Method for simultaneously extracting multiple types of cells from same liver tissue and subculturing

Abstract

The invention discloses a method for simultaneously extracting multiple types of cells from the same liver tissue, which is characterized by comprising the following steps: preparing a single-cell suspension; extracting parenchymal hepatic cells; extracting bile duct epithelial cells; extracting hepatic stellate cells; wherein the extraction step of the parenchymal hepatic cells, the bile duct epithelial cells and the hepatic stellate cells is carried out step by step according to the sequence. According to the technical scheme, efficient extraction of liver tissue cells and culture of functional organoid are achieved, various defects in the prior art are overcome, meanwhile, the technical scheme has a wide application range and is suitable for various liver tissues, and important technical support is provided for research and treatment of liver diseases.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, and in particular to a method for simultaneously extracting multiple types of cells from the same liver tissue and subculture them. Background Art

[0002] The ability to culture mature hepatocyte organoids for long periods of time is an important tool for studying liver-related diseases, such as malaria, hepatitis virus infection, and liver fibrosis. In addition, understanding the basic processes of mature hepatocyte expansion is also crucial for developing new liver regeneration treatments.

[0003] However, existing technologies have problems such as low purity, low cell viability, and complicated procedures when extracting multiple cell types from the same liver tissue. In addition, the technology for culturing functional hepatocyte organoids is not yet mature, making it difficult to maintain their stem cell properties and achieve stable expansion.

[0004] The present invention provides a method for extracting high-purity and high-viability hepatocytes, bile duct epithelial cells and hepatic stellate cells from the same liver tissue in steps through in vitro digestion and single-cell preparation technology, thereby solving the problems in the prior art. Summary of the invention

[0005] In the prior art, hepatocytes are obtained by the following steps:

[0006] Insert the catheter: Clamp the superior vena cava, insert the catheter into the inferior vena cava of the mouse, make sure the catheter is in the blood vessel, and then immediately cut the portal vein;

[0007] Perfuse the liver: perfuse the liver with pre-warmed perfusion medium at a rate of 5-7 ml / min for 10 min;

[0008] Digestive solution perfusion: Use preheated digestive solution (including type IV collagenase and Ca 2+ ) Infuse at a rate of 5 ml / min for 3-5 minutes;

[0009] Cell dissociation: After perfusion, liver tissue was dissociated and cells were filtered through a 70 μm filter;

[0010] Low-speed centrifugation: Hepatocytes were further separated and purified by low-speed centrifugation (50 g, 1-3 min);

[0011] Percoll gradient centrifugation: Percoll gradient centrifugation was used to further purify hepatocytes.

[0012] The above method can obtain liver parenchymal cells with a certain purity and viability, but there are still challenges, such as low cell purity, low viability and cumbersome steps.

[0013] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a method for simultaneously extracting multiple types of cells from the same liver tissue and a method for subculture. A high-concentration single-cell suspension is prepared using RWD universal tissue mild enzymatic digestion solution in combination with an RWD single-cell suspension preparation instrument. The digestion enzyme configuration scheme of this method is simple, suitable for various liver tissues, and the operation process is simple and efficient. By combining low-speed centrifugation, 25% Percoll gradient centrifugation fluid and EpCAM magnetic bead sorting technology, high-purity and high-viability hepatic parenchymal cells can be extracted. From the supernatant retained in the step of extracting hepatic parenchymal cells, high-purity and high-viability bile duct epithelial cells and hepatic stellate cells are extracted respectively by EpCAM magnetic beads and Nycodenz gradient centrifugation fluid. Functional spherical hepatic parenchymal cell organoids and bile duct epithelial cell organoids that can be stably expanded for a long time are cultured. These organoids simulate the functional characteristics of liver tissue in vivo and provide a reliable cell model for the study and treatment of liver-related diseases.

[0014] According to one aspect of the present invention, a method for simultaneously extracting multiple types of cells from the same liver tissue is provided, comprising: preparing a single cell suspension; extracting hepatic parenchymal cells; extracting bile duct epithelial cells; extracting hepatic stellate cells; wherein the steps of extracting the hepatic parenchymal cells, the bile duct epithelial cells and the hepatic stellate cells are performed step by step in a sequential order.

[0015] Preferably, preparing a single cell suspension comprises: decomposing the extracellular matrix and loosening the connection between cells so that some cells in the liver tissue exist in the form of single cells; digesting the liver tissue into single cells to generate a single cell suspension.

[0016] Preferably, after generating a single cell suspension, wash buffer is added to terminate digestion and filtered; the filtered cell suspension C1 is centrifuged at 100g and 4°C for 5 minutes to obtain a precipitate containing hepatic parenchymal cells, and the supernatant S1 is collected for extracting bile duct epithelial cells and hepatic stellate cells.

