like group, and the small group is digested into single cells by trypsin, and after the digestion is terminated, the single cells are obtained by filtration through a 70 μm filter. These cells were transplanted into 8-week-old liver-damaged mice by portal vein injection for treatment. The human serum albumin level in mice was detected by ELISA and the expression of specific human genes was detected by immunohistochemistry. The in vitro identification of biliary cell function mainly includes the uptake and excretion of fluorescein diacetate. In vivo, the expression of cholangiocarcinoma-associated genes was detected by immunohistochemistry mainly by transplanting the above-described hepatocyte-cholangiocarcinoid organ into the kidney under the renal capsule of SCID mice.

Second, the results

The results of cultured fetal liver cells in 29 mediums are shown in Figures 1-5, and Figures 1-5 show the formation of organoids on Day 1, Day 6, Day 12, Day 14, and Day 20, respectively. Happening. The figure shows that the best culture results were obtained using the ninth medium.

2. Establish long-term culture system of embryonic liver cell organs

The isolated primary hepatocytes were mixed with the same volume of matrigel at a density of 1*10 ⁶ per 200 μl at a density of 1*10 ⁶ per 100 μl, planted in a 6-well plate, and then transferred to an incubator for cultivation, until the droplets solidified. 3 ml of medium was added for 3D culture. After 3-5 days to form a typical organ-like structure, it is blown into a single-cell state, re-coated with matrigel, and after it is solidified, it is transferred to a bioreactor for suspension culture, and it takes about 14 days. A typical liver-like tissue is formed, and the organoids that form a complex structure are further cultured.

The results are shown in Figures 6 and 7. Figure 6 shows the formation of organoids in different samples in the bright field after 5-7 days of seeding. Using the medium of the present invention, primary hepatocytes can be cultured in vitro for a long period of time, and each passage is mechanically blown off. Figure 7 shows that after excised tissue blocks were mechanically sheared and collagenase digested into single cells, 3D culture was performed on Matrigel to form organoids, and cells that passed to 25th generation cells still showed good viability. The figure shows the formation of organoids in different samples under bright field after different passages.

The structure of the liver-like organs is a typical epithelioid-like organ structure, that is, a spheroid formed by a single layer of polar epithelium. Liver organ cells have proliferative ability in vitro, and the tumorigenicity of liver organ cells obtained in this experiment was tested. Specifically, the liver-like organ cells were subcutaneously injected into the groin of nude mice. The results showed that the liver-like organ cells were still unable to form tumors after 2 months of injection.

3. Embryonic liver organ formation efficiency

The isolated fetal liver cells were separately seeded into 24-well plates in a classification of 200, 500, 1000, 5000, 10000 per well, and fresh medium (ninth medium) was replaced every 3 days. The organ-forming efficiency was recorded by photographing every two days. When it was 14 days, the number of organoid formation in each well was counted. The above experiment was repeated three times or more, and the results are shown in Figures 8 and 9. Figure 9 shows that the formation efficiency of fetal liver organs is about 9%.

4, embryonic liver organs have liver related gene expression

The isolated primary hepatocytes were cultured in the same manner as in the second point described above, and a typical liver organ structure was formed in about 14 days. The appropriate amount of organs were taken for total RNA extraction, reverse transcription was used to form cDNA, and relevant genes were detected by QPCR. The situation of expression.

The result is shown in Figure 10. From the results of gene expression of ALB, HNF4A, HNF1A, and KRT8, fetal liver can maintain high hepatocyte characteristics. ALB accounts for about 80% of adult liver, and HNF4A, HNF1A, and KRT8 have higher gene expression levels than adult liver.

5. Low expression of AFP in embryonic liver organs

The isolated primary hepatocytes were cultured in the same manner as in the above point 2, and typical liver-like tissues were formed in about 14 days. After formation, an appropriate amount of organs were taken for total RNA extraction, and reverse transcription was used to form cDNA, and the expression of AFP was detected by QPCR.

The result is shown in FIG. The results showed that the expression of AFP in fetal liver tissue was relatively high. With the prolongation of culture time, the expression of AFP decreased gradually, and there was no significant difference between adult liver samples and adult liver samples.

