Construction method of lung organ model based on COPD patient source

Abstract

The invention discloses a construction method of a lung organ model based on a COPD patient source, relates to the technical field of biomedical model construction, and efficiently constructs a lung organ model retaining COPD pathological characteristics by optimizing tissue treatment, cell separation and three-dimensional culture conditions. The method comprises the following specific steps: acquiring fresh surgical resected lung tissues of a COPD patient; primary lung epithelial cells are obtained by combining collagenase IV digestion with cell filtration and lung interstitial cell separation; the method comprises the following steps: culturing cells by a matrigel embedding method, and culturing for 10 days by utilizing an amplification culture medium to form an organ-like structure; and then switching to a differential culture medium and inducing for 7 days to realize functional differentiation of the airway epithelial cells and the pulmonary alveolar cells. According to the method, the construction efficiency and stability of the organoid are remarkably improved, the constructed model can simulate the COPD pathological microenvironment, and the method is suitable for disease mechanism research, drug screening and individualized treatment evaluation and has high reproducibility and clinical transformation potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical model construction technology, and in particular to a method for constructing a lung organoid model based on COPD patients. Background Technology

[0002] Obstructive pulmonary disease (COPD) is a chronic inflammatory respiratory disease with high global morbidity and mortality. It is characterized by progressive, irreversible airflow limitation, heterogeneous endophenotypes, and distinct disease trajectories among individuals (see Reference 1). With approximately 384 million people affected globally and more than 1.28 million deaths annually, COPD poses a significant challenge to public health worldwide. As the complexity and heterogeneity among individual COPD patients become widely recognized, cell-based research remains extremely challenging due to the lack of suitable experimental models to reproduce the disease at the individual level. While commonly used small animal models, including mice, guinea pigs, and rabbits, are widely used in COPD research, they still have significant limitations: these models require artificial induction, cannot fully mimic the genetic and epigenetic characteristics of human COPD, and struggle to reproduce the complete disease spectrum exhibited by clinical patients.

[0003] Organoids are miniature tissues with spatial structures formed through three-dimensional (3D) in vitro culture of adult stem cells or pluripotent stem cells. Although not true human organs, organoids can highly mimic real organs in structure and function and maintain long-term stable passage culture capability, hence the name "miniature organs." This technology provides an excellent model system for pathophysiological research and drug screening. Organoids offer advantages over traditional 2D culture, displaying near-physiological cellular composition and behavior. Compared to animal models, organoids reduce experimental complexity, are suitable for real-time imaging techniques, and can be used to study various aspects of human development and disease. Organoids have been successfully constructed from primary tumors of the colon, prostate, breast, and pancreas. These "tumor organoids" have become preclinical models with the potential to predict individual patient responses to treatment. For example, a live biobank of tumor organoids from patients with metastatic gastrointestinal cancer has reproduced these patients' responses to anticancer drugs in clinical trials.

[0004] Lung organoid technology offers a new avenue for disease research, with some research teams already constructing COPD organoids using clinical samples. However, lung stem cells from COPD patients (especially elderly patients) suffer from low in vitro expansion efficiency and difficulty in maintaining stemness, failing to effectively support long-term expansion and clinicopathological characteristics of COPD stem cells. Therefore, there is an urgent need to develop a lung stem cell expansion culture medium and supporting culture system specifically designed for the COPD pathological microenvironment. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by proposing a method for constructing lung organoid models derived from COPD patients. Its advantage lies in effectively improving the clonal formation rate of COPD organoids, thereby efficiently constructing lung COPD organoid models with stable pathological characteristics.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A method for constructing a lung organoid model derived from COPD patients includes the following steps: Fresh surgically removed lung tissue from COPD patients was obtained and placed in a culture medium containing 2% FBS within 1.5 hours after ex vivo. Necrotic areas were removed and the tissue was minced to less than 1 mm³. Collagenase IV at a final concentration of 1 mg / mL was used for digestion at 37°C with shaking for 45 minutes, followed by filtration through 300 μm and 100 μm cell filters. The filtered cells were cultured in a type I collagen-coated culture dish in basal medium for 1 hour to remove mesenchymal cells. Epithelial cells were cultured in amplification medium under 3D conditions for 10 days, followed by culture in differentiation medium for 7 days to obtain the COPD organoids.

