Liver progenitor organ culture medium and culture method

Abstract

The invention discloses a liver progenitor organoid culture medium and a culture method, and belongs to the technical field of organoid culture. The culture medium comprises a basic culture medium, Jagged-1, Dexamehysone, TGF (Transforming Growth Factor)-alpha, beta-mercaptoethanol, BMP4 (Bone Morphogenetic Protein 4), FGF2 (Fibroblast Growth Factor 2), specific addition factors Glutamax, HEPES (Hydroxyethyl PES), Nicotinamide, Penicillin and the like. The liver progenitor organoid is constructed by using the liver tissue for the first time, continuous growth and efficient proliferation of human liver progenitor cells in vitro can be promoted under the condition that gene editing is not carried out, meanwhile, the pluripotency of the liver progenitor organoid is maintained, and the consistency of specific marker expression is maintained; and a novel batch model is provided for the evaluation of hepatotoxicity at the present stage. From the long-term view, a foundation is laid for constructing a liver organ model with more advanced various differentiated cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and particularly relates to a hepatic progenitor organoid culture medium and a culture method. Background Art

[0002] The liver is the most important metabolic and detoxifying organ in the human body, which is composed of highly complex hepatocyte populations, hepatic sinusoidal endothelial cells, hepatic stellate cells, bile duct epithelial cells, Kupffer cells, etc. The parenchymal cells and surrounding non-parenchymal cells in the liver play crucial roles in forming a hexagonal structure with radiating sinusoids and bile ducts and maintaining hepatocyte function. However, commonly used in vitro liver models can only one-sidedly simulate the liver function and structural complexity, and a liver model that can completely restore intracellular communication in the liver and liver function remains to be studied. Traditionally, in vitro liver research has been carried out using two-dimensional (2D) monolayer cultures of primary human hepatocytes (PHHs). Although these in vitro models are commonly used in basic research and drug development, due to the limitations of 2D technology, they have not fully simulated the gene expression, cell-cell interactions, physiological functions, or complex lobular structures of the liver, resulting in a success rate of only 8% for new drug development.

[0003] Hepatic progenitor cells are oval-shaped cells with a high nuclear-cytoplasmic ratio located in Hering's canals at the junction of hepatocytes and portal areas. They are morphologically and volumetrically similar to bile duct epithelial cells and are considered to have the potential for bidirectional differentiation into hepatocytes and cholangiocytes, high proliferative clonogenic potential, and cell regeneration potential after liver transplantation. Therefore, hepatic progenitor cells can alleviate some practical limitations still faced by existing models, such as the scarcity of primary human hepatocytes (PHHs) and the limited in vitro proliferation of hepatocytes. Although 2D / 3D models based on primary human hepatocytes still serve as the "gold standard" for in vitro drug toxicity evaluation, they not only have problems such as scarce sources and short in vitro survival times. More importantly, the 2D / 3D culture mode can only reflect the drug toxicity response of hepatocytes themselves, rather than the whole liver. Therefore, there is an urgent need for better models to study liver diseases and test the liver's response to drugs.

[0004] Organoids can generally be defined as three-dimensional (3D) tissues with in vitro organ characteristics, which are formed by pluripotent stem cells, adult stem cells, or tissue self-assembly. Organoids derived from human tissues can retain genetic information and exhibit physiological and pathological characteristics of autologous cell sources. Organoid models have become an important platform for studying cell communication and interactions between different organs during biological development and for disease research.

[0005] At present, there are few reports on hepatic progenitor organoids, and the research direction mainly focuses on artificially controlling the differentiation of hepatic progenitor cells under culture media with different dosages and components. However, there are few reports on the culture media with different component compositions of hepatic progenitor organoids for constructing the survival rate of hepatic progenitor organoids. Moreover, the culture conditions of hepatic progenitor organoids are not clear, and hepatic progenitor organoids derived from tissues are usually not easily cultured successfully during the culture process and grow very slowly. Summary of the Invention

[0006] Aiming at the above deficiencies in the prior art, the present invention provides a culture medium and a culture method for hepatic progenitor organoids to solve the problems of low culture success rate, long culture period, poor survival ability and poor passage ability of existing hepatic progenitor organoids during the culture process.

