Mouse lung organ and fibroblast co-culture method

Abstract

The invention discloses a mouse lung organ and fibroblast co-culture method, which comprises the following steps of: performing in-vitro mouse lung organ culture on mouse lung tissue primary epithelial stem cell spheres (pneumospheroids) in a Transwell upper chamber, and after the lung organs in the Transwell upper chamber are differentiated, replacing with a lower chamber paved with mouse lung fibroblasts, therefore, a gas-liquid interface co-culture system of the mouse lung organ (upper chamber) and the fibroblast (lower chamber) is formed, and the hierarchical structure, morphological characteristics and regeneration function of the upper chamber organ and the phenotype, function and related signal path activation level of the lower chamber fibroblast can be dynamically researched.

Description

Technical Field

[0001] This invention belongs to the field of cell biology technology, specifically relating to a method for co-culturing mouse lung organoids and fibroblasts. Background Technology

[0002] Lung organoids are microorganisms formed by the differentiation of lung epithelial stem / progenitor cells in an in vitro 3D culture environment. They have the ability to self-renew and self-organize, and can highly simulate the three-dimensional structure, histological characteristics and physiological state of lung epithelium in vivo. They are an effective tool for studying lung epithelial-related diseases and regeneration.

[0003] Currently, because lung organoids are primarily derived from epithelial stem / progenitor cells, their applications are limited to research related to the lung epithelial system. However, the lung is composed of various cell types, including epithelial cells, fibroblasts, and macrophages. The development and progression of respiratory diseases such as pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease involve the interaction and regulation between epithelial and interstitial cells. Therefore, lung organoid models based on a single primary epithelial cell type significantly limit research into the complex pathological mechanisms of these related diseases. Summary of the Invention

[0004] The purpose of this invention is to overcome the problems existing in the prior art and provide a method for co-culturing mouse lung organoids and fibroblasts.

[0005] To achieve the above technical objectives, the present invention adopts the following technical solution:

[0006] A method for co-culturing mouse lung organoids and fibroblasts, comprising:

[0007] A mixture of mouse lung glomeruli and Matrigel was seeded in the upper chamber of a Transwell chamber; the lung glomeruli were primary epithelial stem cell glomerates from mouse lung tissue.

[0008] After the mixture solidifies, gas-liquid interface differentiation medium is added to both the upper and lower chambers of the Transwell chamber for culture until mouse lung glomeruli differentiate into organoids.

[0009] Lung fibroblasts were seeded in a multi-well plate, the lower chamber was replaced with the multi-well plate seeded with lung fibroblasts, and the gas-liquid interface differentiation medium was added to the new lower chamber for co-culture.

[0010] As a preferred embodiment, mouse lung glomeruli with a diameter of 50-80 μm are taken, and the mixture of mouse lung glomeruli and Matrigel matrix is ​​prepared at a ratio of 200-300 lung glomeruli / 50 μL Matrigel matrix and then implanted into the upper chamber of a Transwell chamber.

[0011] In a preferred embodiment, the pore size of the Transwell chamber is 0.4 μm. In the prior art, the upper chamber of the Transwell in a typical co-culture system of tumor organoids and cancer-associated fibroblasts contains cancer-associated fibroblasts, and the lower chamber contains tumor organoids. The pore size of the Transwell chamber in this co-culture system is 1.0 μm. In this invention, the upper chamber of the Transwell contains lung organoids, and the lower chamber contains fibroblasts. The 0.4 μm pore size of the Transwell permeable membrane ensures that lung epithelial stem cells adhere firmly to the upper chamber, facilitating complete differentiation, while preventing infiltration into the lower chamber before Matrigel solidifies. Simultaneously, it ensures sufficient nutrient supply to the lung organoids in the upper chamber.

[0012] In a preferred embodiment, the lung fibroblasts are third-generation mouse lung fibroblasts.

[0013] Furthermore, the lung fibroblasts were obtained in the following manner:

[0014] Mouse lung tissue was dissociated using digestive enzymes. The resulting single-cell suspension was filtered and induced to adhere and expand using DMEM / F12 complete culture medium. This invention pre-digests the lung tissue with collagenase A solution. Pre-digestion with collagenase A solution loosens the mouse lung tissue, ensuring more complete and thorough subsequent trypsin-EDTA digestion and resulting in a higher yield of lung fibroblasts.

[0015] In a preferred embodiment, the method of dissociating mouse lung tissue using digestive enzymes is as follows: first, the mouse lung tissue is pre-digested using collagenase A digestion solution, and then digested and dissociated using trypsin-EDTA digestion solution.

