A hepatic progenitor organoid culture medium and culture method

Abstract

The present invention discloses a hepatic progenitor organoid culture medium and culture method, which belong to the field of organoid culture technology. It includes basal culture medium, Jagged‑1, Dexamethasone, TGF‑α, β-mercaptoethanol, BMP4, FGF2, specific additive factors Glutamax, HEPES, Nicotinamide, Penicillin streptomycin, etc. The present invention uses liver tissue to construct hepatic progenitor organoids for the first time, which can promote the continuous 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 current evaluation of hepatotoxicity. In the long run, it lays the foundation for the construction of a more advanced hepatic organoid model with multiple types of 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 body's most important metabolic and detoxifying organ, composed of a highly complex population of hepatocytes, including sinusoidal endothelial cells, hepatic stellate cells, bile duct epithelial cells, and Kupffer cells. Parenchymal cells within the liver and surrounding non-parenchymal cells play crucial roles in shaping the hexagonal structure of radiating sinusoids and bile ducts and maintaining hepatocyte function. However, commonly used in vitro liver models only partially simulate liver function and structural complexity, and liver models that fully replicate intracellular communication and liver function remain underdeveloped. Traditionally, in vitro liver studies have been conducted using two-dimensional (2D) monolayer cultures of primary human hepatocytes (PHHs). While these in vitro models are commonly used for basic research and drug development, they are limited by 2D technology and fail to fully replicate the liver's gene expression, cell-cell interactions, physiological functions, or complex lobular architecture, resulting in a low success rate of only 8% for new drug development.

[0003] Hepatic progenitor cells are oval, high-nuclear-to-cytoplasmic ratio cells located in the Hering's canal at the junction of hepatic parenchymal cells and the portal area. They are similar to bile duct epithelial cells in morphology and volume and are believed to have the potential for bidirectional differentiation into hepatocytes and cholangiocytes, high proliferation clone formation potential, and cell regeneration potential after liver transplantation. Therefore, hepatic progenitor cells can alleviate some practical limitations of existing models, such as the scarcity of primary human hepatocytes (PHHs) and limited proliferation of hepatocytes in vitro. Although 2D / 3D models based on primary human hepatocytes are still the "gold standard" for in vitro drug toxicity evaluation, they not only have problems such as scarce sources and short in vitro survival time. More importantly, 2D / 3D culture models can only reflect the drug toxicity responses of hepatocytes themselves, not the entire liver. Therefore, better models are urgently needed to study liver diseases and test the liver's response to drugs.

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

[0005] Currently, research on hepatic progenitor organoids is limited, primarily focusing on artificially controlling the differentiation of hepatic progenitor cells using culture media with varying dosages and compositions. However, there are few reports on the effects of different culture media compositions on the viability of hepatic progenitor organoids. Furthermore, the culture conditions for hepatic progenitor organoids are unclear, and tissue-derived hepatic progenitor organoids are often difficult to culture successfully and grow very slowly. Summary of the Invention

[0006] In response to the above-mentioned deficiencies in the prior art, the present invention provides a hepatic progenitor organoid culture medium and culture method to solve the problems of low culture success rate, long culture cycle, and poor survival and passage ability of existing hepatic progenitor organoids during culture.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0008] The purpose of the present invention is to provide a hepatic progenitor organoid culture medium, which comprises a basal culture medium, specific additive factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4 and FGF2;

[0009] The specific additives include the following components at the following final concentrations:

[0010] 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.

[0011] Furthermore, the specific additive factors include the following components at the final concentrations:

[0012] 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;

[0013] Furthermore, the specific additive factors include the following components at the final concentrations:

[0014] Glutamax, 1×; HEPES, 10mM; Nicotinamide, 10mM; Penicillin streptomycin, 1×; B27, 1×; N2, 1×; A83-01, 500nM; Heparin, 2μg / mL; Y-27632, 10μM; FGF-10, 10n g / mL; Wnt3A, 100ng / mL; R-spondin1, 500ng / mL; Noggin, 100ng / mL; HGF, 20ng / mL and EGF, 50ng / mL.