[0007] The present invention is further characterized in that the basal culture medium comprises AdvancedDMEM / F-12, fetal bovine serum, HEPES, penicillin / streptomycin, L-glutamine, sodium bicarbonate and B27 supplement; the concentrations of each component in the basal culture medium are: FBS: 2%, HEPES: 15mM, penicillin / streptomycin: 1%, L-glutamine: 4mM, sodium bicarbonate: 3.6mM and 1×B27 supplement.

[0008] The present invention further specifies that the amplification culture medium comprises: AdvancedDMEM / F12, 2-5% FBS, EGF: 20-50 ng / mL, FGF7: 20-50 ng / mL, SB431542: 5-15 μM, CHIR99021: 1-5 μM, R-Spondin1: 200-800 ng / mL, Y27632: 5-15 μM, insulin: 5-15 μg / mL, cholera toxin: 0.05-0.2 μg / mL, and Transferrin: 5-20 μg / mL.

[0009] The present invention is further configured such that the components of the differentiation culture medium are: AdvancedDMEM / F12, FGF7: 20-50 ng / mL, FGF10: 20-100 ng / mL, Noggin: 50-200 ng / mL, retinoic acid: 0.5-2 μM, SPC: 5-20 ng / mL, dexamethasone: 30-100 nM and SB202190: 0.5-2 μM.

[0010] The present invention is further configured such that the collagenase IV is gently blown 10-20 times every 10 minutes during digestion.

[0011] The present invention is further configured such that the collagenase IV is a crude extract of enzyme derived from Clostridium histolyticum, and its components not only contain Clostridium protease A, which can degrade natural collagen and reticular fibers, but also contain some proteases, polysaccharides and lipases.

[0012] The present invention is further configured such that collagenase IV effectively hydrolyzes proteins, polysaccharides and lipids present in the extracellular matrix of connective tissue and epithelial tissue, thereby achieving the purpose of cell separation; in addition, type IV collagenase has low pancreatic enzyme activity to reduce damage to membrane proteins and receptors.

[0013] The present invention is further configured such that, during the upper cell expansion and differentiation stage, a basement membrane environment is provided to the cells through a 50% high concentration of Matrigel, thereby promoting epithelial cell polarization and 3D structure formation.

[0014] Application of a lung organoid model derived from COPD patients in precision medicine, organ transplantation, drug screening, and drug mechanism of action research.

[0015] The beneficial effects of this invention are as follows: 1. High-efficiency expansion and stemness maintenance: By optimizing the combination of growth factors and metabolic regulators, the survival rate and proliferation capacity of COPD stem cells are significantly improved, breaking through the bottleneck of in vitro expansion of adult lung stem cells in COPD patients.

[0016] 2. Simulate COPD pathological features: By optimizing the differentiation culture medium formula, cell differentiation is promoted, and organoids are driven to reproduce the core COPD phenotypes such as hypersecretion of mucus, reduced cilia and functional impairment. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the COPD organoid culture process, which is a method for constructing a lung organoid model derived from COPD patients proposed in this invention. Figure 2The images show representative photographs (A), clone formation rate (B), and cell number (C) of COPD organoids cultured for 3, 6, and 9 days in the expansion culture of the method for constructing a lung organoid model based on COPD patients proposed in this invention. Figure 3 This is a schematic diagram of the tracheal and alveolar organoids (A) and qPCR identification diagram (B) formed after amplification and culture of the lung organoid model based on COPD patients proposed in this invention. Figure 4 Morphological images (A) of hollow control group organoids and COPD organoids, and qPCR identification image (B), are provided for the method of constructing lung organoid models based on COPD patients proposed in this invention. Detailed Implementation

[0018] The technical solution of this patent will be further described in detail below with reference to specific embodiments.

[0019] The embodiments of this patent are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this patent, and should not be construed as limiting this patent.

[0020] The English abbreviations used in this invention represent the following meanings: COPD: Chronic Obstructive Pulmonary Disease; 3D: Three-dimensional culture; Matrilgel: Base gel, providing a 3D environment for cell culture; AdvancedDMEM / F-12: A culture medium containing various amino acids and glucose; FBS: Fetal Bovine Serum; PBS: Phosphate Buffered Protein; GlutaMAX: Glutamate additive; NaHCO3: Sodium Bicarbonate; HEPES: A buffer reagent; EGF: Epidermal Growth Factor; FGF7: Fibroblast Growth Factor 7; SPC: Surfactant Protein C; qPCR: Real-time Quantitative PCR.