[0016] As a preferred embodiment, after adding gas-liquid interface differentiation medium to both the upper and lower chambers of the Transwell chamber, the Transwell chamber is placed in a carbon dioxide cell culture incubator for further culture until mouse lung glomeruli differentiate into organoids.

[0017] In a preferred embodiment, the gas-liquid interface differentiation culture medium comprises: PneumaCult with 0.096 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, and 20 ng / mL EGF. TM -Ex Plus medium.

[0018] As a preferred embodiment, the mouse lung glomeruli are prepared as follows:

[0019] a. Take a block of mouse lung tissue and digest it in a preheated collagenase digestion solution;

[0020] b. Add an equal volume of DMEM / F12 medium containing FBS to terminate digestion, mix well by pipetting, and filter through a cell sieve; centrifuge the filtrate and discard the supernatant;

[0021] c. Resuspend the cell pellet in red blood cell lysis buffer, incubate at room temperature, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant;

[0022] d. Repeat step c until the cell pellet turns white;

[0023] e. Resuspend the cell pellet in DNase solution, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant;

[0024] f. Resuspend the cell pellet in DMEM / F12 medium, mix well by pipetting, filter through a cell sieve, and discard the supernatant after centrifugation;

[0025] g. Resuspend the cells in pulmonary glomerular culture medium, adjust the cell density, and then culture in suspension.

[0026] Furthermore, the pulmonary globulin culture medium is a DMEM / F12 medium containing 1×B-27 additive (without vitamin A), 1×penicillin / streptomycin solution, 4 μg / mL heparin, 20 ng / mL EGF, 10 ng / mL FGF2, and 10 μM Y-27632.

[0027] The present invention has the following beneficial effects:

[0028] (1) The system of this invention is not co-cultured throughout the entire process. After the differentiation of lung organoids in the upper chamber is completed, the lower chamber is replaced with one containing mouse lung fibroblasts. This ensures that the differentiation process of lung organoids in the upper chamber is not affected, and allows for dynamic observation of the phenotypic and functional changes of lung organoids and lung fibroblasts before and after co-culture. In addition, once the co-culture stage is underway, the morphological characteristics, tissue structure, phenotype, and function of the culture in the upper or lower chamber can be observed at any time. The co-culture time is flexible, controllable, and unrestricted, meeting the needs of research at different time stages. This system has a wider range of applications and scenarios. The co-culture system of this invention can not only dynamically study the hierarchical structure, morphological characteristics, and regenerative function of lung organoids in the upper chamber of Transwell, but also dynamically detect the phenotype, function, and activation level of related signaling pathways of fibroblasts in the lower chamber. This provides a new technical platform for a comprehensive and in-depth exploration of the cellular and molecular mechanisms of epithelial-mesenchymal cell interactions in related lung diseases.

[0029] (2) In the 3D-Matrigel-ALI co-culture system, the lower chamber culture medium contains cytokines secreted by lung organoids and fibroblasts, but the upper chamber culture medium contains only cytokines secreted by lung organoids. The upper and lower chamber culture media cannot permeate each other. Different interventions can be precisely given to the upper chamber or (and) the lower chamber according to the experimental purpose, and the intervention time can be dynamically adjusted for the study of differences or changes in the content of specific cytokines.

[0030] (3) The gas-liquid interface differentiation culture medium in this invention can be used for the culture of lung organoids and lung fibroblasts. Using the same culture medium has good batch stability and reproducibility, and has a wide range of applications. No additional adjustments to the experimental conditions are required, and the operation is simple and easy.

[0031] (4) Based on the previously established methods for culturing primary epithelial stem cell spheres of mouse lung tissue (CN111534477A) and mouse lung organoids (CN112852709A), this invention successfully established a method for co-culturing mouse lung organoids and fibroblasts using a 3D-Matrigel matrix gel-ALI culture model with a Transwell cell culture chamber as the permeable membrane, and validated the phenotype and function. The establishment of this co-culture method provides strong technical support and an application platform for the study of the pathological mechanisms of respiratory diseases such as pulmonary fibrosis, asthma, and chronic obstructive pulmonary disease, as well as the screening of related drugs. Attached Figure Description

[0032] Figure 1 This is a flowchart of the co-culture process of mouse lung organoids and lung fibroblasts.

[0033] Figure 2 The results are the morphology of mouse third-generation lung fibroblasts under a light microscope (100×) and the identification results of Vimentin immunofluorescence staining.