[0017] Preferably, extracting hepatocytes comprises: adding red blood cell lysate to a precipitate containing hepatocytes, incubating on ice for 3-5 minutes, and then adding wash buffer to terminate the reaction; centrifuging at 100g and 4°C for 5 minutes, discarding the supernatant, and resuspending the cell precipitate with wash buffer to obtain a cell suspension C2; transferring the cell suspension C2 to a 25% percoll gradient centrifuge; centrifuging at 1474g and 4°C for 20 minutes, and resuspending the cell precipitate with a precooled advanced centrifuge. Resuspend the cell pellet with DMEM / F12 basal culture medium to obtain cell suspension C3; centrifuge again and discard the supernatant, add V volume of magnetic column buffer and CD326 magnetic beads, incubate at 4°C in the dark for 15 minutes, and then add 9V volume of magnetic column buffer to terminate the reaction; set up a magnetic bead screening device, a filter column and an EpCAM-negative cell suspension collection tube, add magnetic column buffer to wet the filter column, add the cell-magnetic bead mixture suspension when the liquid flows out, and add magnetic column buffer to rinse the filter column after the suspension flows out; centrifuge the EpCAM-negative cell suspension at 100g and 4°C for 5 minutes, discard the supernatant, and obtain hepatic parenchymal cells.

[0018] Preferably, the bile duct epithelial cells are extracted, including: filtering the supernatant S1 with a 40μm mesh and counting the cells of the filtrate; centrifuging at 580g and 4°C for 10 minutes, obtaining the cell pellet and discarding the supernatant; adding V volume of magnetic bead incubation buffer to the cell pellet to resuspend the cell pellet, and adding CD326 magnetic beads at the same time, incubating at 4°C in the dark for 15 minutes, and adding 9V volume of magnetic bead incubation buffer to terminate the reaction; placing a magnetic bead screening device, a filter column and an EpCAM-negative cell suspension collection tube, adding a magnetic column buffer to wet the filter column, and after the liquid has flowed out, adding a cell-magnetic bead mixture suspension; after the suspension has flowed out, adding a magnetic column buffer to rinse the filter column, and collecting the filtrate L1 for extraction of hepatic stellate cells; removing the filter column and adding a magnetic column buffer, and using the filter column core to push out the cell-magnetic bead mixture suspension; centrifuging the mixture suspension at 300g and 4°C for 10 minutes, discarding the supernatant, and obtaining bile duct epithelial cells.

[0019] Preferably, the hepatic stellate cells are extracted, comprising: centrifuging the filtrate L1 at 580g and 4°C for 10 minutes to obtain a cell pellet; resuspending the cell pellet with 50ml of Grignard balanced salt solution B, and centrifuging again at 580g and 4°C for 10 minutes; discarding the supernatant to leave 10ml of Grignard balanced salt solution B, adding 120μl of DNAse I solution to the remaining liquid to resuspend the cell pellet, then adding Grignard balanced salt solution B to 50ml, and continuing to centrifuge at 580g and 4°C for 10 minutes; discarding the supernatant again to leave 10ml of Grignard balanced salt solution B, adding 120μl of DNAse I solution to resuspend the cell pellet, and adding 22ml of Grignard balanced salt solution B, and mixing; adding 16ml of the mixed cell suspension; Nycodenz gradient centrifugation solution was used, and the tube wall was wetted by turning upside down. 1.5 ml of Grignard balanced salt solution B was added above the surface of the cell suspension. The cell suspension was centrifuged at 1380 g and 4 °C for 17 minutes without brake mode. A white layer appeared at 12 ml, and the hepatic stellate cell layer was obtained.

[0020] According to another aspect of the present invention, a method for subculturing hepatocytes is provided, comprising: resuspending the cell pellet with precooled advanced DMEM / F12 basal medium, adding 100,000 cells to each well in a 24-well plate after counting; centrifuging again at 100g and 4°C for 5 minutes, discarding the supernatant, and resuspending the mixed matrix gel / cells at a ratio of 1:2000 of matrix gel to cells, and planting the mixture in a 24-well plate; after the matrix gel solidifies, adding a hepatocyte organoid culture medium to each well; after the 0th generation grows to 14 days, discarding the culture medium, adding precooled advanced DMEM / F12 basal culture medium, use a pipette to mechanically break the organoid-matrigel mixture and collect it into a centrifuge tube, mix well and place on ice for 30 minutes; centrifuge at 100g and 4°C for 10 minutes, discard the supernatant, resuspend the organoid pellet with matrix gel, and seed it into a 24-well plate at a cell ratio of 1:1; wait for the matrix gel to solidify, and add 500μl of hepatic parenchymal cell organoid culture medium to each well; after the first generation, subculture at a cell ratio of 1:3-1:5, and subculture when each generation grows to 200 organoids.

