Culture method and system for maintaining hepatic parenchymal cell organoid maturation marker albumin and function

Abstract

The invention discloses a culture method and system for maintaining hepatic parenchymal cell organoid maturation marker albumin and functions. By improving a liver organoid culture system, the liver cell organoid capable of stably and highly expressing mature liver cell marker albumin and functions is obtained, and a new possibility is provided for application of the liver cell organoid.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a culture method and system for maintaining albumin, a mature marker of hepatic parenchymal cell organoids, and its function. Background Art

[0002] The ability to culture mature hepatocyte organoids for a long time is the key to developing in vitro toxicology methods and studying hepatotropic infections such as malaria and hepatitis viruses, as well as various genetic and metabolic liver diseases. Culturing and maintaining hepatocyte organoids with high expression of the mature marker albumin and normal functions is still challenging. Currently, the selective culture medium components for hepatocyte organoids are numerous, but it is difficult to maintain highly expressed albumin, and albumin is one of the important markers of mature hepatic parenchymal cells.

[0003] Currently, there are many selective culture medium systems for hepatocyte organoids, but it is difficult to maintain a stable and highly expressed pattern of albumin in hepatocyte organoids and hepatocyte functions for a long time. As a result, culturing hepatocyte organoids requires a large amount of liver tissue. However, due to the lack of liver sources, the production of hepatocyte organoids is limited. At the same time, the rapidly decreasing albumin also affects the large-scale expansion of organoids. These all directly limit the wide application of hepatocyte organoids.

[0004] It is of great practical significance to provide a culture method and system for maintaining albumin, a mature marker of hepatic parenchymal cell organoids, and its function. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. For this purpose, the present invention provides a culture method and system for maintaining albumin, a mature marker of hepatic parenchymal cell organoids, and its function.

[0006] According to the first aspect of the present invention, there is provided a culture method for maintaining albumin, a mature marker of hepatic parenchymal cell organoids, and its function, wherein the hepatic parenchymal cell organoids are treated with dexamethasone and / or γ-secretase inhibitor.

[0007] According to another aspect of the present invention, there is provided a culture medium for maintaining albumin, a mature marker of hepatic parenchymal cell organoids, and its function, wherein the culture medium contains dexamethasone and / or γ-secretase inhibitor.

[0008] Preferably, the basic component of the culture medium is advanced DMEM / F12 basal medium.

[0009] Beneficial effects: Currently, it is difficult for various selective hepatocyte organoid culture media to maintain a stable high-expression pattern of albumin (ALB), resulting in the rapid loss of the protein synthesis function of hepatocytes in organoids. As a result, a large amount of liver tissue is required for organoid culture. The rapid loss of function also affects the large-scale expansion of organoids for drug screening or the clinical application of the preparation of functional organoids. By improving the liver organoid culture system, the present invention obtains hepatocyte organoids that stably and highly express mature hepatocyte markers and have functions, providing a new possibility for the application of hepatocyte organoids. Brief Description of the Drawings

[0010] The drawings described herein are used to provide a further understanding of the present invention and form a part of this application. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0011] Figure 1 It is the experimental result provided by the embodiment of the present invention for proving that the simultaneous addition of dexamethasone and γ-secretase inhibitor can promote more mature hepatic parenchymal cell organoids.

[0012] Figure 2 It is the experimental result provided by the embodiment of the present invention for proving that dexamethasone and γ-secretase inhibitor need to be added to the culture system for a long time. Detailed Embodiments

[0013] The following listed embodiments are to enable those skilled in the art to understand the present invention more clearly. It should be noted that the following embodiments do not limit the protection scope required by the present invention and are only illustrative embodiments. The raw materials, reagents or devices mentioned in the following embodiments can be obtained from commercial channels or by known existing methods without special instructions.

[0014] Definition

[0015] (1) Organoids: Organoids are three-dimensional cell clusters generated by in vitro culture, which are miniaturized and simplified versions of specific organs, mimicking the key functions, structures and biological complexities of the organs. The culture of organoids can originate from pluripotent stem cells such as embryonic stem cells or adult stem cells, induced pluripotent stem cells, and cancer stem cells. The self-renewal and differentiation potential of these cells endow them with the ability to self-assemble under three-dimensional culture conditions.