[0007] To achieve the above object, the technical solution adopted by the present invention to solve its technical problems is: The object of the present invention is to provide a culture medium for hepatic progenitor organoids, which comprises a basal medium, a specific additive factor, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4 and FGF2; The specific additive factor comprises components with the following final concentrations: Glutamax, 0.5-2×; HEPES, 10-15 mM; Nicotinamide, 10-15 mM; Penicillinstreptomycin, 0.5-2×; B27, 0.5-3×; N2, 0.5-3×; A83-01, 400-650 nM; Heparin, 1-5 μg / mL; Y-27632, 10-15 μM; FGF-10, 8-12 ng / mL; Wnt3A, 100-160 ng / mL; R-spondin1, 400-550 ng / mL; Noggin, 80-120 ng / mL; HGF, 20-30 ng / mL and EGF, 40-55 ng / mL.

[0008] Further, the specific additive factor comprises components with the following final concentrations: Glutamax, 0.5 - 1×; HEPES, 10 - 12 mM; Nicotinamide, 10 - 12 mM; Penicillin streptomycin, 0.5 - 1×; B27, 1 - 2×; N2, 1 - 1.5×; A83 - 01, 500 - 550 nM; Heparin, 1 - 3 μg / mL; Y - 27632, 10 - 15 μM; FGF - 10, 8 - 10 ng / mL; Wnt3A, 100 - 120 ng / mL; R - spondin1, 450 - 500 ng / mL; Noggin, 100 - 120 ng / mL; HGF, 20 - 25 ng / mL and EGF, 40 - 50 ng / mL.

[0009] Furthermore, the specific addition factors include components with the following final concentrations: Glutamax, 1×; HEPES, 10 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1×; B27, 1×; N2, 1×; A83 - 01, 500 nM; Heparin, 2 μg / mL; Y - 27632, 10 μM; FGF - 10, 10 ng / mL; Wnt3A, 100 ng / mL; R - spondin1, 500 ng / mL; Noggin, 100 ng / mL; HGF, 20 ng / mL and EGF, 50 ng / mL.

[0010] Furthermore, the basal medium is Williams' E medium.

[0011] Furthermore, the final concentration of Jagged - 1 in the hepatic progenitor organoid medium is 1 - 5 μM; The final concentration of Dexamethasone is 1 - 5 μM; The final concentration of TGF - α is 8 - 15 ng / mL; The final concentration of β - mercaptoethanol is 5 - 10 ng / mL; The final concentration of BMP4 is 10 - 15 ng / mL; The final concentration of FGF2 is 50 - 80 μM.

[0012] Furthermore, the final concentration of Jagged - 1 in the hepatic progenitor organoid medium is 1 - 3 μM; The final concentration of Dexamethasone is 1 - 3 μM; The final concentration of TGF - α is 8 - 10 ng / mL; The final concentration of β - mercaptoethanol is 5 - 8 ng / mL; The final concentration of BMP4 is 10-12 ng / mL; The final concentration of FGF2 is 50-65 μM.

[0013] Furthermore, the final concentration of Jagged-1 in the hepatic progenitor organoid medium is 1 μM; The final concentration of Dexamethasone is 1 μM; The final concentration of TGF-α is 10 ng / mL; The final concentration of β-mercaptoethanol is 5 ng / mL; The final concentration of BMP4 is 10 ng / mL; The final concentration of FGF2 is 50 μM.

[0014] Another object of the present invention is to provide the use of the above-mentioned hepatic progenitor organoid medium in culturing hepatic progenitor organoids.

[0015] Another object of the present invention is to provide a method for culturing hepatic progenitor organoids with the above-mentioned hepatic progenitor organoid medium, which comprises the following steps: (1) Reprogramming hepatic parenchymal cells into hepatic progenitor cells; (2) Add 1 mL of 0.25% Tryspin-EDTA to each culture flask to digest hepatic progenitor cells cultured for 5 minutes for 7 days, add reprogramming medium to terminate digestion, centrifuge at 1000 rpm for 3 minutes, discard the supernatant after centrifugation, and collect the cell pellet at the bottom of the centrifuge tube.

[0016] (3) Resuspend the collected cell pellet, that is, add 1.5-2 volumes of undiluted Matrigel (Corning, 356231), and gently pipette up and down 10 times to fully mix, avoiding the generation of bubbles during this period.