[0021] According to another aspect of the present invention, a method for subculturing bile duct epithelial cells is provided, comprising: resuspending the cell pellet with precooled advanced DMEM / F12 basal medium, counting and adding 50,000 cells per well to a 24-well plate; centrifuging the cell suspension again at 300g and 4°C for 10 minutes, discarding the supernatant, and resuspending and fully mixing the matrix gel / cells at a ratio of 1:1000 of matrix gel to cells, and planting the cell-matrix gel mixture in a 24-well plate; after the matrix gel solidifies, adding bile duct epithelial cell organoid culture medium to each well; after the 0th generation grows to 10 days, discarding the culture medium, adding precooled advanced DMEM / F12 basal culture medium, use a pipette to mechanically break the organoid-matrigel mixture and collect it into a centrifuge tube, mix well and place on ice for 30 minutes; centrifuge at 300g and 4°C for 10 minutes, discard the supernatant, resuspend the organoid pellet with matrix gel, and seed it into a 24-well plate at a subculture ratio of 1:4-1:8; wait for the matrix gel to solidify, add 500μl of bile duct epithelial cell organoid culture medium to each well, and subculture when 200 organoids are grown in each generation.

[0022] Compared with the prior art, the present invention has the following beneficial effects

[0023] Simplified and efficient digestion method: The present invention uses RWD universal tissue mild enzymatic digestion solution and combines it with RWD single cell suspension preparation instrument to prepare high-concentration single cell suspension. The digestion enzyme configuration scheme of this method is simple and suitable for liver tissues in various disease states. The operation process is simple and efficient, and the digestion procedure is fixed, which helps to obtain high-concentration and active single cell suspension.

[0024] High-purity and high-viability cell extraction: The present invention can extract high-purity and high-viability hepatocytes by combining low-speed centrifugation, 25% Percoll gradient centrifugation fluid, and EpCAM magnetic bead sorting technology. In addition, from the supernatant retained in the step of extracting hepatocytes, high-purity and high-viability bile duct epithelial cells and hepatic stellate cells are extracted by EpCAM magnetic beads and Nycodenz gradient centrifugation fluid, respectively. These technologies ensure high-quality extraction of different cell types and meet the needs of subsequent research and application.

[0025] Improve sample utilization: The method of the present invention is suitable for small-volume liver tissue samples. Through efficient digestion and single-cell preparation technology, the utilization of samples is maximized. Hepatic parenchymal cells, bile duct epithelial cells and hepatic stellate cells can be extracted simultaneously, providing a variety of cell types to meet different research needs.

[0026] Cultivation of functional organoids: The present invention can cultivate functional spherical hepatocyte organoids and bile duct epithelial cell organoids that can be stably expanded for a long time. These organoids simulate the functional characteristics of liver tissue in vivo and provide a reliable cell model for the research and treatment of liver-related diseases.

[0027] Through the above advantages, the present invention technically realizes the efficient extraction of liver tissue cells and the cultivation of functional organoids, overcoming the various deficiencies existing in the prior art, such as low purity of cell extraction, low viability, and complex operation. At the same time, the technical solution has a wide range of applications and is suitable for various liver tissues, providing important technical support for the research and treatment of liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0029] Figure 1 A cell extraction flow chart according to an embodiment of the invention is shown.

[0030] Figure 2 A light microscopic image of a hepatocyte organoid provided according to an embodiment of the invention is shown.

[0031] Figure 3 An immunofluorescence image of a hepatocyte organoid provided according to an embodiment of the invention is shown.

[0032] Figure 4 A diagram showing the functional detection of hepatocyte organoids provided according to an embodiment of the invention is shown.

[0033] Figure 5 A light microscopic image of a bile duct epithelial cell organoid provided according to an embodiment of the invention is shown.

[0034] Figure 6 Shown are immunofluorescence and functional detection images of bile duct epithelial cell organoids provided according to an embodiment of the invention.

[0035] Figure 7 Light microscopic images of hepatic stellate cells in resting and activated states provided according to an embodiment of the invention are shown.

[0036] Figure 8 The immunofluorescence results of hepatic stellate cells in the quiescent state and activated state provided according to the embodiments of the invention are shown. DETAILED DESCRIPTION

[0037] The following examples are provided to allow those skilled in the art to more clearly understand the present invention. It should be noted that the raw materials, reagents or devices mentioned in the following examples, unless otherwise specified, can be obtained from commercial sources or by known existing methods.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The terms "include", "comprising", "having", "containing", etc. used herein are all open-ended terms, which mean including but not limited to. The percentages of ethanol described in the present invention are all volume fractions unless otherwise specified. The technical scheme of the present invention is further described below by way of examples.

[0042] Explanation of terms

[0043] Organoids: Organoids are three-dimensional cell clusters generated by in vitro culture. They are miniature and simplified versions of specific organs, mimicking the key functions, structures, and biological complexity of the organ. Organoid culture can originate from pluripotent stem cells such as embryonic stem cells or adult stem cells, artificially induced pluripotent stem cells, and cancer stem cells. The self-renewal and differentiation potential of these cells give them the ability to self-assemble under three-dimensional culture conditions.

[0044] Hepatocytes: Refers to the main functional cells in the liver, also known as hepatocytes. These cells are responsible for most of the liver's metabolic functions, including detoxification, protein synthesis, and biochemical breakdown.