[0016] (2) Hepatic parenchymal cells: Refers to the main functional cells in the liver, also known as hepatocytes. These cells are responsible for most of the metabolic functions of the liver, including detoxification, protein synthesis, and biochemical decomposition, etc.

[0017] Example 1: Cell Extraction

[0018] Preparation of liver single-cell suspension: Take the mouse liver, rinse it 2 - 3 times with pre-cooled advanced DMEM / F12 basal medium, and cut the liver into pieces of 0.5 mm 3 fragments with scissors. Rinse the fragments 2 - 3 times with 10 ml of pre-cooled wash buffer, add 5 ml of RWD Universal Tissue Gentle Enzymatic Digestion Solution and transfer it to a single-cell tube. Start the RWD Single-Cell Suspension Preparation Instrument and select the Liver_Heater mode to prepare the liver single-cell suspension.

[0019] Extraction of hepatocytes: After the procedure, add 5 ml of wash buffer to terminate the digestion, and filter through a 100 μm sieve. Centrifuge the cell suspension at 100 g and 4 °C for 5 minutes. The precipitate is the population of hepatocytes to be further purified. Transfer the supernatant to another tube for subsequent extraction of cholangiocytes and hepatic stellate cells. Add 1 ml of red blood cell lysis solution to the hepatocyte precipitate, mix well and incubate on ice for 3 - 5 minutes, then 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, carefully transfer the cell suspension to the surface of the percoll solution, and at this time, layering can be seen between the two. Centrifuge the cell suspension at 1474 g and 4 °C for 20 minutes. Resuspend the cell precipitate with 1 ml of pre-cooled advanced DMEM / F12 basal medium and count. Centrifuge again, discard the supernatant, add 100 μl of magnetic column buffer, and add 10 μl of CD326 magnetic beads for every 10 7 cells. Incubate in the dark at 4 °C for 15 minutes, then add 900 μl of magnetic column buffer to terminate the reaction. Place the magnetic bead screening device, filter column, and EpCAM-negative cell suspension collection tube, add 3 ml of magnetic column buffer to moisten the filter column. When the liquid is about to run out, add the cell-magnetic bead mixture suspension. When the suspension is about to run out, add 3 ml of magnetic bead incubation buffer to wash the filter column three times. Centrifuge the EpCAM-negative cell suspension at 100 g and 4 °C for 5 minutes, discard the supernatant, resuspend the cell precipitate with 2 ml of pre-cooled advanced DMEM / F12 basal medium, count, and seed 100,000 cells per well in a 24-well plate. Centrifuge again at 100 g and 4 °C for 5 minutes, discard the supernatant, resuspend and mix well the matrix gel / cells at a ratio of matrix gel:cells 1:2000, and seed the cell-matrix gel mixture into the 24-well plate. Wait for the matrix gel to solidify, and then add 500 μl of hepatocyte organoid medium to each well.

[0020] Reagents required for cell extraction: Wash buffer: Advanced DMEM / F12 basal medium, 1% penicillin-streptomycin mixture. 25% Percoll gradient centrifugation solution is 100% Percoll: 1×PBS = 1:3. 100% Percoll is Percoll cell dispersion solution: 10×PBS = 9:1. Magnetic column buffer: 2.5 ml MACS BSA solution, 47.5 ml autoMACS rinse solution. The patent No. 2024116146257 is incorporated into the present invention by reference and regarded as a part of the disclosure of the present invention.

[0021] Example 2: Subculture of Hepatocyte Organoids

[0022] When the 0th generation grows to 14 days, discard the culture medium, add 1 ml of pre-cooled advanced DMEM / F12 basal medium, use a 1 ml pipette to mechanically break the organoid-matrix gel mixture and collect it into a centrifuge tube, add 4 ml of pre-cooled advanced DMEM / F12 basal medium, mix well and place on ice for 30 minutes. Centrifuge at 100 g and 4 °C for 10 minutes, discard the supernatant, resuspend the organoid precipitate with matrix gel, and seed it into a 24-well plate at a cell ratio of 1:1, regarded as the 1st generation. Wait for the matrix gel to solidify, and add 500 μl of hepatocyte organoid culture medium to each well. After the 1st generation, passage at a cell ratio of 1:3 - 1:5, and passage can be carried out when each generation grows to 200 organoids.