[0034] Figure 3 The results are H&E and PAS staining of paraffin sections of upper chamber lung organoids after 0 hours and 72 hours of co-culture of mouse lung organoids and lung fibroblasts.

[0035] Figure 4 The images show the morphology (100×) and EdU staining results of lower ventricular lung fibroblasts after 0 hours and 72 hours of co-culture with mouse lung organoids and lung fibroblasts. Detailed Implementation

[0036] The main materials used in the embodiments and their sources are as follows:

[0037] Transwell cell culture chamber: Corning Incorporated, USA, catalog number 3460.

[0038] TrypLE cellular digestive enzyme: Thermo Fisher Scientific (China) Co., Ltd., item number 12604021.

[0039] Gas-liquid interface differentiation medium: [added with 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, 4 μg / mL heparin, 20 ng / mL EGF PneumaCult] TM -ALI culture medium]: Hydrocortisone solution: STEMCELL Ltd., Canada, catalog number 07925; Penicillin and streptomycin solution: ScienCell Ltd., USA, catalog number 0503; Heparin: APExBIO Ltd., USA, catalog number B3602; EGF: PeproTech Ltd., USA, catalog number 315-09; PneumaCult TM -ALI culture medium: STEMCELL Ltd, Canada, catalog number 05001.

[0040] Collagenase A digestion solution (2 mg / mL collagenase A in DMEM / F12 medium): Collagenase A: Shanghai Sigma-Aldrich Trading Co., Ltd., catalog number C0130; DMEM / F12 medium: Thermo Fisher Scientific (China) Co., Ltd., catalog number 11039021.

[0041] Trypsin-EDTA digestion solution (0.25%): Shanghai Beyotime Biotechnology Co., Ltd., product number C0203.

[0042] DMEM / F12 Complete Medium: [DMEM / F12 medium supplemented with 10% FBS (fetal bovine serum)]: FBS: ScienCell, Inc., USA, catalog number 0010; DMEM / F12 medium: Thermo Fisher Scientific (China) Co., Ltd., catalog number 11039021.

[0043] 40μm cell sieve: Hefei Biosharp Co., Ltd., product number BS-40-XBS.

[0044] 12-well cell culture plate: Corning Incorporated, USA, catalog number 3513.

[0045] T25 cell culture flask: Corning Incorporated, USA, catalog number 430639.

[0046] Sterile PBS (phosphate buffered solution): Beijing Solarbio Science & Technology Co., Ltd., catalog number P1020.

[0047] Example 1

[0048] The example first uses the method shown in CN111534477A to culture mouse lung glomeruli (i.e., steps 1 to 10 below), and then uses Transwell chambers for culture. The procedure is as follows: Figure 1 As shown, the specific steps include the following:

[0049] (1) Thoroughly disinfect the skin of mice with 75% alcohol, aseptically separate the lung tissue and rinse it in pre-cooled sterile PBS (phosphate buffer solution) to remove connective tissue and the main bronchus in the lungs. At the same time, separate the lung lobes and wash them 2-3 times to remove blood.

[0050] (2) Use sterile forceps to transfer the cleaned lung lobes to a new cell culture dish, remove any residual PBS, and use ophthalmic surgical scissors to cut the lung tissue into pieces of approximately 1 mm. 3 The tissue blocks were transferred to a preheated collagenase digestion solution and digested at 37°C in a shaker (100 rpm) for 45-60 minutes.

[0051] (3) Add an equal amount of DMEM / F12 medium containing 10% FBS to stop digestion, mix well by pipetting, and filter through a 100μm cell sieve; centrifuge the filtrate at 700-900g for 10 minutes at 4℃ and discard the supernatant;

[0052] (4) Add 2-3 mL of red blood cell lysis buffer to resuspend the cell pellet, incubate at room temperature for 1-2 minutes, add 6 mL of DMEM / F12 medium and mix well, centrifuge at 700-900g at 4℃ for 10 minutes, and discard the supernatant;

[0053] (5) Repeat step (4) 1-2 times until the cell pellet turns white;

[0054] (6) Add 4 mL of DNase solution (20 U / mL) to resuspend the cell pellet, shake by hand for 3-5 minutes at room temperature, add 6 mL of LDM / F12 medium and mix well, centrifuge at 700-900g for 10 minutes at 4℃, and discard the supernatant;

[0055] (7) Add 6 mL of DMEM / F12 medium to resuspend the cell pellet, mix thoroughly by pipetting, filter through a 40 μm cell sieve, centrifuge at 700-900 g for 10 minutes at 4℃, and discard the supernatant;