[0045] Biliary epithelial cells: Forming the lining of the bile ducts, these cells are primarily responsible for the secretion and transport of bile and play important roles in the liver and digestive system.

[0046] Hepatic stellate cells: interstitial cells present in the liver, which have the ability to store vitamin A and transform into myofibroblasts after liver damage, and play an important role in the development of liver fibrosis.

[0047] CD326 magnetic beads: a cell sorting technology, CD326 can specifically mark and separate EpCAM-positive cells. EpCAM (epithelial cell adhesion molecule) is a protein on the cell surface and is often used to extract bile duct epithelial cells and stem cells.

[0048] Percoll gradient centrifugation: A cell purification technique that uses Percoll solutions of different densities for centrifugation to separate and purify cells by layering cells of different densities in the gradient.

[0049] Single-cell suspension: refers to a cell solution composed of individual single cells suspended in a liquid medium, usually prepared by methods such as tissue dissociation and filtration, and used for cell sorting, culture and experimental research.

[0050] Required reagents and main technical means

[0051] Wash buffer: advanced DMEM / F12 basal medium, 1% penicillin-streptomycin mixture.

[0052] The 25% percoll gradient centrifugation buffer is 100% percoll: 1×PBS = 1:3.

[0053] 100% percoll is percoll cell dispersion: 10×PBS=9:1.

[0054] Magnetic column buffer: 2.5 ml MACS BSA solution, 47.5 ml autoMACS wash solution.

[0055] DNAse I solution: Dissolve 100 mg DNAse I in 50 ml Grignard balanced salt solution B, mix gently, and the prepared DNAse I should be aliquoted and stored at -20°C until use. Avoid repeated freezing and thawing, and it is stable for up to 18 months.

[0056] Nycodenz density gradient centrifugation medium: Dissolve 4.94 g of Nycodenz powder in 15 ml of Grignard balanced salt solution A.

[0057] Grignard balanced salt solution A: 370 mg / L KCl, 210 mg / L MgCl 2 6H 2 O, 70mg / LMgSO 4 7H 2 O, 59.6 mg / L Na 2 HPO 4 , 30mg / L KH 2 PO 4 , 991mg / L glucose, 227mg / L NaHCO 3 , 225mg / L CaCl 2 ·2H 2 O. Dissolve the above ingredients in 1 L ddHO 2 Prepare the solution in 4% CO. Adjust pH to between 7.35-7.4 and filter through a 0.22 μm syringe filter. Can be prepared in advance and stored at 4°C for 6 months.

[0058] Grignard balanced salt solution B: 8000mg / L NaCl, 370mg / L KCl, 210mg / LMgCl 2 6H 2 O, 70mg / LMgSO 4 7H 2 O, 59.6 mg / L Na 2 HPO 4 , 30mg / L KH 2 PO 4 , 991mg / L glucose, 227mg / L NaHCO 3 , 225mg / LCaCl 2 ·2H 2 O. Dissolve the above ingredients in 1 L ddHO 2 Prepare the solution in 4% CO. Adjust the pH to between 7.35 and 7.4 and filter through a 0.22 μm syringe filter.

[0059] Hepatocyte organoid culture medium: advanced DMEM / F12 basal medium, 1% penicillin-streptomycin mixture, 1% GlutaMAX, 10mM HEPES, 1:50B27 supplement, 1.25mM N-acetylcysteine, 10mM nicotinamide, 1.5μg / ml R-spodin 1, 3μM ChIR99021, 10nM human recombinant gastric mucosal regeneration factor, 50ng / ml human recombinant epidermal growth factor, 50ng / ml recombinant fibroblast growth factor-7, 50ng / ml human recombinant fibroblast growth factor-10, 25ng / ml human recombinant hepatocyte growth factor, 1μM A83-01, 10μM ROCK pathway inhibitor Y-27632.

[0060] Biliary epithelial cell organoid culture medium: advanced DMEM / F1 basal medium, 1% penicillin-streptomycin mixture, 1% GlutaMAX, 25mM HEPES, 1:50B27 supplement, 1:100N2 supplement, 1.25mM N-acetylcysteine, 10mM nicotinamide, 10nM human recombinant gastric mucosal regeneration factor, 50ng / ml human recombinant epidermal growth factor, 100ng / ml human recombinant fibroblast growth factor-10, 50ng / ml human recombinant hepatocyte growth factor, 5μM A83-01, 10μM forskolin, 1μg / ml R-spodin 1, 100ng / ml Noggin, 100ng / ml Wnt3a. 10μM ROCK pathway inhibitor Y-27632 should be added on the first six days of each generation.

[0061] Complete culture medium for hepatic stellate cells: high glucose DMEM, 10% fetal bovine serum, 1% penicillin-streptomycin mixture.