[0023] Example 3: Different Formulation Systems

[0024] Formulation system 1 (advanced DMEM / F12 basal medium, cell-med): 1% penicillin-streptomycin mixture, 1% GlutaMAX, 10 mM HEPES, 1:50 B27 supplement, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1.5 μg / ml R-spodin 1, 3 μM ChIR99021, 10 nM human recombinant gastric mucosal regenerative factor, 50 ng / ml human recombinant epidermal growth factor, 50 ng / ml recombinant fibroblast growth factor-7, 50 ng / ml human recombinant fibroblast growth factor-10, 25 ng / ml human recombinant hepatocyte growth factor, 1 μM A83-01, 10 μM ROCK pathway inhibitor Y-27632.

[0025] Formulation system 2 (DAPT + / Dexamethasone +): 1% Penicillin-Streptomycin mixture, 1% GlutaMAX, 10 mM HEPES, 1:50 B27 supplement, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1.5 μg / ml R-spodin1, 3 μM ChIR99021, 10 nM human recombinant gastromucin, 50 ng / ml human recombinant epidermal growth factor, 50 ng / ml recombinant fibroblast growth factor-7, 50 ng / ml human recombinant fibroblast growth factor-10, 25 ng / ml human recombinant hepatocyte growth factor, 1 μM A83-01, 10 μM ROCK pathway inhibitor Y-27632, 3 μM dexamethasone, 10 μM γ-secretase inhibitor.

[0026] Formulation system 3 (Dexamethasone + ): 1% Penicillin-Streptomycin mixture, 1% GlutaMAX, 10 mM HEPES, 1:50 B27 supplement, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1.5 μg / ml R-spodin 1, 3 μM ChIR99021, 10 nM human recombinant gastromucin, 50 ng / ml human recombinant epidermal growth factor, 50 ng / ml recombinant fibroblast growth factor-7, 50 ng / ml human recombinant fibroblast growth factor-10, 25 ng / ml human recombinant hepatocyte growth factor, 1 μM A83-01, 10 μM ROCK pathway inhibitor Y-27632, 3 μM dexamethasone.

[0027] Formulation system 4 (DAPT + ): 1% Penicillin-Streptomycin mixture, 1% GlutaMAX, 10 mM HEPES, 1:50 B27 supplement, 1.25 mM N-acetylcysteine, 10 mM nicotinamide, 1.5 μg / ml R-spodin 1, 3 μM ChIR99021, 10 nM human recombinant gastromucin, 50 ng / ml human recombinant epidermal growth factor, 50 ng / ml recombinant fibroblast growth factor-7, 50 ng / ml human recombinant fibroblast growth factor-10, 25 ng / ml human recombinant hepatocyte growth factor, 1 μM A83-01, 10 μM ROCK pathway inhibitor Y-27632, 10 μM γ-secretase inhibitor.

[0028] Example 4: Albumin ELISA Detection (ALB ELISA kit)

[0029] Washing solution (1×): If crystals precipitate in the washing solution (20×), heat it at 37 °C until all the crystals dissolve. Dilute it at a dilution factor of 1:20: For example, take 30 ml of the concentrated washing solution (20×) and add 570 ml of ultrapure water or deionized water to obtain the washing solution (1×).

[0030] HRP-labeled detection antibody (1×): Centrifuge briefly before opening the cap and dilute it at a ratio of 1:100. Prepare it according to the total amount required for the experiment calculated in advance (100 μL / well) before dilution. When actually preparing, an additional 0.1 - 0.2 mL should be prepared. For example, prepare it by adding 10 μL of HRP-labeled detection antibody concentrate (100×) to 990 μL of antibody diluent and mix gently.