[0015] Furthermore, the basal culture medium is Williams' E medium.

[0016] Furthermore, the final concentration of Jagged-1 in the hepatic progenitor organoid culture medium was 1–5 μM;

[0017] The final concentration of dexamethasone was 1–5 μM;

[0018] The final concentration of TGF-α was 8–15 ng / mL;

[0019] The final concentration of β-mercaptoethanol was 5–10 ng / mL;

[0020] The final concentration of BMP4 was 10–15 ng / mL;

[0021] The final concentration of FGF2 is 50~80μM.

[0022] Furthermore, the final concentration of Jagged-1 in the hepatic progenitor organoid culture medium was 1–3 μM;

[0023] The final concentration of dexamethasone was 1–3 μM;

[0024] The final concentration of TGF-α was 8–10 ng / mL;

[0025] The final concentration of β-mercaptoethanol was 5–8 ng / mL;

[0026] The final concentration of BMP4 was 10–12 ng / mL;

[0027] The final concentration of FGF2 is 50~65μM.

[0028] Furthermore, the final concentration of Jagged-1 in the hepatic progenitor organoid culture medium was 1 μM;

[0029] The final concentration of dexamethasone was 1 μM;

[0030] The final concentration of TGF-α was 10 ng / mL;

[0031] The final concentration of β-mercaptoethanol was 5 ng / mL;

[0032] The final concentration of BMP4 was 10 ng / mL;

[0033] The final concentration of FGF2 was 50 μM.

[0034] Another object of the present invention is to provide use of the above-mentioned hepatic progenitor organoid culture medium in culturing hepatic progenitor organoids.

[0035] Another object of the present invention is to provide a method for culturing hepatic progenitor organoids using the above-mentioned hepatic progenitor organoid culture medium, comprising the following steps:

[0036] (1) Reprogramming hepatocytes into hepatic progenitor cells;

[0037] (2) Add 1 mL of 0.25% Tryspin-EDTA to each culture flask and digest the hepatic progenitor cells cultured for 7 days for 5 minutes. Add reprogramming medium to terminate the digestion and centrifuge at 1000 rpm for 3 minutes. After centrifugation, discard the supernatant and collect the cell pellet at the bottom of the centrifuge tube.

[0038] (3) Resuspend the collected cell pellet by adding 1.5-2 times the volume of undiluted Matrigel (Corning, 356231) and mixing. Carefully pipette up and down 10 times to mix thoroughly, avoiding the generation of bubbles.

[0039] (4) Pipette 50 μL of cell suspension and add it to the center of each well of a preheated 24-well plate, so that the sample forms a dome-shaped gel droplet in the center of each well.

[0040] (5) Place the inoculated culture dish in a CO2 incubator and let it stand at 37°C for 5 minutes. Gently shake the gel droplet until there is no obvious flow. Carefully turn it upside down and wait for it to fully solidify for 25 minutes. Then add 500 μL of the hepatic progenitor organoid culture medium prepared above. After microscopic examination and photography, place it in an incubator at 37°C and 5% CO2. Observe it daily, replace the culture medium every 2 days, and take pictures under a microscope after 7 days of culture.

[0041] Furthermore, the reprogramming medium used in the hepatocyte reprogramming process includes Williams' E medium and the following components at the following final concentrations:

[0042] 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.

[0043] 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; and human recombinant HGF protein 20 ng / mL.

[0044] Furthermore, the hepatocytes are derived from primary human liver tissue.

[0045] Furthermore, the culture conditions in step (2) are 37°C and 5% CO2.

[0046] Beneficial effects of the present invention:

[0047] 1. The present invention isolates and extracts parenchymal cells from liver tissue and then reprograms the parenchymal cells into proliferating hepatic progenitor cells. Furthermore, the present invention does not require gene editing, avoiding its common off-target effects that can lead to unexpected mutations in non-target genes, reducing genetic risks, and maintaining the stability of the source genome, which is beneficial for consistency research. Furthermore, the serum-free culture method is used, which has clear ingredients, high reproducibility, low contamination risk, and complies with ethical and regulatory requirements, making it easier to connect with modern biomedical and biopharmaceutical research.