[0017] (4) Aspirate 50 μL of the cell suspension and add it to the center of each well of a pre-warmed 24-well plate, so that the sample forms a dome-shaped gel droplet in the center of each well.

[0018] (5) Place the inoculated culture dish into a CO 2 incubator, let it stand at 37 °C for 5 minutes, gently shake the gel droplet without obvious flow and then carefully invert it. After it has fully solidified for 25 minutes, add 500 μL of the above-prepared hepatic progenitor organoid medium. After microscopic examination and photographing, place it in the incubator at 37 °C and 5% CO 2 for culturing under the conditions. Observe every day, change the fresh medium every 2 days during this period, and photograph under the microscope after culturing for 7 days.

[0019] Furthermore, the reprogramming medium used in the process of reprogramming hepatic parenchymal cells includes Williams' E medium and the following components at the final concentrations: B27 1~3×; L-ascorbic acid 100~160μM; Dexamethasone 10~15μM; Forskolin 15~20μM; A83-01 10~15μM; UK-383367 1~3μM; human recombinant Wnt3A protein 100~150ng / mL; human recombinant EGF protein 50~80ng / mL; human recombinant HGF protein 15~20ng / mL.

[0020] Furthermore, the final concentrations of the above components are: B27 1×; L-ascorbic acid 100 μM; Dexamethasone 10 μM; Forskolin 20 μM; A83-01 10 μM; UK-383367 1 μM; human recombinant Wnt3A protein 100 ng / mL; human recombinant EGF protein 50 ng / mL; human recombinant HGF protein 20 ng / mL.

[0021] Furthermore, the hepatocytes are derived from primary human liver tissue.

[0022] Furthermore, the culture conditions in step (2) are 37°C, 5% CO 2 .

[0023] Beneficial effects of the present invention: 1. The present invention extracts parenchymal cells from liver tissue and then reprograms the parenchymal cells into liver progenitor cells that can proliferate in large quantities. In addition, the present invention does not require gene editing, avoiding its common off-target effects, leading to unexpected mutations of non-target genes, reducing genetic risks, and keeping the source genome stable, which is conducive to consistency research. At the same time, the serum-free culture method is adopted, which has clear ingredients, high repeatability, low contamination risk, and meets ethical and regulatory requirements, making it easier to connect with modern biomedical and biopharmaceutical research.

[0024] 2. Traditional cell culture relies on animal serum (such as fetal bovine serum, bovine serum albumin, etc.), which provides necessary growth factors and nutrients, but also has problems such as batch differences, pathogen risks, and ethical issues, which increase technical risks and accuracy burdens. The culture medium of the present invention does not add any serum components, and the method of increasing or decreasing specific factors can obtain stably expanded and passaged liver progenitor organoids.

[0025] 3. This invention uses liver tissue to construct hepatic progenitor organoids for the first time. It can promote the sustained growth and efficient proliferation of human hepatic progenitor cells in vitro without gene editing, while maintaining the pluripotency of hepatic progenitor organoids and the consistency of specific marker expression, providing a new batch model for the evaluation of hepatotoxicity at this stage. In the long run, it lays the foundation for the construction of a more advanced hepatic organoid model with multiple types of differentiated cells. Brief Description of the Drawings

[0026] Figure 1 It is a flow chart for the extraction and isolation of hepatic progenitor cells from liver tissue; Figure 2 It is a culture diagram of hepatic progenitor cells after reprogramming; Figure 3 It is a culture diagram of hepatic progenitor organoids in Example 1; Figure 4 It is a culture diagram of hepatic progenitor organoids in Example 2; Figure 5 It is a culture diagram of hepatic progenitor organoids in Example 3; Figure 6 It is a culture diagram of hepatic progenitor organoids in Comparative Example 1; Figure 7 It is a culture diagram of hepatic progenitor organoids in Comparative Example 2; Figure 8 It is a culture diagram of hepatic progenitor organoids in Comparative Example 3; Figure 9 It is a culture diagram of hepatic progenitor organoids in Comparative Example 4; Figure 10 It is a culture diagram of hepatic progenitor organoids in Comparative Example 5; Figure 11 It is a culture diagram of hepatic progenitor organoids in Comparative Example 6; Figure 12 It is a culture diagram of hepatic progenitor organoids in Comparative Example 7; Figure 13 It is a culture diagram of hepatic progenitor organoids in Comparative Example 8; Figure 14 It is a culture diagram of hepatic progenitor organoids in Comparative Example 9; Figure 15 It is a HE staining identification result diagram of hepatic progenitor organoids in Example 1; Figure 16 It is an immunofluorescence staining identification result diagram of hepatic progenitor organoids in Example 1; Figure 17 It is an RT-qPCR identification result diagram of hepatic progenitor organoids in Example 1. Detailed Description of the Invention