[0031] Add samples, and set zero wells, standard wells, and test sample wells respectively. Add 100 μL of sample diluent to the zero well, and add 100 μL of gradient-diluted standard or test sample to the remaining wells respectively, taking care not to generate bubbles (it is recommended to perform duplicate wells for both the standard and the sample to minimize experimental errors as much as possible, ensure continuous loading, and complete loading within 5 - 10 minutes);

[0032] Cover the enzyme-linked immunosorbent assay (ELISA) plate with a plastic film and incubate at 37 °C for 2 h;

[0033] Washing: Remove the sealing film (gently to avoid liquid overflow and cross-contamination due to excessive movement), discard the liquid, and pat dry; Wash the plate strips with washing solution (1×), 350 μL per well. After washing, shake off the liquid and pat dry the plate strips. Repeat this step 4 times, avoiding foreign matter entering the wells and keeping the plate strips from drying; Add 100 μL of HRP-labeled detection antibody (1×) to each well, cover with the sealing film, and incubate at 37 °C for 40 min; Repeat the washing step;

[0034] Color development: Add 100 μL of TMB color development solution to each well and develop color at 37 °C in the dark for 20 min; Keep the color development substrate in the dark at all times;

[0035] Termination: Add 100 μL of termination solution to each well, and the color changes from blue to yellow. The addition order of the termination solution is the same as that of the TMB color development solution;

[0036] Reading: Use a microplate reader to measure the optical density (OD value) of each well at a wavelength of 450 nm with a correction wavelength of 630 nm; Read within 5 min after adding the termination solution;

[0037] Data analysis: The OD value of each standard and sample needs to subtract the OD value of the zero well. Set duplicate wells and take the average value. Use the concentration of the standard as the abscissa and the OD value as the ordinate, and perform four-parameter fitting (4-PL) using professional software (such as Origin, ELISACalc, etc.). Calculate the fitted concentration of the sample from the standard curve based on the OD value of the sample.

[0038] Example 5: Cell Culture and Grouping Treatment

[0039] Collect cell supernatant: Passage the cells on the 14th day of passage 0, which is regarded as passage 1. After cell passage, wait for the Matrigel to solidify, and add 500 μl of 4 types of organoid culture medium formula systems to each well, namely formula system 1 (cell-med), formula system 2 (DAPT + / Dexamethasone + ), formula system 3 (Dexamethasone + ), formula system 4 (DAPT + ). On the 6th day of passage 1, collect the cell supernatants corresponding to the wells of each formula system for albumin concentration detection ( Figure 1 as shown).

[0040] The detection results show that after adding dexamethasone and γ-secretase inhibitor simultaneously, in hepatic parenchymal cell organoids, the albumin expression level is 58.25 times that without adding dexamethasone and γ-secretase inhibitor. After only adding dexamethasone, the albumin expression level is 7.3 times that without adding dexamethasone and γ-secretase inhibitor. After only adding γ-secretase inhibitor, the albumin expression level is 3.25 times that without adding dexamethasone and γ-secretase inhibitor, indicating that adding dexamethasone and γ-secretase inhibitor simultaneously can promote the generation of more mature hepatic parenchymal cell organoids.

[0041] In the second generation, continue to culture using various culture medium formula systems of the first generation, and collect the supernatant on the 6th day of culture. When passaging to the third generation, one well continues to use the corresponding culture medium formula system of the second generation, and the other well uses formula system 1 (cell-med), and collect the supernatant on the 6th day of culture. Use an ELISA kit to detect the albumin concentration in the cell supernatants corresponding to all different formula systems of the second and third generations ( Figure 2 as shown).

[0042] The detection results show that in the second generation (P2), when culturing hepatic parenchymal cell organoids using a culture medium supplemented with dexamethasone and γ-secretase inhibitor, and removing the two key components of dexamethasone and γ-secretase inhibitor in the third generation (P3), the expression level of the hepatic parenchymal cell maturation marker albumin is significantly reduced. It shows that dexamethasone and γ-secretase inhibitor need to be added to the culture system for a long time.

[0043] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for maintaining albumin, a mature marker of hepatocyte organoids, and its function, characterized in that: The hepatocyte organoids were treated with dexamethasone and or a γ-secretase inhibitor.

2. A culture medium for maintaining albumin, a maturation marker of hepatocyte organoids, and its function, characterized in that: Contains dexamethasone and or a gamma-secretase inhibitor.

3. The culture medium according to claim 2, characterized in that The basic component of the culture medium is advanced DMEM / F12 basic culture medium.

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