6. Embryonic liver organs have histological features of the liver

The isolated primary hepatocytes were cultured in the same manner as in the above point 2, and typical liver-like tissues were formed in about 14 days. After the formation, an appropriate amount of organs were taken to fix the paraformaldehyde, and then subjected to paraffin embedding, 4 μm serial sectioning, H&E staining and the like.

The result is shown in FIG. The results showed that from the early liver organs to the one-month-old organ-like process, the organ-like organs had the characteristics of mature liver and showed typical hepatocyte characteristics: the nuclear large; the polygonal few were round, occasionally dual-nuclear The cells are in contact with each other with clear boundaries and arranged in a hepatic cord-like structure.

7. Embryonic liver organs express markers of mature hepatocytes

The isolated primary hepatocytes were cultured in the same manner as in the second point described above, and a typical liver organ structure was formed in about 14 days. The appropriate amount of organs were taken out and fixed with paraformaldehyde, embedded in paraffin, and serially sectioned at 4 μm. Antigen retrieval, blocking, primary antibody overnight at 4 ° C, secondary antibody at room temperature for 1 h, DAPI staining for 7 minutes, and mounting.

The result is shown in FIG. The results showed that the pre-cultured organs showed high expression of ALB, HNF4A, CDH1 and low expression of KRT19, which were characteristic of mature hepatocytes.

8. Embryonic liver organs express functional genes of mature hepatocytes

The isolated fetal liver cells were cultured in the same manner as in the second point described above, and typical liver-like tissues were formed in about 14 days. The appropriate amount of organs were taken for total RNA extraction, reverse transcription into cDNA, and mature hepatocytes were performed by QPCR. Functional gene testing.

The results are shown in Figures 14 and 15. From the gene expression results of SERPINA, CDH1, CYP3A4, SOX17, it can be seen that the liver organs can maintain relatively high liver function gene expression; from the gene expression results of CYP2B6, CYP2C18, CYP2C8, CYP2D6, CYP3A4, the liver can be seen The organoids can maintain relatively high levels of liver detoxification function.

9. Embryonic liver organs have mature liver function

The isolated fetal liver cells were cultured in the same manner as in the second point described above, and a typical complex organ-like structure was formed in about 21 days. The relevant functional experiments were taken out and studied according to the oil red staining method and the glycogen staining method. Functional staining.

The result is shown in Fig. 16. It can be seen from the results of oil red staining and glycogen staining that the liver-like organs have the function of mature liver.

10. Embryonic liver organs have the function of repairing liver of mice with liver injury

The organ-like organs that have formed a complex structure according to the above-mentioned point 2 are digested into single cells, and 1*10 ⁶ cells are transplanted into each mouse to 8-12 weeks of FRG mice, and the cells are injected by portal vein transplantation. After 48 hours, blood was taken to detect the secretion of human ALB, and then blood was taken every two weeks to detect the expression of human ALB until 2 months. Two months later, the mice were sacrificed, and the liver was removed, and paraformaldehyde fixation, paraffin embedding, serial sectioning, and H&E staining and immunohistochemical staining were performed.

The result is shown in Figure 17-20. The results show that liver-like organs can prolong the lifespan of FRG liver injury model mice. Specifically, transplantation of liver tube into immunodeficient FRG mice with liver injury revealed that the control group died within 1 month after withdrawal of NTBC water, while mice transplanted with liver-like organs generally lived. By 2 months, liver damage was cured and the difference was significant (Fig. 17). Furthermore, it can be seen from the results of immunofluorescence that the expression of the human gene ALB can be detected in the liver of FRG mice, suggesting that human hepatocytes are integrated into the liver of the injured mouse (Fig. 18). From the results of H&E staining, it can be seen that the damaged FRG mice can be seen after transplanting the liver-like organs, and the polygonal hepatocytes can be formed into a cord-like hepatocyte cord structure, while the negative control has a large number of necrotic structures; from FAH The results of immunohistochemistry showed that the liver's organoids were integrated into the damaged liver structure (Fig. 19). The content of human ALB in the blood of mice was measured on the 30th and 60th day respectively. From the experimental results, the amount of ALB was stably maintained in the range of 100-300 ng/ml within 2 months of transplantation (Fig. 20).