[0062] Identification of hepatocyte organoids: Immunofluorescence was used to detect hepatocyte markers albumin (ALB), alpha-fetoprotein (AFP) and bile duct epithelial cell markers cytokeratin 19 (CK19) and SRY-Box Transcription Factor 9 (SOX9).

[0063] Functional testing of parenchymal cell organoids: Exogenous low-density albumin was added to the culture medium to test the absorption of low-density albumin (LDL) by organoids. Rhodamine 123 (Rho) experiments were performed to test whether organoids have bile duct function, that is, transporting rhodamine 123. Periodic acid-Schiff staining was used to detect the presence of glycogen in the cytoplasm of hepatic parenchymal cell organoids.

[0064] Identification of bile duct epithelial cell organoids: Immunofluorescence was used to detect bile duct epithelial cell markers CK19, CK7 and hepatocyte parenchymal cell marker ALB.

[0065] Functional testing of bile duct epithelial cell organoids: The Rhodamine 123 experiment was used to test whether the organoids had the same transport function as in vivo.

[0066] Identification of hepatic stellate cell activation: Cell immunofluorescence was used to detect the hepatic stellate cell quiescence marker Retinoid binding protein 1 and activation marker Desmin.

[0067] Example: Cell extraction and subculture

[0068] like Figure 1 The detailed steps of cell extraction are as follows:

[0069] (1) Preparation of liver single cell suspension: Take mouse liver, rinse it 2-3 times with pre-cooled advanced DMEM / F12 basal medium, and cut the liver into 0.5 mm pieces with scissors. 3 Fragments. Wash the fragments 2-3 times with pre-cooled wash buffer, add RWD Universal Tissue Gentle Enzymatic Digestion Buffer and transfer to a single cell tube. Start the RWD Single Cell Suspension Preparation Instrument to prepare liver single cell suspension.

[0070] (2) Extraction of hepatocytes: After the procedure is completed, add 5 ml of wash buffer to terminate digestion and filter with a 100 μm mesh. Centrifuge the cell suspension at 100 g and 4 °C for 5 minutes to precipitate the hepatocytes to be further purified. Transfer the supernatant to another tube for subsequent extraction of bile duct epithelial cells and hepatic stellate cells. Add 1 ml of red blood cell lysis buffer to the hepatocytes precipitate, mix thoroughly and incubate on ice for 3-5 minutes, and add 4 ml of wash buffer to terminate the reaction. Centrifuge the cell suspension at 100 g and 4 °C for 5 minutes, discard the supernatant, and resuspend the cell precipitate with 5 ml of wash buffer. Pre-add 15 ml of 25% percoll gradient centrifugation solution to the bottom of another centrifuge tube, and carefully transfer the cell suspension to the percoll liquid surface. At this time, the two can be seen to be layered. Centrifuge the cell suspension at 1474 g and 4 °C for 20 minutes. Resuspend the cell precipitate with pre-cooled advanced DMEM / F12 basal medium and count. Centrifuge again, discard the supernatant and add 100 μl of magnetic column buffer. 7 Add 10μl CD326 magnetic beads to each cell, incubate at 4℃ in the dark for 15 minutes, and then add 900μl magnetic column buffer to terminate the reaction. Place the magnetic bead screening device, filter column and EpCAM-negative cell suspension collection tube, add 3ml magnetic column buffer to wet the filter column, and add the cell-magnetic bead mixture suspension when the liquid is about to flow out. When the suspension is about to flow out, add 3ml magnetic bead incubation buffer three times to rinse the filter column. Centrifuge the EpCAM-negative cell suspension at 100g and 4℃ for 5 minutes, discard the supernatant, resuspend the cell pellet with pre-cooled advancedDMEM / F12 basal medium, count, and plant 100,000 cells per well of a 24-well plate. Centrifuge again at 100g and 4℃ for 5 minutes, discard the supernatant, resuspend and fully mix the matrix gel / cells at a ratio of 1:2000 for matrix gel: cells, and plant the cell-matrix gel mixture in a 24-well plate. When the matrix gel solidifies, add 500μl of hepatocyte organoid culture medium to each well.

[0071] (3) Extraction of bile duct epithelial cells: Filter the cell supernatant retained in step (2) with a 40 μm mesh, count, and centrifuge at 580 g and 4°C for 10 minutes. Discard the supernatant, add 100 μl of magnetic bead incubation buffer to resuspend the cell pellet, and 7Add 10μl CD326 magnetic beads to each cell and incubate at 4℃ in the dark for 15 minutes. Add 900μl magnetic bead incubation buffer to terminate the reaction. Place the magnetic bead screening device, filter column and EpCAM negative / positive cell suspension collection tube, add 3ml magnetic column buffer to wet the filter column, and add the cell-magnetic bead suspension when the liquid is about to flow out. When the suspension is about to flow out, add 3ml magnetic column buffer three times. The filtrate is the EpCAM negative cell suspension for subsequent hepatic stellate cell extraction. Remove the filter column and keep it away from the magnetic bead screening device, place it on another new centrifuge tube, add 5ml magnetic column buffer, and use the filter column core to quickly push out the cell-magnetic bead mixture suspension. Centrifuge the cell suspension at 300g and 4℃ for 10 minutes, discard the supernatant, resuspend the cell pellet with pre-cooled advanced DMEM / F12 basal medium, count, and seed 50,000 cells per well of a 24-well plate. Centrifuge the cell suspension again at 300g and 4°C for 10 minutes, discard the supernatant, resuspend and fully mix the matrix gel / cells at a matrix gel: cell ratio of 1:1000, and seed the cell-matrix gel mixture in a 24-well plate. After the matrix gel solidifies, add 500 μl of bile duct epithelial cell organoid culture medium to each well.