[0056] (8) Resuspend the pulmonary glomeruli in culture medium and adjust the cell density to 2×10⁻⁶. 6 - 3×10 6 / mL;

[0057] (9) Culture in poly-HEMA-coated T25 cell culture flasks, 3-5 mL of pulmonary glomeruli culture medium / flask;

[0058] (10) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium every three days;

[0059] (11) After culturing for 3-4 days, collect lung glomeruli with a diameter of 50-80 μm, centrifuge at 200-300g for 5-10 minutes at 4℃, and discard the supernatant;

[0060] (12) Add 5 mL of DMEM / F12 medium to resuspend the lung glomeruli, centrifuge at 200-300g for 5-10 minutes at 4℃, and discard the supernatant;

[0061] (13) Repeat step (12) 1-2 times;

[0062] (14) Add Matrigel according to the number of lung cells (200-300 lung cells / 50μL Matrigel), and mix thoroughly with a pre-cooled 200μL pipette tip to avoid generating air bubbles (operate on ice).

[0063] (15) Take 50 μL of the mixture of pulmonary bulb and Matrigel into the center of the upper chamber of the Transwell, gently shake to spread the mixture evenly on the bottom of the upper chamber, and incubate at 37°C for 30-45 minutes to allow the Matrigel to solidify.

[0064] (16) After the mixture solidifies, 0.5 mL and 1 mL of gas-liquid interface differentiation medium are added to the upper and lower chambers of the Transwell, respectively, and the mixture is transferred to a carbon dioxide cell culture incubator (37℃, 5% CO2). The medium is changed every two days, and the formation and morphological changes of mouse lung organoids are dynamically observed.

[0065] (17) Thoroughly disinfect the skin of mice with 75% alcohol, aseptically separate the lung tissue and rinse it in pre-cooled sterile PBS (phosphate buffer solution) to remove connective tissue and the main bronchus in the lungs. At the same time, separate the lung lobes and wash them 2-3 times to remove blood.

[0066] (18) Use sterile forceps to transfer the cleaned lung lobes to a new cell culture dish, remove any residual PBS, and use ophthalmic surgical scissors to cut the lung tissue into pieces of approximately 1 mm. 3 The tissue blocks were transferred to a preheated collagenase A digestion solution and digested at 37°C on a shaker (100 rpm) for 30 minutes. Then, they were centrifuged at 700-900g for 10 minutes at 4°C, and the supernatant was discarded.

[0067] (19) Add 1 mL of sterile PBS to wash the precipitate, centrifuge at 700-900g for 10 minutes at 4℃, and discard the supernatant;

[0068] (20) Add 1 mL of trypsin-EDTA digestion solution (0.25%), digest at 37°C in a shaker (100 rpm) for 20 minutes, and terminate digestion with an equal volume of DMEM / F12 complete medium;

[0069] (21) Filter the cells through a 40μm cell sieve to remove undigested cell clumps, centrifuge at 900g for 5 minutes at 4℃, and discard the supernatant;

[0070] (22) Resuspend in DMEM / F12 complete medium and adjust cell density to 1×10⁻⁶. 5 - 2×10 5 / mL, seeded in T25 cell culture flasks;

[0071] (23) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium every three days;

[0072] (24) Based on the cell proliferation and fusion status, use trypsin-EDTA digestion solution (0.25%) to digest and separate the cells, and passage them at a ratio of 1:3;

[0073] (25) Third generation lung fibroblasts were seeded in 12-well plates. After the cells adhered, the culture medium was aspirated and the Transwell chamber containing airway-like structures was inserted into the 12-well plates. Then, gas-liquid interface differentiation medium was added to both the upper and lower chambers.

[0074] (26) Transfer to a carbon dioxide cell culture incubator (37℃, 5% CO2), and change the culture medium every two days. During this period, the phenotypic and functional changes of lung organoids (upper chamber) and lung fibroblasts (lower chamber) in this co-culture system under different intervention conditions can be dynamically detected according to the research objectives.

[0075] Example: Cultured third-generation mouse lung fibroblasts, such as Figure 2 As shown, under a light microscope (100×), mouse lung fibroblasts with large, multi-protruding, spindle-shaped or star-shaped flat cells and regular oval nuclei with indistinct cell outlines are visible (left), and identified by immunofluorescence staining with the fibroblast marker Vimentin (right).

[0076] Figure 3 It can be seen that after 0 hours and 72 hours of co-culture of mouse lung organoids and lung fibroblasts, histological staining of lung organoids in the upper chamber of Transwell showed mucus secreted by ciliated cells (H&E staining, left) and goblet cells (PAS staining, right).