[0048] 2. Traditional cell culture relies on animal serum (such as fetal bovine serum and bovine serum albumin). While this provides essential growth factors and nutrients, it also presents issues such as batch variability, pathogen risks, and ethical concerns, increasing technical risk and accuracy burdens. The culture medium of this invention does not contain any serum components and utilizes a method of adding or removing specific factors to produce stable expansion and passage of hepatic progenitor organoids.

[0049] 3. This invention, for the first time, utilizes liver tissue to construct hepatic progenitor organoids. This method promotes the sustained growth and efficient proliferation of human hepatic progenitor cells in vitro without gene editing, while maintaining their pluripotency and consistent expression of specific markers. This provides a novel, mass-produced model for evaluating hepatotoxicity. Long-term, it lays the foundation for the construction of more advanced hepatic organoid models with diverse differentiated cell types. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Flow chart for the extraction and separation of hepatic progenitor cells from liver tissue;

[0051] Figure 2 This is a diagram of the culture of hepatic progenitor cells after reprogramming;

[0052] Figure 3 This is a diagram of the hepatic progenitor organoid culture in Example 1;

[0053] Figure 4 This is a diagram of the hepatic progenitor organoid culture in Example 2;

[0054] Figure 5 This is a diagram of the hepatic progenitor organoid culture in Example 3;

[0055] Figure 6 This is a picture of the hepatic progenitor organoid culture in Comparative Example 1;

[0056] Figure 7 This is a picture of the hepatic progenitor organoid culture in Comparative Example 2;

[0057] Figure 8 This is a picture of the hepatic progenitor organoid culture in Comparative Example 3;

[0058] Figure 9 This is a picture of the hepatic progenitor organoid culture in Comparative Example 4;

[0059] Figure 10 This is a picture of the hepatic progenitor organoid culture in Comparative Example 5;

[0060] Figure 11 This is a picture of the hepatic progenitor organoid culture in Comparative Example 6;

[0061] Figure 12 This is a picture of the hepatic progenitor organoid culture in Comparative Example 7;

[0062] Figure 13 This is a picture of the hepatic progenitor organoid culture in Comparative Example 8;

[0063] Figure 14 This is a diagram of the hepatic progenitor organoid culture in Comparative Example 9;

[0064] Figure 15 This is the HE staining identification result of liver progenitor organoids in Example 1;

[0065] Figure 16 This is the result of immunofluorescence staining of hepatic progenitor organoids in Example 1;

[0066] Figure 17 This is a diagram showing the RT-qPCR identification results of hepatic progenitor organoids in Example 1. DETAILED DESCRIPTION

[0067] The specific embodiments of the present invention are described below to facilitate understanding of the present invention by those skilled in the art. However, it should be clear that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the appended claims, these changes are obvious, and all inventions and creations utilizing the concepts of the present invention are protected.

[0068] Example 1

[0069] A hepatic progenitor organoid culture medium comprising Williams' E medium, specific supplemental factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4, and FGF2;

[0070] The specific additive factors include the following components at the final concentrations:

[0071] Glutamax, 1×; HEPES, 10mM; Nicotinamide, 10mM; Penicillin streptomycin, 1×; B27, 1×; N2, 1×; A83-01, 500nM; Heparin, 2μg / mL; Y-27632, 10μM; FGF-10, 10n g / mL; Wnt3A, 100ng / mL; R-spondin1, 500ng / mL; Noggin, 100ng / mL; HGF, 20ng / mL and EGF, 50ng / mL.

[0072] The final concentrations of Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4, and FGF2 in the hepatic progenitor organoid culture medium were 1 μM, 1 μM, 10 ng / mL, 5 ng / mL, and 10 ng / mL, respectively.