11. The tendency of embryonic liver organs to differentiate into bile duct cells in the later stage

Fig. 21 shows that the long-term in vitro culture of liver-like organs formed according to the second point culture has a tendency to transdifferentiate into biliary cells. From the results of immunofluorescence, it can be seen that during the pre-culture and P3 generation and before, ALB is in a state of high expression, and KRT19 is in a state of low or weak expression. At the late stage of culture, KRT19 slowly aggregates to the bile duct and the expression of ALB. Also reduced accordingly.

12. Establish adult liver organ culture system

The obtained adult liver (from the adjacent tissues of liver cancer patients) was subjected to mechanical shearing and type IV collagenase digestion. After no obvious blocky structure, the reaction was terminated, centrifuged at 800 rpm/min for 5 min, the supernatant was discarded, and 10% was added. The DMEM-high glucose medium of fetal calf serum was resuspended, filtered through a 70 μm mesh sieve, and the cell suspension was centrifuged at 800 rpm/min for 3 min at 4 ° C, and the supernatant was centrifuged. The deposited cells were lysed with red blood cell lysate, centrifuged, resuspended in the above medium, and assayed for hepatocyte viability by trypan blue staining. The adult hepatocytes isolated as described above were cultured as described in the above second point. The results showed that adult liver organs were cultured in vitro and exhibited a typical organoid structure for about one week and could be stably passaged (Fig. 22).

The EPCAM-positive and negative cell populations from the sorting were cultured separately, and it was found that typical organ-like structures were formed, but the negative efficiency was slightly lower than that of unsorted and EPCAM-positive cells (Fig. 23).

Individual hepatocytes isolated from the tissues were plated into 200-well plates at 200, 500, 1000, 5000, 10000 per well, and the organ-forming efficiency was photographed every two days. When it was 14 days, the number of organs in each well was counted. Figures 24 and 25 show the efficiency of adult liver organ formation. Adult liver organ formation efficiency is about 1%.

13. Adult liver-like organs have liver-related gene expression

Mature liver organs were cultured in the same manner as in the above-mentioned 12th point. At the third passage, appropriate amount of organs were taken to extract RNA, and cDNA was reverse transcribed to detect the expression of related genes by QPCR.

The result is shown in Fig. 26. From the results of gene expression of ALB, HNF4A, APOE and SERPINA, it can be seen that mature liver organs can maintain high hepatocyte characteristics, and the expression level of SERPINA is higher than that of adult liver tissues. ALB, HNF4A and APOE have higher expression levels. .

14. Markers expressing mature hepatocytes in adult liver organs

The isolated mature hepatocytes were cultured in the same manner as in the above point 12. The appropriate amount of organs were removed and fixed with paraformaldehyde, embedded in paraffin, serially sectioned at 4 μm, antigen-repaired, blocked, primary antibody at 4 ° C overnight, two Anti-room temperature 1 h, DAPI staining for 7 minutes, mounting.

The result is shown in Fig. 27. The results showed that the pre-cultured organs expressed HNF4A and CDH1 with high expression of mature hepatocytes.

15. Functional genes of mature liver cells expressed by adult liver organs

As can be seen from Fig. 28, the mature liver-like organs highly express the genes CYP3A4, CYP2B6, CYP2C8, CYP2C18, and CYP2D6 of the cytochrome P450 family. It is indicated that the mature liver organs cultured have the detoxification function of the liver.

16. Early adult liver organs are mainly composed of liver cells and liver stem cells.

Mature liver organs were cultured in the same manner as in the above-mentioned point 12. The appropriate amount of the apparatus was taken out and digested into single cells at the third and previous passages, and single cell sequencing was performed by the method of 10×Genomics to observe the cell composition.

As a result, as shown in Fig. 29, the early liver-like organs are mainly composed of hepatocytes and hepatic stem cells. Among them, hepatocytes account for the majority and high expression of ALB.

17. Adult liver organs have the function of mature liver cells

The mature liver-like organs were cultured in the same manner as in the above-mentioned 12th point, and complex organ-like structures were formed, and the relevant functional experiments were carried out, and the related functions were carried out according to the oil red staining method, the glycogen staining method, and the phthalocyanine green staining method. dyeing.