[0077] Figure 4It can be seen that after 0 hours and 72 hours of co-culture of mouse lung organoids and lung fibroblasts, Transwell lower chamber lung fibroblasts showed typical light microscopic morphology (left) and cell proliferation (EdU staining, right).

[0078] As can be seen from the attached diagram, the co-culture system does not affect the normal growth of airway-like cells and fibroblasts, and is indeed feasible.

[0079] The co-culture system of mouse lung organoids and fibroblasts established by the method described in the examples uses a Transwell chamber with a permeable membrane of 0.4 μm pore size to separate the two cell types. This non-contact co-culture model can better simulate the paracrine effect or synergistic effect between cells in the native environment of lung tissue in vivo, with stronger physiological relevance. At the same time, it can also be used to study the phenotype, function and activation level of related signaling pathways of lung organoids (upper chamber) and lung fibroblasts (lower chamber) separately, which is particularly suitable for the study of respiratory diseases caused by the combined action of epithelial and mesenchymal cells.

Claims

1. A method for co-culturing mouse lung organoids and fibroblasts, characterized in that, include: Mouse lung bulbs were seeded in the upper chamber of a Transwell chamber as a mixture of Matrigel and glomeruli. The lung spheres are primary epithelial stem cell spheres from mouse lung tissue. After the mixture solidifies, gas-liquid interface differentiation medium is added to both the upper and lower chambers of the Transwell chamber for culture until mouse lung glomeruli differentiate into organoids. Lung fibroblasts were seeded in a multi-well plate, the lower chamber was replaced with the multi-well plate seeded with lung fibroblasts, and the gas-liquid interface differentiation medium was added to the new lower chamber for co-culture.

2. The method according to claim 1, characterized in that, Take lung bulbs with a diameter of 50-80 μm, mix mouse lung bulbs with Matrigel at a rate of 200-300 lung bulbs / 50 μL Matrigel, and then implant them into the upper chamber of a Transwell chamber.

3. The method according to claim 1, characterized in that, The membrane pore size of the Transwell chamber is 0.4 μm.

4. The method according to claim 1, characterized in that, The lung fibroblasts mentioned are third-generation lung fibroblasts from mice.

5. The method according to claim 1 or 4, characterized in that, The lung fibroblasts were obtained in the following manner: Mouse lung tissue was dissociated using digestive enzymes. The resulting single-cell suspension was filtered and then induced to adhere and expand using DMEM / F12 complete culture medium to obtain lung fibroblasts.

6. The method according to claim 5, characterized in that, The method for dissociating mouse lung tissue using digestive enzymes is as follows: first, the mouse lung tissue is pre-digested using collagenase A digestion solution, and then digested and dissociated using trypsin-EDTA digestion solution.

7. The method according to claim 1, characterized in that, After adding gas-liquid interface differentiation medium to both the upper and lower chambers of the Transwell chamber, the Transwell chamber was placed in a carbon dioxide cell culture incubator for further culture until the mouse lung glomeruli differentiated into organoids.

8. The method according to claim 1, characterized in that, The gas-liquid interface differentiation culture medium consisted of: 0.48 μg / mL hydrocortisone solution, 100 U / mL penicillin solution, 100 μg / mL streptomycin solution, 4 μg / mL heparin, and 20 ng / mL EGF in PneumaCult. TM -ALI medium.

9. The method according to claim 1, characterized in that, The mouse lung glomeruli were prepared as follows: a. Take a block of mouse lung tissue and digest it in a preheated collagenase digestion solution; b. Add an equal volume of DMEM / F12 medium containing FBS to terminate digestion, mix well by pipetting, and filter through a cell sieve; centrifuge the filtrate and discard the supernatant; c. Resuspend the cell pellet in red blood cell lysis buffer, incubate at room temperature, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant; d. Repeat step c until the cell pellet turns white; e. Resuspend the cell pellet in DNase solution, add DMEM / F12 medium, mix well, centrifuge and discard the supernatant; f. Resuspend the cell pellet in DMEM / F12 medium, mix well by pipetting, filter through a cell sieve, and discard the supernatant after centrifugation; g. Resuspend the cells in pulmonary glomerular culture medium, adjust the cell density, and then culture in suspension.

10. The method according to claim 9, characterized in that, The pulmonary glomeruli culture medium was DMEM / F12 medium containing 1×B-27 additive (without vitamin A), 1×penicillin / streptomycin solution, 4 μg / mL heparin, 20 ng / mL EGF, 10 ng / mL FGF2, and 10 μM Y-27632.

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