[0073] Example 2

[0074] A hepatic progenitor organoid culture medium comprising Williams' E medium, specific supplemental factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4, and FGF2;

[0075] The specific additive factors include the following components at the final concentrations:

[0076] Glutamax, 2×; HEPES, 15mM; Nicotinamide, 15mM; Penicillin streptomycin, 2×; B27, 3×; N2, 3×; A83-01, 650nM; Heparin, 5μg / mL; Y-27632, 12μM; FGF-10, 12n g / mL; Wnt3A, 160ng / mL; R-spondin1, 550ng / mL; Noggin, 120ng / mL; HGF, 30ng / mL and EGF, 55ng / mL.

[0077] The final concentration of Jagged-1 in the hepatic progenitor organoid culture 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; and the final concentration of FGF2 is 65 μM.

[0078] Example 3

[0079] A hepatic progenitor organoid culture medium comprising Williams' E medium, specific supplemental factors, Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4, and FGF2;

[0080] The specific additive factors include the following components at the final concentrations:

[0081] Glutamax, 0.5×; HEPES, 12mM; Nicotinamide, 12mM; Penicillin streptomycin, 0.5×; B27, 0.5×; N2, 0.5×; A83-01, 400nM; Heparin, 1μg / mL; Y-27632, 12μM; FGF-10 , 8ng / mL; Wnt3A, 120ng / mL; R-spondin1, 400ng / mL; Noggin, 80ng / mL; HGF, 25ng / mL and EGF, 40ng / mL.

[0082] The final concentration of Jagged-1 in the hepatic progenitor organoid culture 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; and the final concentration of FGF2 is 60 μM.

[0083] Example 4

[0084] A method for culturing hepatic progenitor organoids, comprising the following steps:

[0085] (1) Reprogramming of hepatocytes into hepatic progenitor cells

[0086] 1) Rinse the fresh human primary liver tissue with pre-cooled PBS three times until the tissue preservation solution is completely rinsed out; use sterile surgical instruments to cut away necrotic or excess tissue, leaving at least 1.0*1.0*1.0cm 3 Volume of liver tissue.

[0087] 2) Cut the processed liver tissue into pieces of about 1 mm 3 Transfer the tissue fragments to a 1.5 mL EP tube, add 200 μL type IV collagenase (0.5 mg / mL), and use a Pasteur pipette to completely transfer the tissue fragments and digestion solution to a glass tissue grinder and grind them thoroughly into a cell homogenate.

[0088] 3) Filter the cell homogenate through a 70 μm cell sieve and collect the filtrate into a 50 mL centrifuge tube. Rinse the glass tissue grinder with an appropriate amount of DPBS, sieve the cell homogenate rinse, and collect it into the same centrifuge tube.

[0089] 4) Centrifuge at 1000 rpm for 3 min, remove the supernatant, resuspend the cell pellet in 5 mL of type I collagenase, transfer to a 15 mL centrifuge tube, and incubate at 37°C with shaking for 30 min. Centrifuge at 1000 rpm for 3 min, remove the supernatant, resuspend the cell pellet in 2 mL of 0.25% trypsin, incubate at 37°C with shaking for 5-8 min, centrifuge at 1000 rpm for 3 min, remove the supernatant, and resuspend the cell pellet in 20 mL of William's E medium for later use.

[0090] 5) In a 50 mL centrifuge tube, slowly add 20 mL of 75% Percoll separation buffer. Slowly add the cell suspension collected in the previous step to the upper layer of the separation buffer (upper layer cell suspension: bottom layer 75% Percoll separation buffer = 1:1). Centrifuge at 1000 rpm for 10 minutes at 4°C. Carefully transfer the top cell layer to a new 15 mL centrifuge tube for later use. From top to bottom, separate the tube into three layers: the top layer contains Williams's E medium containing mixed hepatocytes and dead cells, the middle layer contains hepatocytes, and the bottom layer contains Percoll separation buffer.