The result is shown in FIG. From the results of oil red staining, glycogen staining, and phthalocyanine green staining, it can be seen that the functional functions of the adult liver take up low-density lipoprotein, store glycogen, absorb indocyanine green, and have the function of mature liver.

18, adult liver organs will have a tendency to differentiate into biliary cells in the later stage

The mature liver organs were cultured in the same manner as in the above-mentioned point 12. From the experimental results shown in Fig. 31, it was revealed that the expression of the biliary cell marker KRT19 was observed in the adult liver organs after the P3 generation in the late culture stage. The level gradually increased and the expression level of ALB gradually decreased. It is indicated that adult liver organs have a tendency to differentiate into cholangiocarcinoma cells.

19. In the late stage of differentiation, liver organs form hepatocytes - biliary cell organs

The mature liver-like organs were cultured in the same manner as in the above-mentioned point 12, and differentiation was induced with the six differentiation media described in Table 4 when the adult liver-like organs in the latter stage of the third passage were as long as about 80% (about 7 days). The medium was changed every 3 days and the growth of the cells was recorded. After 14 days of induction, organs were collected, RNA was extracted and reverse transcription was performed to detect the expression of genes related to hepatocytes, biliary cells and stem cells.

The result is shown in Figures 32-33. It can be seen from Fig. 32 that after induction with 14 different differentiation media, the proliferation of liver-like organs was inhibited and there was no significant difference between the different treatment groups. Among them, the expression level of hepatocyte markers corresponding to the organoid induced by the differentiation medium No. 4 was the highest. Therefore, medium No. 4 was used as a differentiation medium for liver-like organs (Fig. 33).

20, mature hepatocytes - bile duct cell organs have a variety of cellular components

The above-mentioned 19-point culture method was used to induce differentiation by the fourth medium to form a mature hepatocyte-cholangiocarcinoid-like organ structure. Appropriate organs were taken, digested into single cells, and single cell sequencing was performed using 10×Genomics to observe the cell composition.

As a result, as shown in Fig. 34, the differentiated mature hepatocytes-cholangiocarcinoid organs contained hepatocytes, biliary cells, and hepatic stem cells. Hepatocytes express high levels of hepatocyte markers ALB, TTR and RBP4. Stem cells express EpCAM, ALB, KRT19 and SOX9 genes, and biliary cells express KRT19, EpCAM and SOX9 genes.

21. Mature hepatocytes - biliary cell-like organs are similar to liver tissue in gene expression profiles.

The mature hepatocyte-cholangiocarcinoid organ structure was obtained by the aforementioned 19-point culture method. Take appropriate amount of organs for RNA construction and sequencing. Detect its gene expression.

As can be seen from Fig. 35, the differentiated mature hepatocyte-cholangiocarcinoid organ is closer to the adult liver tissue in the gene expression profile than the later liver organ, and the matured hepatocyte is found by gene enrichment analysis. - The bile duct cell-like organs are highly enriched with genes involved in cell metabolism and biooxidation, which are consistent with the functions of mature hepatocytes.

22. Mature hepatocytes - biliary cell organs have the function of liver organs

The mature hepatocyte-cholangiocarcinoid organ structure was obtained by the aforementioned 19-point culture method. Appropriate organs were taken for glycogen staining, oil red staining, and phthalocyanine green staining, and the viability of CYP3A4 was identified to determine whether it has hepatocyte function.

The results are shown in Figures 36-37. From the results of Figure 36, it can be seen that the mature hepatocyte-cholangiocarcinogen functionally ingests low density lipoprotein, stores glycogen, and absorbs indocyanine green. Moreover, this type of organ has higher CYP3A4 activity than the undifferentiated late liver type organ, which is closer to the level of liver tissue, and the activity of CYP3A4 can be further enhanced after the substrate rifampicin induction (Fig. 37). , indicating the function of mature hepatocytes - biliary cell-organic mature liver cells

At the same time, an appropriate amount of organ was taken out, fluorescein diacetate was added, and its uptake and excretion were observed and recorded to detect whether or not the function of the biliary cell was present.

As a result, as shown in Fig. 38, after 5 minutes of addition, the organoids began to ingest fluorescein and reached a maximum value at about 20-30 minutes, and then began to be discharged, and all of the fluorescein was discharged at 90 minutes. This type of organ has the function of biliary cells.