[0027] The following is a description of the specific embodiments of the present invention to facilitate those skilled in the art to understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions created using the concept of the present invention are within the scope of protection.

[0028] Example 1 A hepatic progenitor organoid culture medium, which comprises Williams' E medium, specific addition factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4 and FGF2; Among them, the specific addition factors include components with the following final concentrations: Glutamax, 1×; HEPES, 10 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1×; B27, 1×; N2, 1×; A83-01, 500 nM; Heparin, 2 μg / mL; Y-27632, 10 μM; FGF-10, 10 ng / mL; Wnt3A, 100 ng / mL; R-spondin1, 500 ng / mL; Noggin, 100 ng / mL; HGF, 20 ng / mL and EGF, 50 ng / mL.

[0029] The final concentration of Jagged-1 in the hepatic progenitor organoid culture medium is 1 μM, the final concentration of Dexamethasone is 1 μM, the final concentration of TGF-α is 10 ng / mL; the final concentration of β-mercaptoethanol is 5 ng / mL; the final concentration of BMP4 is 10 ng / mL; the final concentration of FGF2 is 50 μM.

[0030] Example 2 A hepatic progenitor organoid culture medium, which comprises Williams' E medium, specific addition factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4 and FGF2; Among them, the specific addition factors include components with the following final concentrations: Glutamax, 2×; HEPES, 15 mM; Nicotinamide, 15 mM; Penicillin streptomycin, 2×; B27, 3×; N2, 3×; A83-01, 650 nM; Heparin, 5 μg / mL; Y-27632, 12 μM; FGF-10, 12 ng / mL; Wnt3A, 160 ng / mL; R-spondin1, 550 ng / mL; Noggin, 120 ng / mL; HGF, 30 ng / mL and EGF, 55 ng / mL.

[0031] The final concentration of Jagged-1 in the hepatic progenitor organoid medium is 3 μM; the final concentration of Dexamethasone is 3 μM; the final concentration of TGF-α is 8 ng / mL; the final concentration of β-mercaptoethanol is 8 ng / mL; the final concentration of BMP4 is 12 ng / mL; the final concentration of FGF2 is 65 μM.

[0032] Example 3 A hepatic progenitor organoid medium, which comprises Williams' E medium, specifically added factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4 and FGF2; Among them, the specifically added factors include components with the following final concentrations: Glutamax, 0.5×; HEPES, 12 mM; Nicotinamide, 12 mM; Penicillin streptomycin, 0.5×; B27, 0.5×; N2, 0.5×; A83-01, 400 nM; Heparin, 1 μg / mL; Y-27632, 12 μM; FGF-10, 8 ng / mL; Wnt3A, 120 ng / mL; R-spondin1, 400 ng / mL; Noggin, 80 ng / mL; HGF, 25 ng / mL and EGF, 40 ng / mL.

[0033] The final concentration of Jagged-1 in the hepatic progenitor organoid medium is 2 μM; the final concentration of Dexamethasone is 2 μM; the final concentration of TGF-α is 8.5 ng / mL; the final concentration of β-mercaptoethanol is 6.5 ng / mL; the final concentration of BMP4 is 10 ng / mL; the final concentration of FGF2 is 60 μM.

[0034] Example 4 A method for culturing hepatic progenitor organoids, comprising the following steps: (1) Reprogramming hepatocytes into hepatic progenitor cells 1) Rinse the obtained fresh human primary liver tissue with pre-cooled PBS 3 times until the tissue preservation solution is completely rinsed away; use sterilized surgical instruments to cut off necrotic or redundant tissue, and retain at least 1.0*1.0*1.0 cm 3 volume of liver tissue.