[0091] 6) Aspirate the top layer of mixed hepatocytes and resuspend in 15 mL of Williams' E medium. In a 50 mL centrifuge tube, carefully add the bottom layer of 50% Percoll, the middle layer of 25% Percoll, and the top layer of mixed hepatocyte suspension in a 1:1:1 ratio. Minimize turbulence and centrifuge at 1000 rpm for 10 minutes at 4°C. The bottom layer of hepatocytes is pelleted. Aspirate the top layer and resuspend in Williams' E medium.

[0092] 7) Combine the hepatocytes collected from the two centrifugations and centrifuge at 1000 rpm for 10 min at 4°C. Remove the supernatant and resuspend the cells in 2 mL of Williams' E medium. Pipette 20 μL of the cell suspension and mix with 20 μL of AO / PI. Pipette 20 μL into a cell counting plate and count using a cell counter.

[0093] 8) Press 1×10 per hole 6 The cells were seeded in a 6-well plate coated with 10 μg / mL type I collagen. After 24 hours, the cell morphology was observed under an inverted microscope. After 24 hours, the medium was replaced with hepatic progenitor cell reprogramming medium (as shown in Table 1). 2 mL of medium was changed every other day per well. The flow chart of the above process is shown in Figure 1 The pictures of the hepatic progenitor cells obtained by culture are as follows Figure 2 shown.

[0094] Table 1 Composition of hepatic progenitor cell reprogramming medium

[0095]

[0096] (2) Add 1 mL of 0.25% Tryspin-EDTA to each culture flask and digest the hepatic progenitor cells cultured for 7 days for 5 minutes. Add reprogramming medium to terminate the digestion and centrifuge at 1000 rpm for 3 minutes. After centrifugation, discard the supernatant and collect the cell pellet at the bottom of the centrifuge tube.

[0097] (3) Resuspend the collected cell pellet by adding 1.5-2 times the volume of undiluted Matrigel (Corning, 356231) and mixing. Carefully pipette up and down 10 times to mix thoroughly, avoiding the generation of bubbles.

[0098] (4) Pipette 50 μL of cell suspension and add it to the center of each well of a preheated 24-well plate, so that the sample forms a dome-shaped gel droplet in the center of each well.

[0099] (5) Place the inoculated culture dish in a CO2 incubator and let it stand at 37°C for 5 minutes. Gently shake the gel droplet until there is no obvious flow. Carefully turn it upside down and wait for it to fully solidify for 25 minutes. Then add 500 μL of the hepatic progenitor organoid culture medium prepared above. After microscopic examination and photography, place it in an incubator at 37°C and 5% CO2. Observe it daily, replace the culture medium every 2 days, and take pictures under a microscope after 7 days of culture.

[0100] Figure 1 shows the hepatic progenitor organoids cultured in the culture medium prepared in Example 1. Figure 3 shown.

[0101] Comparative Example 1

[0102] The difference from Example 1 is that Jagged-1 is removed from the culture medium, and the rest is the same as Example 1.

[0103] Comparative Example 2

[0104] The difference from Example 1 is that Dexamethasone is removed from the culture medium, and the rest is the same as Example 1.

[0105] Comparative Example 3

[0106] The difference from Example 1 is that TGF-α is removed from the culture medium, and the rest is the same as Example 1.

[0107] Comparative Example 4

[0108] The difference from Example 1 is that β-mercaptoethanol is removed from the culture medium, and the rest is the same as Example 1.

[0109] Comparative Example 5

[0110] The difference from Example 1 is that BMP4 is removed from the culture medium, and the rest is the same as Example 1.

[0111] Comparative Example 6

[0112] The difference from Example 1 is that FGF2 is removed from the culture medium, and the rest is the same as Example 1.

[0113] Comparative Example 7

[0114] 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 Example 1.

[0115] Comparative Example 8

[0116] The difference from Example 1 is that BMP4 and TGF-α are removed from the culture medium, and the rest is the same as Example 1.

[0117] Comparative Example 9

[0118] The difference from Example 1 is that FGF2 and TGF-α are removed from the culture medium, which is actually the same as Example 1.