23. Mature hepatocytes - biliary cell-like organs have the function of repairing liver of mice with acute liver injury

The mature hepatocyte-cholangiocarcinoid organ structure was digested into single cells by the above-mentioned 19-point culture method, and 1*10 ⁶ cells were transplanted per mouse, and transplanted to carbon tetrachloride for 24 hours of SCID by spleen transplantation. In mice. The blood of the mice was continuously taken through the eyelids for 7 days after transplantation to measure aspartate aminotransferase (AST) and alanine aminotransferase (ALT). At the same time, some mice were transplanted for 48h, and were fixed by paraformaldehyde, paraffin-embedded and 4μm serial sections for immunohistochemical staining of human-specific liver tissue markers ALB, APOE and CYP3A4.

As can be seen from Fig. 39, the mice transplanted into the adult liver-like organs showed a significant decrease in the level of the body after one day of transplantation as compared with the control group. Figure 40 further demonstrates that adult liver organs enter the liver of injured mice at 48 h of transplantation and function as mature hepatocytes.

24. Mature hepatocytes-cholangiocarcinoid organs have the function of repairing mice with chronic liver injury.

The mature hepatocyte-cholangioblastic organ structure was digested into single cells by the above-mentioned 19-point culture method, and 1*10 ⁶ cells were transplanted per mouse, and transplanted into 8-12w FRG mice by portal vein transplantation. At the same time withdraw NTBC water. Two months after transplantation, blood was taken to detect the expression of human ALB. At the same time, the mice were sacrificed, and the liver was removed, and paraformaldehyde fixation, paraffin embedding, serial sectioning, and immunohistochemical staining were performed.

The results are shown in Figures 41-44. FRG mice transplanted with adult liver-like organs had a greater survival advantage after withdrawal of NTBC water (Figure 41). Furthermore, the presence of human ALB was detected in the serum of mice at a level of 695.86 μg ml ^-1 , which was comparable to that of mature hepatocyte transplantation (Fig. 42). Immunohistochemical detection of human liver-specific markers ALB, APOE, FAH (Fig. 43, 44) further demonstrates that adult liver-like organs successfully colonize damaged mice and function as mature hepatocytes.

25. Mature hepatocytes - bile duct cell type functionalities form bile duct structures in vivo

The mature hepatocyte-cholangioblastic organ structure will be digested into single cells by the aforementioned 19-point culture method and mixed with an equal amount of mesenchymal stem cells, and the collagen is mixed and added to the plate at 10 μl/pack. A small bag of about 1 mm ^{2 was} formed, and it was planted under the renal capsule of SCID mice the next day. Two months later, the mice were sacrificed, and the tissues under the renal capsule were taken out for paraffin embedding and tissue staining to observe the expression of genes in the biliary cells.

As can be seen from Fig. 45, the mature hepatocyte-cholangiocarcinoid organ structure in vivo can form a homologous structure similar to the bile duct and highly express the markers of cholangiocytes, KRT19 and KRT7. This indicates that mature hepatocytes - biliary cell-like organs have the potential of biliary cells.

As described above, the present invention finds a suitable method for long-term culture of hepatocyte-like organs in embryonic and adult stages, and verifies the efficiency of cell-like passage, cell viability, gene expression level and histological level, and proves that it has been fed. Liver cells can maintain hepatocyte characteristics within one month and transform into bile ducts during long-term culture. The differentiation medium of the present invention can be used to differentiate transdifferentiated cholangiocarcinoma cells into mature hepatocytes to form a complex structure of hepatocyte-cholangiocarcinoma cells. By single cell sequencing, transcriptome analysis confirmed that the complex structure has a cellular composition similar to that of liver cells and a gene expression lineage. In addition, liver functional organs and in vivo experiments have confirmed that the liver-like organs cultured in vitro are functional, have the functions of mature liver cells and biliary cells, and can not only repair liver-damaged mice. It also forms a bile duct structure in the body.

