Differentiation culture methods for bronchial epithelial stem / progenitor cells to differentiate into bronchial epithelial organoids

Abstract

This invention provides a method for differentiating bronchial epithelial stem / progenitor cells into bronchial epithelial organoids. Specifically, this invention provides a differentiation culture medium for bronchial epithelial organoids, characterized in that the culture medium includes a basal culture medium and additives; wherein the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof, and the additives include glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum. The differentiation culture method of this invention can stably obtain bronchial epithelial organoids using bronchial stem / progenitor cells as the cell source, with morphological structure consistent with human bronchial epithelial tissue, and can be used to prepare drug screening models, lung toxicity detection models, or microbial infection models.

Description

Technical Field

[0001] This invention relates to the field of biotechnology. Specifically, it relates to a method for differentiating bronchial epithelial stem / progenitor cells into bronchial epithelial organoids. Background Technology

[0002] Current research on bronchial epithelial organoids faces challenges such as cell source, culture medium composition, differentiation condition control, cell-cell interactions, and functional verification.

[0003] Currently, there is no differentiation culture medium that can differentiate bronchial epithelial organoids from bronchial stem / progenitor cells.

[0004] Therefore, there is an urgent need in the field to develop a differentiation culture medium and differentiation method that can stably obtain bronchial epithelial organoids. This invention uses bronchial stem / progenitor cells as the cell source, provides a specific culture medium formula, and with controlled differentiation conditions, can stably obtain bronchial epithelial organoids with the same morphology and structure as human bronchial epithelial tissue. These organoids can be co-cultured with fibroblasts for screening antifibrotic drug candidates. Summary of the Invention

[0005] This invention discloses a differentiation culture medium that allows bronchial stem / progenitor cells to differentiate into bronchial epithelial organoids. With controlled differentiation conditions, bronchial epithelial organoids can be stably obtained, with a structure consistent with human bronchial epithelial tissue. These organoids can be co-cultured with fibroblasts for screening antifibrotic drug candidates.

[0006] In a first aspect of the present invention, a differentiation culture medium for bronchial epithelial organoids is provided, the culture medium comprising a basal culture medium and additives; wherein the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof, and the additives comprise glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum.

[0007] In another preferred embodiment, the basal culture medium comprises DMEM and Ham's F-12.

[0008] In another preferred embodiment, the concentration (volume percentage) of the basal medium is 90-99% based on the total volume of the culture medium.

[0009] In another preferred embodiment, the volume ratio of DMEM to Ham's F-12 is 40-60:45-55, preferably 50:49, based on the total volume of the culture medium.

[0010] In another preferred embodiment, the concentration (volume percentage) of the fetal bovine serum is 0.5-4%, more preferably 0.8-2%, based on the total volume of the culture medium.

[0011] In another preferred embodiment, the concentration of glucose is 1000-6000 mg / L, more preferably 1500-5000 mg / L, and even more preferably 3000-4500 mg / L, based on the total volume of the culture medium.

[0012] In another preferred embodiment, the concentration of insulin is 0.05-10 μg / mL, more preferably 0.08-8 μg / mL, and even more preferably 0.1-5 μg / mL, based on the total volume of the culture medium.

[0013] In another preferred embodiment, the concentration of EGF is 0.05-5 μg / mL, more preferably 0.08-1 μg / mL, and even more preferably 0.1-0.5 μg / mL, based on the total volume of the culture medium.

[0014] In another preferred embodiment, the concentration of hydrocortisone is 0.1-20 μg / mL, more preferably 0.2-10 μg / mL, and even more preferably 0.5-5 μg / mL, based on the total volume of the culture medium.

[0015] In another preferred embodiment, the concentration of RA is 0.001-10 μM, more preferably 0.01-8 μM, and even more preferably 0.08-5 μM, based on the total volume of the culture medium.

[0016] In another preferred embodiment, the differentiation culture medium is used for the differentiation of bronchial epithelial stem / progenitor cells into bronchial epithelial organoids.