[0119] Test example

[0120] 1. The hepatic progenitor organoids obtained after 7 days of culture in Examples 1 to 3 and Comparative Examples 1 to 9 were observed. Figure 3-14 ;in, Figure 3 This is a diagram showing the culture results of hepatic progenitor organoids in Example 1; Figure 4 This is a diagram showing the culture results of hepatic progenitor organoids in Example 2; Figure 5 This is a diagram showing the culture results of hepatic progenitor organoids in Example 3; Figure 6 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 1; Figure 7 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 2; Figure 8 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 3; Figure 9 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 4; Figure 10 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 5; Figure 11 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 6; Figure 12 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 7; Figure 13 This is a diagram showing the culture results of hepatic progenitor organoids in Comparative Example 8; Figure 14 This is a diagram showing the culture results of hepatic progenitor organoids in Example 1.

[0121] Depend on Figure 3-14 It can be seen that the hepatic progenitor organoids cultured using the method of the present invention are numerous and have good activity.

[0122] 2. The hematoxylin-eosin (HE) staining method was used to embed and stain the liver progenitor organoids obtained in Example 1. The results are shown in Figure 15 .

[0123] Depend on Figure 15 As can be seen, the hepatic progenitor organoids are vacuolar, with thin cyst walls and round or oval nuclei. This indicates that the HE histological characteristics of the hepatic progenitor organoids constructed in the present invention are consistent with those of hepatic progenitor cells.

[0124] 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 .

[0125] 4. Detection of liver progenitor-specific expression markers

[0126] RT-qPCR was used to detect the relative expression levels of mRNA for marker identification in hepatic progenitor organoids. 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), and the resulting cDNA was amplified by PCR using primers (Table 2). The experimental results are shown in Figure 2. Figure 17 As shown, the expression of SOX9, CK19, HNF1β, HNF4α, ALB and AFP was significantly increased in the hepatic progenitor organoid model.

[0127] Table 2 Primers for PCR amplification of hepatic progenitor cells

[0128]

[0129] 5. Liver progenitor organoid viability assay

[0130] The hepatic progenitor organoids were cultured using the culture medium in Examples 1-3 and Comparative Examples 1-9 for 7 days. The morphology of the hepatic progenitor organoids obtained in Comparative Examples 1-9 was as follows: Figure 6-14 The number and viability of organoids formed in Examples 1 to 3 and Comparative Examples 1 to 9 were detected, and the results are shown in Table 3.

[0131] Table 3 Number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-9

[0132]

[0133] From Table 3 and Figures 3 to 14The test results show that when Jagged-1, Dexamethasone, TGF-α, β-mercaptoethanol, BMP4, FGF2 and other components in the culture medium are deleted respectively, the growth and viability of the organoids are significantly affected.

[0134] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A hepatic progenitor organoid culture medium, characterized in that The invention comprises a basal culture medium, specific additive factors, Jagged-1 with a final concentration of 1 to 5 μM, Dexamethasone with a final concentration of 1 to 5 μM, TGF-α with a final concentration of 8 to 15 ng / mL, β-mercaptoethanol with a final concentration of 5 to 10 ng / mL, BMP4 with a final concentration of 10 to 15 ng / mL, and FGF2 with a final concentration of 50 to 80 μM; the basal culture medium is Williams' E medium; 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, wherein The final concentration of Jagged-1 in the hepatic progenitor organoid culture medium was 1–3 μM; The final concentration of dexamethasone was 1–3 μM; 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.

4. Use of the hepatic progenitor organoid culture medium according to any one of claims 1 to 3 in culturing hepatic progenitor organoids.

5. A method for culturing hepatic progenitor organoids using the hepatic progenitor organoid culture medium according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) Reprogramming hepatocytes into hepatic progenitor cells; (2) Resuspend the hepatic progenitor cells and mix them with matrix gel. After solidification, add hepatic progenitor organoid culture medium for culture to obtain hepatic progenitor organoids.

6. The method according to claim 5, characterized in that The reprogramming medium used during hepatocyte reprogramming includes Williams' E medium and the following components at the following 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.

7. The method according to claim 5, characterized in that Hepatocytes were derived from primary human liver tissue.

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

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