Claims

A serum-free cell culture medium based on a medium for mammalian cell growth, a reagent supplemented with L-glutamine, a pH adjuster for maintaining a stable pH of the culture medium, and a primary cell Culture antibiotics, serum replacements, N-acetylcysteine and optionally nicotinamide, as well as addition of BMP inhibitors, Wnt agonists, growth factors, Rock signaling pathway inhibitors, P38 signaling pathway inhibitors, Notch signaling pathways One or any of a plurality of inhibitors, dexamethasone, BMP7, and cAMP activators.
The serum-free cell culture medium according to claim 1, wherein the serum-free cell culture medium is a medium based on a medium for growth of mammalian cells, and a reagent supplemented with L-glutamine is added. Maintain pH-stabilized pH regulators, primary cell culture antibiotics, serum replacements, N-acetylcysteine, growth factors, and Rock signaling pathway inhibitors, optionally with nicotinamide, BMP inhibitors One or more of a Wnt agonist, a P38 signaling pathway inhibitor, a Notch signaling pathway inhibitor, dexamethasone, BMP7, and a cAMP activator.
The serum-free cell culture medium according to claim 1, wherein the cell culture medium is a cell proliferation medium, and a medium based on a medium for growth of mammalian cells is supplemented with L-glutamine. Amide reagents, pH regulators that maintain medium pH stability, primary cell culture antibiotics, serum replacements, N-acetylcysteine, and nicotinamide, plus BMP inhibitors, Wnt agonists, growth factors , one or more of a Rock signaling pathway inhibitor, a P38 signaling pathway inhibitor, and a cAMP activator; preferably, the cell culture medium is supplemented with a BMP inhibitor, a Wnt agonist, a growth factor, and a Rock signaling pathway inhibitor And optionally added with one or both of a P38 signaling pathway inhibitor and a cAMP activator; or

The cell culture medium is a cell differentiation medium, a medium based on a medium for growth of mammalian cells, a reagent supplemented with L-glutamine, a pH adjuster for maintaining a stable pH of the culture medium, and the original medium. Substituting cells for antibiotics, serum replacements, and N-acetylcysteine, and adding one or more of BMP7, growth factors, Rock signaling pathway inhibitors, Notch signaling pathway inhibitors, and dexamethasone; preferably The medium is supplemented with BMP7, growth factors, Notch signaling pathway inhibitors, dexamethasone and Rock signaling pathway inhibitors.
The serum-free cell culture medium according to any one of claims 1 to 3, wherein the basal medium is modified DMEM/F12 or modified RPMI medium.
The serum-free cell culture medium according to any one of claims 1 to 4, wherein

The BMP inhibitor is selected from one or more of Noggin, A-83-01, DAN and DAN-like proteins; preferably, the final concentration of the BMP inhibitor in the medium is between 0.5 and 800 ng/ml Within the scope of the medium;

The Wnt agonist is selected from one or more of R spondin, a GSK inhibitor, and Wnt3 (such as Wnt 3a); preferably, each Wnt agonist has a final concentration of 1-1500 ng/ml of medium;

The growth factor is selected from one or more of epidermal growth factor, transforming growth factor beta, basic fibroblast growth factor, hepatocyte growth factor, brain-derived neurotrophic factor, and keratinocyte growth factor; preferably The final concentration of the growth factor is 1-1000 ng / ml of the medium;

The Rock signaling pathway inhibitor is selected from one or more of Y 27632, HA 1077 and H 1152; preferably, the final concentration of the Rock inhibitor is in the range of 0.5-50 μM;

The Notch signaling pathway inhibitor is selected from one or more of DAPT (GSI-IX), MK-0752, RO4929097, Semagacestat (LY450139), LY411575, Dibenzazepine (YO-01027), Avagacestat, Crenigacestat, NGP 555; Preferably, the final concentration of the Notch signaling pathway inhibitor is 0.1-50 μM;

The P38 signaling pathway inhibitor is selected from one or more of SB203580, Doramapimod, SB202190, LY2228820, VX-702, PH-797804, VX-745, TAK-715, BMS-582949, Losmapimod, Pexmetinib and Skepinoe-L. Preferably, the final concentration of the P38 signaling pathway inhibitor is 1-20 μM;