[0035] 2) Cut the processed liver tissue into tissue fragments of about 1 mm 3 and transfer them to a 1.5 mL EP tube. Add 200 μL of type IV collagenase (0.5 mg / mL), and use a Pasteur pipette to completely transfer the tissue fragments and the digestive solution to a glass tissue grinder, and grind them into cell homogenate.

[0036] 3) Filter the cell homogenate through a 70-μm cell strainer, collect the filtrate into a 50-mL centrifuge tube, then rinse the glass tissue grinder with an appropriate amount of DPBS, and filter the cell homogenate rinse solution, collecting it into the same centrifuge tube.

[0037] 4) Centrifuge at 1000 rpm for 3 min to remove the supernatant. Resuspend the cell pellet with 5 mL of type I collagenase, transfer it to a 15-mL centrifuge tube, and incubate with shaking at 37 °C for 30 min. Centrifuge at 1000 rpm for 3 min to remove the supernatant. Resuspend the cell pellet with 2 mL of 0.25% trypsin, incubate with shaking at 37 °C for 5 - 8 min, centrifuge at 1000 rpm for 3 min to remove the supernatant, and resuspend the cell pellet with 20 mL of William’s E medium for later use.

[0038] 5) Take a 50-mL centrifuge tube, slowly add 20 mL of 75% Percoll separation solution, and slowly add the cell suspension collected in the previous step to the upper layer of the separation solution (upper layer cell suspension: bottom layer 75% Percoll separation solution = 1:1). Centrifuge at 4 °C and 1000 rpm for 10 min. Carefully aspirate the top cell layer into a new 15-mL centrifuge tube for later use. There are three layers from top to bottom. The top layer is William’s E medium containing mixed hepatocytes and dead cells, the middle layer is hepatocytes, and the bottom layer is Percoll separation solution.

[0039] 6) Aspirate the top layer of mixed hepatocytes and resuspend with 15 mL of William’s E medium. Take a 50-mL centrifuge tube, and carefully add the bottom layer 50% Percoll separation solution, the middle layer 25% Percoll separation solution, and the top layer mixed hepatocyte suspension in a ratio of 1:1:1 in sequence, minimizing shaking. Centrifuge at 4 °C and 1000 rpm for 10 min. The bottom layer precipitate is hepatocytes. Aspirate the upper layer liquid and resuspend with William’s E medium.

[0040] 7) Mix the hepatocytes collected by the two centrifugations, centrifuge at 4 °C and 1000 rpm for 10 min; remove the supernatant, add 2 mL of William’s E medium to resuspend. Aspirate 20 μL of the cell suspension and mix it with 20 μL of AO / PI, aspirate 20 μL and pipette it onto a cell counting chamber, and count with a cell counter.

[0041] 8) Seed at 1×10 6 per well in a 6-well plate coated with 10 μg / mL type I collagen. Observe the cell morphology under an inverted microscope after 24 h. After 24 h, change to hepatocyte progenitor cell reprogramming medium (as shown in Table 1), 2 mL per well, and change the medium every other day. The flow chart of the above process is shown inFigure 1 , the pictures of hepatic progenitor cells obtained by its culture are as Figure 2 shown.

[0042] Table 1 Composition of the reprogramming medium for hepatic progenitor cells

[0043] (2) Add 1 mL of 0.25% Tryspin-EDTA to each culture flask to digest the hepatic progenitor cells cultured for 7 days for 5 minutes, add the reprogramming medium to terminate the digestion, centrifuge at 1000 rpm for 3 minutes, discard the supernatant after centrifugation, and collect the cell pellet at the bottom of the centrifuge tube.

[0044] (3) Resuspend the collected cell pellet, that is, add 1.5 - 2 volumes of undiluted Matrigel (Corning, 356231) and mix well by gently pipetting up and down 10 times, avoiding generating bubbles during this process.

[0045] (4) Pipette 50 μL of the cell suspension and add it to the center of each well of a pre-warmed 24-well plate, so that the sample forms a dome-shaped gel droplet in the center of each well.

[0046] (5) Place the inoculated culture dish into the CO 2 incubator, let it stand at 37 °C for 5 minutes, carefully invert it after gently shaking the gel droplet without obvious flow, and after it has fully solidified for 25 minutes, add 500 μL of the prepared hepatic organoid medium. After microscopic examination and taking pictures, place it in the incubator and culture it at 37 °C under the condition of 5% CO 2 . Observe it every day, change the fresh medium every 2 days during this period, and take pictures under the microscope after culturing for 7 days.