[0021] Reference Figure 1-4 A method for constructing lung organoid models based on COPD patients includes the following steps:

[0022] Step 1: Obtain surgically removed lung tissue from a COPD patient (preserve on ice and process within 1.5 hours after removal from the body).

[0023] Step 2: Place the tissue in a culture dish containing pre-cooled basal medium (Advanced DMEM / F-12 + 2% FBS + 1% penicillin and streptomycin) and cut it into ≤1mm³ fragments with a sterile scalpel.

[0024] Step 3: Add collagenase IV to a final concentration of 1 mg / mL, and digest at 37°C with shaking for 40 minutes, gently blowing and swishing 10-20 times every 10 minutes.

[0025] Step 4: First, filter the cell suspension through a 300μm cell filter, rinse the filter with 1mL of basal culture medium, centrifuge at 4°C, 400×g for 5 minutes, discard the supernatant, resuspend in 400μL of basal culture medium, then filter the cell suspension through a 100μm cell filter, rinse the filter with 1mL of basal culture medium, centrifuge at 4°C, 1000×g for 5 minutes, and discard the supernatant.

[0026] Step 5: Add 200 μL of erythrocyte lysis buffer, incubate at room temperature for 5 minutes, centrifuge, wash, and resuspend in basal culture medium.

[0027] Step 6: Wash the cells with 1 mL of pre-cooled basal culture medium, centrifuge at 4°C, 400×g for 5 minutes, and discard the supernatant.

[0028] Step 7: Resuspend cells in 100 μL of basal culture medium, count cells, centrifuge at 4°C, 400 × g for 5 minutes, and discard the supernatant. Step 8: Add 1 mL of L1 type rat tail collagen coating solution (formula) to a 35 mm culture dish. After 2 hours at room temperature, remove the coating solution and allow it to air dry at room temperature for at least 5 minutes. Step 9: Wash the top and bottom surfaces of the culture dish three times with 1 mL of sterile PBS to remove unbound collagen; Step 10: Inoculate the cell suspension prepared in step 7 into collagen-coated culture dishes and incubate at 37°C for 1 hour.

[0029] Step 11: Carefully collect the non-adherent cells into a new 15 mL centrifuge tube. Centrifuge at 1000 × g for 5 minutes. Discard the supernatant.

[0030] Step 12: Resuspend the cells in 1 mL of basal culture medium.

[0031] Step 13: Use a cell counter to count the number of cells and adjust to a suitable cell density.

[0032] Step 14: Mix the cell suspension with Matrigel at a 1:1 ratio on ice.

[0033] Note: The mixed cell-Matrigel mixture should be placed on ice to prevent the Matrigel from solidifying.

[0034] Step 15: Seed the mixture into the center region of a Matrigel-coated 48-well plate at a ratio of 800 cells per well, forming 3D droplets containing 50% Matrigel. Incubate at 37°C for 30 minutes.

[0035] Step 16: After Matrigel solidifies into a gel, slowly add 200 μL of amplification medium along the well wall.

[0036] Step 17: Incubate at 37℃ in a 5% CO2 incubator. Take photos on days 3, 6, and 9 to observe the organoid formation and enlargement process and to detect the organoid clonal formation rate (see attached). Figure 2 (AB). The isolated cells were cultured in expansion medium for 10 days, enabling rapid cell expansion (see appendix). Figure 2 C).

[0037] Step 18: After 10 days of expansion culture under 3D conditions, hollow airway organoids and non-hollow alveolar organoids can be observed (see appendix). Figure 3 A), qPCR analysis further confirmed that the formed organoids expressed proximal airway epithelial marker genes (SOX2, P63) and distal epithelial marker genes (SOX9, SPC) (see appendix). Figure 3 A).

[0038] Step 19: The expanded organoids were induced to differentiate on differentiation medium for 7 days. Numerous secretions or cells appeared within the hollow COPD airway organoid lumen. qPCR analysis showed a significant increase in the expression of MUC5AC in the COPD organoids, and a significant decrease in the expression of the ciliated cell marker gene AC-TUB. (See appendix) Figure 4 (AB), which simulates the clinical characteristics of high MUC5AC secretion and reduced ciliated cells in COPD patients.