[0072] (4) Hepatic stellate cell extraction: Centrifuge the EpCAM-negative cell suspension in step (3) at 580g and 4°C for 10 minutes. Resuspend the cell pellet with 50ml Grignard balanced salt solution B and centrifuge at 580g and 4°C for 10 minutes. Discard the supernatant until 10ml Grignard balanced salt solution B remains, add 120μl DNAse I solution and resuspend the cell pellet, add Grignard balanced salt solution B to 50ml, and centrifuge at 580g and 4°C for 10 minutes. Discard the remaining 10ml Grignard balanced salt solution B again, add 120μl DNAse I solution and resuspend the cell pellet, add 22ml Grignard balanced salt solution B, mix well and add 16ml Nycodenz gradient centrifugation solution. Divide into 4 tubes of 12ml each, turn the centrifuge tube upside down to wet the wall, and slowly add 1.5ml Grignard balanced salt solution B to the surface of the cell suspension in each tube with a syringe. Centrifuge the cell suspension at 1380g and 4°C for 17 minutes without braking mode. A white layer can be seen at 12 ml, which is the hepatic stellate cell layer. Pipette the cells into another centrifuge tube, count them, and seed 100,000 cells per 24-well plate. Centrifuge at 580 g for 10 minutes at 4°C, resuspend the cells in complete hepatic stellate cell culture medium and seed them into the well plate.

[0073] (5) Passaging of hepatocyte organoids: When the 0th generation has grown to 14 days, discard the culture medium, add pre-cooled advanced DMEM / F12 basal culture medium, use a pipette to mechanically break up the organoid-matrigel mixture and collect it in a centrifuge tube, mix well and place on ice for 30 minutes. Centrifuge at 100g and 4°C for 10 minutes, discard the supernatant, resuspend the organoid pellet with matrix gel, and plant it in a 24-well plate at a cell ratio of 1:1. After the matrix gel solidifies, add 500μl of hepatocyte organoid culture medium to each well. After the first generation, pass the cells at a cell ratio of 1:3-1:5. Each generation can be passaged when 200 organoids have grown, and can currently be passaged to more than 10 generations.

[0074] (6) Passaging of bile duct epithelial cell organoids: When the 0th generation has grown to 10 days, discard the culture medium, add pre-cooled advanced DMEM / F12 basal culture medium, use a pipette to mechanically break up the organoid-matrigel mixture and collect it in a centrifuge tube, mix well and place on ice for 30 minutes. Centrifuge at 300g and 4°C for 10 minutes, discard the supernatant, resuspend the organoid pellet with matrix gel, and seed it into a 24-well plate at a passaging ratio of 1:4-1:8. After the matrix gel solidifies, add 500μl of bile duct epithelial cell organoid culture medium to each well. Each generation can be passaged when 200 organoids have grown, and currently it can be passaged to more than 10 generations.

[0075] (7) Activation of hepatic stellate cells: After the primary hepatic stellate cells in step (4) have adhered to the wall for 24 hours, the culture medium is discarded and 500 μl of complete culture medium for mouse hepatocytes containing cytokines is added. The medium is changed every 3 days and the cells are collected on the 5th to 8th day of culture.