[0047] The pictures of hepatic organoids cultured based on the medium prepared in Example 1 are as Figure 3 shown.

[0048] Comparative Example 1 The difference from Example 1 is that Jagged-1 is removed from the medium, and the rest is the same as in Example 1.

[0049] Comparative Example 2 The difference from Example 1 is that Dexamethasone is removed from the medium, and the rest is the same as in Example 1.

[0050] Comparative Example 3 The difference from Example 1 is that TGF-α is removed from the medium, and the rest is the same as in Example 1.

[0051] Comparative Example 4 The difference from Example 1 is that β-mercaptoethanol is removed from the medium, and the rest is the same as in Example 1.

[0052] Comparative Example 5 The difference from Example 1 is that BMP4 is removed from the culture medium, and the rest is the same as in Example 1.

[0053] Comparative Example 6 The difference from Example 1 is that FGF2 is removed from the culture medium, and the rest is the same as in Example 1.

[0054] Comparative Example 7 The difference from Example 1 is that Jagged-1 and TGF-α are removed from the culture medium, and the rest is the same as in Example 1.

[0055] Comparative Example 8 The difference from Example 1 is that BMP4 and TGF-α are removed from the culture medium, and the rest is the same as in Example 1.

[0056] Comparative Example 9 The difference from Example 1 is that FGF2 and TGF-α are removed from the culture medium, and actually the rest is the same as in Example 1.

[0057] Test Example 1. Observe the hepatic progenitor organoids obtained after culturing Examples 1 to 3 and Comparative Examples 1 to 9 for 7 days. The results are shown in Figure 3-14 ; among them, Figure 3 is the culture result diagram of the hepatic progenitor organoids in Example 1; Figure 4 is the culture result diagram of the hepatic progenitor organoids in Example 2; Figure 5 is the culture result diagram of the hepatic progenitor organoids in Example 3; Figure 6 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 1; Figure 7 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 2; Figure 8 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 3; Figure 9 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 4; Figure 10 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 5; Figure 11 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 6; Figure 12 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 7; Figure 13 is the culture result diagram of the hepatic progenitor organoids in Comparative Example 8; Figure 14 is the culture result diagram of the hepatic progenitor organoids in Example 1.

[0058] It can be seen from Figure 3-14 that the number of hepatic progenitor organoids cultured by the method of the present invention is large and the activity is good.

[0059] 2. The liver progenitor organoids obtained in Example 1 were embedded, sectioned, and stained using hematoxylin-eosin staining (HE). The results are shown in Figure 15 .

[0060] As can be seen from Figure 15 , the liver progenitor organoids were vacuolated with a thin cyst wall, and the cell nuclei were round or oval. Thus, it can be seen that the HE histological characteristics of the liver progenitor organoids constructed in the present invention are consistent with the characteristics of liver progenitor cells.

[0061] 3. Immunofluorescence staining was used to identify the liver progenitor organoids obtained in Example 1, and the liver-specific markers albumin (ALB) and alpha-fetoprotein (AFB) were detected. The results are shown in Figure 16 .

[0062] 4. Detection of liver progenitor-specific expression markers The relative expression levels of marker identification mRNAs in liver progenitor organoids were detected using RT-qPCR. RNA was extracted using the TRIcomReagent Total RNA Extraction Kit (Jianshi Biotechnology, TR201-50), reverse-transcribed into cDNA using the RevertAid First Stand cDNA Synthesis Kit (Thermo Scientific, K1622) kit, and then the obtained cDNA was subjected to PCR amplification detection using primers (Table 2). The experimental results are as Figure 17 shown. The expression of SOX9, CK19, HNF1β, HNF4α, ALB, and AFP was significantly increased in the liver progenitor organoid model.

[0063] Table 2 Primer table for PCR amplification detection of liver progenitor cells

[0064] 5. Detection of the viability of liver progenitor organoids The liver progenitor organoids were cultured in the media of Examples 1-3 and Comparative Examples 1-9 for 7 days. The morphologies of the liver progenitor organoids obtained in Comparative Examples 1-9 are as Figure 6-14 shown, and the number and viability of the organoids formed in Examples 1-3 and Comparative Examples 1-9 were detected. The results are shown in Table 3.