[0039] The collagenase IV used in this invention is Sigma C9407, the erythrocyte lysis buffer is Biosharp BL503A, the Advanced DMEM / F-12 is Gibco C11330500BT, the FBS is Gibco 10099-141, the penicillin-streptomycin is Life Technologies 15140-163, the Matrigel is Corning 354230, the HEPES is Gibco 15630-080, the L-glutamine is Gibco 25030081, the PBS is Thermo Fisher 20012027, the sodium bicarbonate is Qiagen ZY-F1491, and the B27 supplement is Invitrogen 17504. 044, EGF catalog number Corning354001, Y27632 catalog number Tocris1254, Insulin catalog number SigmaI6634, Transferrin catalog number Sigma-AldrichT1147-100mg, Cholera toxin catalog number 9012-63-9, FGF7 catalog number 251-GMP-050, SB431542 catalog number MCEHY-10431, CHIR99021 catalog number Tocris4423, R-Spondin1 catalog number BT4645, FGF10 catalog number 345-FG-025 / CF, Noggin catalog number AF719, Retinol catalog number Sigma302-79-4, SPC catalog number RPU52575, Dexamethasone catalog number D4902.

[0040] (1) Basal culture medium The basic culture medium used in this invention is AdvancedDMEM / F-12, with each component added at the following final concentrations: FBS: 2% (v / v), HEPES: 15mM (pH 7.4), penicillin / streptomycin (1%), L-glutamine: 4mM, sodium bicarbonate (NaHCO3): 3.6mM, and 1×B27 supplement (without vitamin A).

[0041] (2) Amplification medium The expansion medium used in this invention is suitable for long-term expansion culture of human lung adult stem cells and organoids, supporting efficient expansion and maintenance of stem cell stemness. The specific formulation is as follows: Based on AdvancedDMEM / F12, the following components are added to the final concentrations: FBS: 5% (v / v), EGF: 25 ng / mL, Y27632: 10 μM, insulin: 10 μg / mL, transferrin: 5 μg / mL, cholera toxin: 0.1 μg / mL, 100 μg / mL, FGF7 (50 ng / mL), SB431542 (10 μM), CHIR99021 (3 μM), R-Spondin1 (500 ng / mL).

[0042] (3) Differentiation medium The differentiation medium used in this invention is suitable for COPD organoid differentiation culture and supports the effective differentiation of lung adult stem cells. The specific formulation is as follows: Based on Advanced DMEM / F12 medium, the following components are added to the final concentrations: FGF7 (50 ng / mL), FGF10 (50 ng / mL), Noggin (100 ng / mL), retinoic acid (RA) (0.5 μM), SPC (10 ng / mL), SB202190 (1 μM), and dexamethasone (50 nM).

[0043] Preferably, the concentration range of the factors added to the amplification medium is as follows: EGF: 20-50 ng / mL, Y27632: 5-15 μM, insulin: 5-15 μg / mL, transferrin: 5-20 μg / mL, cholera toxin: 0.05-0.2 μg / mL, FGF7: 20-50 ng / mL, SB431542: 5-15 μM, CHIR99021: 1-5 μM, R-Spondin1: 200-800 ng / mL.

[0044] COPD organoid amplification culture medium is supplemented with different small molecules and growth factors according to the maintenance and proliferation characteristics of adult lung stem cells. Among them, FGF7 can specifically promote the proliferation of alveolar and airway epithelial cells; EGF can continuously promote the continuous synthesis of DNA, thereby maintaining the proliferation and differentiation potential of airway epithelial cells; SB431542 is a TGFβ inhibitor that inhibits epithelial mesenchymalization; the Wnt agonist CHIR99021 is used to maintain lung stem cell characteristics and promote adult stem cell proliferation; and R-Spondin1 enhances Wnt signaling and promotes epithelial proliferation.

[0045] Preferably, the concentration range of the factors added to the differentiation medium is: FGF7: 20-50 ng / mL, FGF10: 20-100 ng / mL, Noggin: 50-200 ng / mL, retinoic acid: 0.5-2 μM, SPC: 5-20 ng / mL, dexamethasone: 30-100 nM, SB202190 (0.5-2 μM).