The cAMP agonist is Forskolin; preferably, the cAMP agonist has a final concentration of 1-200 [mu]M.
The serum-free cell culture medium according to claim 3, wherein the cell culture medium is a cell proliferation medium comprising Noggin and A-83-01 as BMP inhibitors, and EGF and FGF10 as mitotic growth factors. And FGF2, R spondin as a Wnt agonist, and Y 27632 as a rock signaling pathway inhibitor, supplemented with GlutaMAX-I, a pH regulator that maintains the pH of the culture medium, primary cell antibiotics, B27 serum substitute Nicotinamide and N-acetylcysteine; preferably, the cell culture medium further comprises a GSK inhibitor such as CHIR99021, and/or Wnt3a as a Wnt agonist.
The serum-free cell culture medium according to claim 6, wherein the medium further comprises any one, any two or all three of a P38 inhibitor such as SB202190, a cAMP activator such as Forskolin, and BMP7. .
The serum-free cell culture medium according to claim 6 or 7, wherein when included, the final concentration of Noggin is 5-15 ng/ml; the final concentration of A-83-01 is 300-800 ng/ml; EGF The final concentration is 20-80 ng/ml; the final concentration of FGF10 is 5-15 ng/ml; the final concentration of FGF2 is 0.1-2 ng/ml; the final concentration of R spondin is 50-150 ng/ml; the final concentration of Y 27632 is 5-15 μM; the final concentration of SB202190 is 5-15 μM; the final concentration of cAMP activator is 5-15 μM; the final concentration of GSK inhibitor is 1-5 μM; the final concentration of BMP7 is 10-40 ng/ml; the final concentration of Wnt3a It is 300-600 ng/ml.
The serum-free cell culture medium according to claim 3, wherein the cell culture medium is a cell differentiation medium containing BMP7, a growth factor, a Notch signaling pathway inhibitor, a Rock signaling pathway inhibitor, and dexamethasone. And supplemented with GlutaMAX-I, a pH regulator that maintains a stable pH of the culture medium, a primary cellular antibiotic, a B27 serum replacement, and N-acetylcysteine; preferably, the growth factors include FGF10, FGF2, and HGF And optionally FGF19, the Notch signaling pathway inhibitor is DAPT and the Rock signaling pathway inhibitor is Y27632.
The serum-free medium according to claim 9, wherein said BMP7 has a final concentration of 10 to 40 ng/ml, said growth factor has a final concentration of 50 to 200 ng/ml, and said Notch signaling pathway inhibitor The final concentration is 1-15 μM, the final concentration of the Rock signaling pathway inhibitor is 5-15 μM, and the final concentration of the dexamethasone is 0.01-30 μM;

Preferably, the final concentration of FGF10 is 5-15 ng/ml; the final concentration of FGF2 is 0.1-2 ng/ml; the final concentration of HGF is 10-40 ng/ml; when included, the final concentration of FGF19 is 10-100 ng/ml. The final concentration of Y 27632 is 5-15 μM; the final concentration of dexamethasone is 1-10 μM; and the final concentration of DAPT is 5-15 μM.
A kit containing a medium for growth of mammalian cells as a basal medium, a reagent for supplementing L-glutamine, a pH adjuster for maintaining a stable pH of the culture medium, a primary cell culture antibiotic, serum Substitutes, N-acetylcysteine and optionally nicotinamide, as well as BMP inhibitors, Wnt agonists, growth factors, Rock signaling pathway inhibitors, P38 signaling pathway inhibitors, Notch signaling pathway inhibitors, dexamethasone , one or more of BMP7 and cAMP activators;

Preferably, the kit further comprises an extracellular matrix;

Preferably, the kit comprises the medium of any one of claims 2 to 10, or a component thereof, which can be formulated into a medium according to any one of claims 2 to 10; preferably, The kit contains the cell proliferation medium and a cell differentiation medium.
A cell culture comprising the medium of any one of claims 1 to 10 and liver cells;

Preferably, the cell culture is a cell culture containing the medium and organoids.
A method for culturing a hepatocyte, the method comprising the steps of: preparing a hepatocyte suspension using the cell proliferation medium according to any one of claims 3-8, mixing the suspension with an extracellular matrix, and then performing culturing, And/or the step of differentiating the cells using the cell differentiation medium of any one of claims 3-5 and 9-10.
Use of the organoid according to claim 12 or an organoid prepared by the method of claim 13 for drug development, drug screening and toxicity determination of food supplements.