[0076] Figure 2 Light microscopic images of hepatocyte organoids from passage 0 to passage 10. Figure 2 It can be seen that the hepatocyte organoids of passage 0 were grape-shaped on days 1-3, and gradually turned into spherical organoids with thick walls as the culture time increased. They can be continuously passaged for more than 10 generations and always maintain a stable spherical structure, indicating that hepatocyte organoids can be stably expanded in vitro. Figure 3 This is the immunofluorescence result of hepatocyte organoids. Figure 3 It can be seen that the hepatocyte organoids of passage 1 (3A), passage 5 (3B), and passage 10 (3C) all highly expressed ALB and AFP, but did not express CK19 and SOX9. The above results indicate that this method can isolate and culture hepatocyte organoids that are stably expanded and have liver characteristics. Figure 4 The results of the functional test of hepatocyte organoids are shown in Figure 4. The hepatocyte organoids of the 1st (4A), 5th (4B), and 10th (4C) generations can take up LDL and contain a large amount of glycogen in the cytoplasm, but they do not have the function of transporting rhodamine. Figure 5Light microscopic images of bile duct epithelial cell organoids from passage 0 to passage 10. Figure 5 It can be seen that bile duct epithelial cell organoids have thin walls and a spherical structure. They can be continuously passaged for more than 10 generations and always maintain a stable spherical structure, indicating that bile duct epithelial cell organoids can be stably expanded in vitro. Figure 6 The results of immunofluorescence and functional testing of bile duct epithelial cell organoids are shown in Figure 2. Figure 6 It can be seen that the 1st generation (6A), 5th generation (6B), and 10th generation (6C) bile duct epithelial cell organoids all highly expressed CK19 and CK7, but did not express ALB. The 1st generation (6D), 5th generation (6E), and 10th generation (6F) organoids were able to transport rhodamine into the lumen, and after using the ATP Binding Cassette Subfamily B Member 1 (MDR1) inhibitor verapamil (VP), rhodamine was excreted from the lumen. The above results show that this method can isolate and culture bile duct epithelial cell organoids that are stably expanded and functional. Figure 7 Light microscopic images of resting and activated hepatic stellate cells. Figure 7 It can be seen that the resting hepatic stellate cells are polygonal and contain a large number of lipid droplets (7A). After activation, the lipid droplets disappear, the vitamin A content decreases, and they become myofibroblasts (7B). It is preliminarily determined that this cell is a hepatic stellate cell and can be activated. Figure 8 The following are the immunofluorescence results of resting and activated hepatic stellate cells. Figure 8 It can be seen that resting hepatic stellate cells highly express retinoid binding protein 1 and do not express desmin (8A), while activated hepatic stellate cells show the opposite (8B). This further confirms that this method can isolate functional hepatic stellate cells.

[0077] It should be noted that the RWD tissue digestion enzyme used in the present invention can be replaced by a tissue digestion enzyme kit of Miltenyi or other brands. These kits can also efficiently digest liver tissue and provide a high-concentration single-cell suspension. The RWD single-cell suspension preparation instrument can be replaced by a single-cell suspension preparation instrument of Miltenyi or other brands. These instruments can also efficiently and quickly prepare single-cell suspensions to ensure cell activity and concentration.

[0078] Although the present invention uses Percoll gradient centrifugation fluid, other brands or formulas of gradient centrifugation fluids can also be substituted, such as Ficoll, Histopaque, etc., which can also efficiently purify hepatic parenchymal cells, bile duct epithelial cells and hepatic stellate cells.

[0079] CD326 magnetic beads can be replaced by other brands or magnetic beads with different labels, such as Dynabeads, LgR5, etc., which can also efficiently sort out high-purity hepatocytes and bile duct epithelial cells.

[0080] Changing the brand of instruments or consumables, such as using centrifuges, incubators or filtration equipment of different brands, can also be considered valid alternatives as long as they can achieve the same functions and ensure cell activity and purity.

[0081] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for simultaneously extracting multiple types of cells from the same liver tissue, characterized in that: include: Prepare single cell suspension; Extracting hepatic parenchymal cells; Extraction of bile duct epithelial cells; Extraction of hepatic stellate cells; The steps of extracting the hepatic parenchymal cells, the bile duct epithelial cells and the hepatic stellate cells are performed step by step in chronological order.

2. The method according to claim 1, characterized in that: Prepare a single cell suspension, including: Break down the extracellular matrix and loosen the connections between cells, so that some cells in the liver tissue exist as single cells; Liver tissue was digested into single cells to generate single cell suspension.

3. The method according to claim 2, characterized in that After generating a single-cell suspension, wash buffer was added to terminate digestion and filtered; The filtered cell suspension C1 was centrifuged at 100 g and 4° C. for 5 minutes to obtain a precipitate containing hepatic parenchymal cells, and the supernatant S1 was collected for extracting bile duct epithelial cells and hepatic stellate cells.

4. The method according to claim 3, characterized in that Extraction of hepatic parenchymal cells, including: Add red blood cell lysis buffer to the pellet containing hepatocytes, incubate on ice for 3-5 minutes, and then add wash buffer to terminate the reaction; Centrifuge at 100 g and 4 °C for 5 min, discard the supernatant, and resuspend the cell pellet with wash buffer to obtain cell suspension C2; Transfer cell suspension C2 onto 25% percoll gradient centrifugation medium; Centrifuge at 1474 g and 4 °C for 20 min, and resuspend the cell pellet in pre-cooled advanced DMEM / F12 basal medium to obtain cell suspension C3; After centrifugation again, discard the supernatant, add V volume of magnetic column buffer and CD326 magnetic beads, incubate at 4°C in the dark for 15 minutes, and then add 9V volume of magnetic column buffer to terminate the reaction; Set up a magnetic bead screening device, a filter column, and an EpCAM-negative cell suspension collection tube, add a magnetic column buffer to wet the filter column, add a cell-magnetic bead mixture suspension when the liquid has run out, and add a magnetic column buffer to rinse the filter column after the suspension has run out; The EpCAM-negative cell suspension was centrifuged at 100 g and 4° C. for 5 minutes, and the supernatant was discarded to obtain hepatic parenchymal cells.