[0046] The COPD organoid differentiation culture medium used, by adding FGF7, FGF10, Noggin, retinoic acid, recombinant pulmonary surfactant protein (SPC), and dexamethasone, promotes lung epithelial cell differentiation, mimicking the pathological characteristics of COPD. Specifically: FGF7 promotes epithelial proliferation; FGF10 synergistically works with FGF7 to maintain progenitor cell self-renewal; Noggin inhibits BMP signaling and promotes airway epithelial cell differentiation; retinoic acid maintains proximal epithelial cell characteristics; SB202190 promotes stem cell self-renewal and induces cell differentiation; SPC specifically supports the maturation of type II alveolar cells; and dexamethasone has anti-inflammatory effects and promotes lung epithelial cell maturation.

[0047] During cell proliferation and differentiation, a basement membrane environment is provided to cells using a 50% high concentration of Matrigel, promoting epithelial cell polarization and 3D structure formation.

[0048] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for constructing a lung organoid model derived from COPD patients, characterized in that, Includes the following steps: Fresh surgically removed lung tissue from COPD patients was obtained and placed in a culture medium containing 2% FBS within 1.5 hours after ex vivo. Necrotic areas were removed and the tissue was minced to less than 1 mm³. Collagenase IV at a final concentration of 1 mg / mL was used for digestion at 37°C with shaking for 45 minutes, followed by filtration through 300 μm and 100 μm cell filters. The filtered cells were cultured in a type I collagen-coated culture dish in basal medium for 1 hour to remove mesenchymal cells. Epithelial cells were cultured in amplification medium under 3D conditions for 10 days, followed by culture in differentiation medium for 7 days to obtain the COPD organoids.

2. The method for constructing a lung organoid model based on COPD patients according to claim 1, characterized in that, The basal culture medium includes Advanced DMEM / F-12, fetal bovine serum, HEPES, penicillin / streptomycin, L-glutamine, sodium bicarbonate, and B27 supplement; the concentrations of each component in the basal culture medium are: FBS: 2%, HEPES: 15mM, penicillin / streptomycin: 1%, L-glutamine: 4mM, sodium bicarbonate: 3.6mM, and 1×B27 supplement.

3. The method for constructing a lung organoid model based on COPD patients according to claim 1, characterized in that, The amplification medium consisted of: AdvancedDMEM / F12, 2-5% FBS, EGF: 20-50 ng / mL, FGF7: 20-50 ng / mL, SB431542: 5-15 μM, CHIR99021: 1-5 μM, R-Spondin1: 200-800 ng / mL, Y27632: 5-15 μM, insulin: 5-15 μg / mL, cholera toxin: 0.05-0.2 μg / mL, and Transferrin: 5-20 μg / mL.

4. The method for constructing a lung organoid model based on COPD patients according to claim 1, characterized in that, The differentiation medium consisted of: AdvancedDMEM / F12, FGF7: 20-50 ng / mL, FGF10: 20-100 ng / mL, Noggin: 50-200 ng / mL, retinoic acid: 0.5-2 μM, SPC: 5-20 ng / mL, dexamethasone: 30-100 nM, and SB202190: 0.5-2 μM.

5. The method for constructing a lung organoid model based on COPD patients according to claim 1, characterized in that, During collagenase IV digestion, gently blow on the surface 10-20 times every 10 minutes.

6. The method for constructing a lung organoid model based on COPD patients according to claim 5, characterized in that, The collagenase IV is a crude extract of enzymes derived from Clostridium histolyticum. Its components not only contain Clostridium protease A, which can degrade natural collagen and reticular fibers, but also contain some proteases, polysaccharides and lipases.

7. The method for constructing a lung organoid model based on COPD patients according to claim 6, characterized in that, The collagenase IV effectively hydrolyzes proteins, polysaccharides, and lipids present in the extracellular matrix of connective tissue and epithelial tissue, thereby achieving the purpose of cell separation; in addition, type IV collagenase has low pancreatic enzyme activity to reduce damage to membrane proteins and receptors.

8. The method for constructing a lung organoid model based on COPD patients according to claim 1, characterized in that, The upper cell expansion and differentiation stage provides a basement membrane environment for cells with a 50% high concentration of Matrigel, promoting epithelial cell polarization and 3D structure formation.

9. The application of a lung organoid model derived from COPD patients as described in any one of claims 1-8 in precision medicine, organ transplantation, drug screening, and drug mechanism of action research.

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