[0065] Table 3 Number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-9

[0066] As can be seen from Table 3 and the detection results of Figure 3~14 , when the components such as Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4, and FGF2 in the medium were deleted separately, the growth and viability of the organoids were significantly affected.

[0067] Finally, it should be noted that the above specific embodiments are only used to illustrate the technical solutions of the present invention rather than to limit them. Although the present invention has been described in detail with reference to the examples, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention, and they should all be covered within the scope of the claims of the present invention.

Claims

1. A hepatic progenitor organoid culture medium, characterized in that Includes basal medium, specific supplemental factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4 and FGF2; The specific additive factors include the following components at the final concentrations: Glutamax, 0.5~2×; HEPES, 10~15mM; Nicotinamide, 10~15mM; Penicillinstreptomycin, 0.5~2×; B27, 0.5~3×; N2, 0.5~3×; A83-01, 400~650nM; Heparin, 1~5μ g / mL; Y-27632, 10~15μM; FGF-10, 8~12ng / mL; Wnt3A, 100~160ng / mL; R-spondin1, 400~550ng / mL; Noggin, 80~120ng / mL; HGF, 20~30ng / mL and EGF, 40~55ng / mL.

2. The hepatic progenitor organoid culture medium according to claim 1, characterized in that The specific additive factors include the following components at the final concentrations: Glutamax, 0.5~1×; HEPES, 10~12mM; Nicotinamide, 10~12mM; Penicillinstreptomycin, 0.5~1×; B27, 1~2×; N2, 1~1.5×; A83-01, 500~550nM; Heparin, 1~3μg / mL; Y-27632, 10~15μM; FGF-10, 8~10ng / mL; Wnt3A, 100~120ng / mL; R-spondin1, 450~500ng / mL; Noggin, 100~120ng / ml; 3. The hepatic progenitor organoid culture medium according to claim 1, characterized in that The basic culture medium was Williams' E medium.

4. The hepatic progenitor organoid culture medium according to claim 1, characterized in that The final concentration of Jagged-1 in the hepatic progenitor organoid culture medium was 1~5μM; The final concentration of dexamethasone is 1~5μM; The final concentration of TGF-α was 8-15 ng / mL; The final concentration of β-mercaptoethanol was 5-10 ng / mL; The final concentration of BMP4 was 10-15 ng / mL; The final concentration of FGF2 is 50~80μM.

5. The hepatic progenitor organoid culture medium according to claim 4, characterized in that The final concentration of Jagged-1 in the hepatic progenitor organoid culture medium was 1~3μM; The final concentration of dexamethasone is 1~3μM; The final concentration of TGF-α was 8-10 ng / mL; The final concentration of β-mercaptoethanol was 5-8 ng / mL; The final concentration of BMP4 was 10-12 ng / mL; The final concentration of FGF2 is 50~65μM.

6. Use of the hepatic progenitor organoid culture medium according to any one of claims 1 to 5 in culturing hepatic progenitor organoids.

7. A method for culturing hepatic progenitor organoids using the hepatic progenitor organoid culture medium according to any one of claims 1 to 5, characterized in that: The following steps are involved: (1) Reprogramming hepatocytes into hepatic progenitor cells; (2) Resuspend the hepatic progenitor cells and mix with the matrix gel. After solidification, add hepatic progenitor organoid culture medium for culturing to obtain hepatic progenitor organoids.

8. The method according to claim 7, characterized in that The reprogramming medium used in the hepatocyte reprogramming process includes Williams' E medium and the following components at the final concentrations: B27 1~3×; L-ascorbic acid 100~160μM; Dexamethasone 10~15μM; Forskolin 15~20μM; A83-01 10~15μM; UK-383367 1~3μM; Human recombinant Wnt3A protein 100~150ng / mL; human recombinant EGF protein 50~80ng / mL; human recombinant HGF protein 15~20ng / mL.

9. The method according to claim 7, characterized in that: Hepatocytes are derived from primary human liver tissue.

10. The method according to claim 7, characterized in that The culture conditions in step (2) are 37°C and 5% CO2.

Images