5. The method according to claim 1, characterized in that Extraction of bile duct epithelial cells, including: Filter the supernatant S1 using a 40 μm mesh, and count the cells in the filtrate; Centrifuge at 580 g and 4 °C for 10 min to obtain the cell pellet and discard the supernatant; Add V volume of magnetic bead incubation buffer to the cell pellet to resuspend the cell pellet, add CD326 magnetic beads at the same time, incubate at 4°C in the dark for 15 minutes, and add 9V volume of magnetic bead incubation buffer to terminate the reaction; Place the magnetic bead screening device, filter column and EpCAM negative cell suspension collection tube, add magnetic column buffer to wet the filter column, and add the cell-magnetic bead mixture suspension after the liquid has drained away; After the suspension has flowed away, add magnetic column buffer to rinse the filter column, and collect the filtrate L1 for the extraction of hepatic stellate cells; Remove the filter column and add magnetic column buffer, and use the filter column core to push out the cell-magnetic bead mixture suspension; The mixture suspension was centrifuged at 300 g and 4° C. for 10 minutes, and the supernatant was discarded to obtain bile duct epithelial cells.

6. The method according to claim 1, characterized in that Extraction of hepatic stellate cells, including: The filtrate L1 was centrifuged at 580 g and 4 °C for 10 min to obtain a cell pellet; Resuspend the cell pellet in 50 ml of Grignard balanced salt solution B and centrifuge again at 580 g and 4 °C for 10 min; Discard the supernatant until 10 ml of Grignard balanced salt solution B remains, add 120 μl of DNAse I solution to the remaining liquid to resuspend the cell pellet, then add Grignard balanced salt solution B to 50 ml, and continue centrifugation at 580g and 4°C for 10 minutes; Discard the supernatant again until 10 ml of Grignard balanced salt solution B remains, add 120 μl of DNAse I solution to resuspend the cell pellet, and add 22 ml of Grignard balanced salt solution B and mix well; Add 16 ml of Nycogenz gradient centrifugation solution to the mixed cell suspension and turn the centrifuge tube upside down to wet the wall of the tube; Add 1.5 ml of Grignard balanced salt solution B above the cell suspension surface; The cell suspension was centrifuged at 1380 g and 4° C. for 17 minutes without braking and without acceleration or deceleration. A white layer appeared at 12 ml, and the hepatic stellate cell layer was obtained.

7. A method for subculturing hepatocytes obtained according to the method of claim 1, characterized in that: include: Resuspend the cell pellet in pre-cooled advanced DMEM / F12 basal medium, count and add 100,000 cells per well to a 24-well plate; Centrifuge again at 100 g and 4 °C for 5 min, discard the supernatant, resuspend the mixed Matrigel / cells at a Matrigel to cell ratio of 1:2000, and seed the mixture in a 24-well plate; After the matrix gel solidified, hepatocyte organoid culture medium was added to each well; When the 0th generation cells grow to 14 days, discard the culture medium, add pre-cooled advanced DMEM / F12 basal medium, use a pipette to mechanically break up the organoid-matrigel mixture and collect it into a centrifuge tube, mix well and place on ice for 30 minutes; Centrifuge at 100 g and 4 °C for 10 min, discard the supernatant, resuspend the organoid pellet in Matrigel, and seed into a 24-well plate at a 1:1 cell ratio; After the matrix gel solidifies, add 500 μl of hepatocyte organoid culture medium to each well; After the first generation, the cells were passaged at a ratio of 1:3-1:5, and each generation was passaged when 200 organoids were grown.

8. A method for subculturing bile duct epithelial cells obtained according to the method of claim 1, characterized in that: include: Resuspend the cell pellet in pre-chilled advanced DMEM / F12 basal medium, count and add 50,000 cells per well into a 24-well plate; The cell suspension was centrifuged again at 300 g and 4 °C for 10 min, the supernatant was discarded, and the matrix gel / cells were resuspended and mixed thoroughly at a matrix gel to cell ratio of 1:1000, and the cell-matrix gel mixture was seeded in a 24-well plate; After the matrix gel solidified, bile duct epithelial cell organoid culture medium was added to each well; When the 0th generation cells grow to 10 days, discard the culture medium, add pre-cooled advanced DMEM / F12 basal medium, use a pipette to mechanically break up the organoid-matrigel mixture and collect it into a centrifuge tube, mix well and place on ice for 30 minutes; Centrifuge at 300 g and 4 °C for 10 min, discard the supernatant, resuspend the organoid pellet in Matrigel, and seed into 24-well plates at a subculture ratio of 1:4-1:8; After the matrix gel solidified, 500 μl of bile duct epithelial cell organoid culture medium was added to each well, and each generation was passaged when 200 organoids were grown.

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