[0017] A second aspect of the present invention provides a bronchial epithelial organoid differentiation culture medium kit, comprising:

[0018] (a) A first container and additives located within the first container, said additives including: glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum.

[0019] In another preferred embodiment, the culture medium kit further includes:

[0020] (b) The second container and the basal culture medium located within the second container.

[0021] In another preferred embodiment, the culture medium kit further includes:

[0022] (d) The third container and the bronchial epithelial stem / progenitor cells located within the third container.

[0023] In another preferred embodiment, the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof.

[0024] In another preferred embodiment, the culture medium kit is used to prepare the differentiation culture medium for bronchial epithelial organoids as described in the first aspect of the invention.

[0025] In another preferred embodiment, the culture medium kit further includes: an instruction manual that describes the concentrations of each component in the culture medium kit and a method for preparing the differentiation culture medium for bronchial epithelial organoids as described in the first aspect of the present invention.

[0026] In another preferred embodiment, the specification also describes the recommended concentrations of each component in the culture medium kit when preparing the differentiation culture medium for bronchial epithelial organoids as described in the first aspect of the present invention.

[0027] In another preferred embodiment, the first container, the second container, and the third container may be the same or different containers.

[0028] A third aspect of this invention provides a method for differentiating bronchial epithelial stem / progenitor cells into bronchial epithelial organoids, the method comprising the steps of:

[0029] (S1) Provide a bronchial epithelial stem / progenitor cell, and mix the bronchial epithelial stem / progenitor cell with a culture medium for culturing bronchial epithelial stem / progenitor cells to obtain a bronchial epithelial stem / progenitor cell suspension.

[0030] (S2) Under suitable culture conditions, culture medium for bronchial epithelial stem / progenitor cells and the suspension of the bronchial epithelial stem / progenitor cells are added to the cell culture chamber for culture;

[0031] (S3) On days 3-5 (preferably day 4) the culture medium for culturing bronchial epithelial stem / progenitor cells is removed, and the culture medium described in the first aspect of the present invention is added for differentiation culture to obtain differentiated bronchial epithelial organoids.

[0032] In another preferred embodiment, the bronchial epithelial stem / progenitor cells are derived from human bronchial epithelial stem / progenitor cells.

[0033] In another preferred embodiment, the bronchial epithelial stem / progenitor cells are obtained by the following method:

[0034] (a) Provide an isolated bronchial epithelial sample and trophoblast cells, the trophoblast cells being obtained by irradiating fibroblasts;

[0035] (b) The sample was centrifuged, washed, and digested to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0036] (c) The cell suspension containing bronchial epithelial stem / progenitor cells is inoculated onto the trophoblast cells and cultured in the presence of a culture medium for bronchial epithelial stem / progenitor cells to obtain the bronchial epithelial stem / progenitor cells.

[0037] In another preferred embodiment, the culture medium for culturing bronchial epithelial stem / progenitor cells comprises a basal medium and additives; wherein the basal medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof, and the additives include: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

[0038] In another preferred embodiment, the basal culture medium comprises DMEM and Ham's F-12.

[0039] In another preferred embodiment, the concentration (volume percentage) of the basal medium is 80-90% based on the total volume of the culture medium.

[0040] In another preferred embodiment, the volume ratio of DMEM to Ham's F-12, based on the total volume of the culture medium, is 0.5-2:0.5-2, preferably 1:1.

[0041] In another preferred embodiment, the concentration (volume percentage) of the fetal bovine serum is 10-20% based on the total volume of the culture medium.

[0042] In another preferred embodiment, the concentration of glucose is 1000-5000 mg / L, more preferably 1500-4500 mg / L, more preferably 2000-4000 mg / L, and even more preferably 2500-3500 mg / L, based on the total volume of the culture medium.

[0043] In another preferred embodiment, the concentration of insulin, based on the total volume of the culture medium, is 0.1-100 μg / mL, more preferably 0.3-80 μg / mL, even more preferably 0.5-50 μg / mL, even more preferably 0.8-30 μg / mL, even more preferably 1-10 μg / mL, and even more preferably 3-10 μg / mL.

[0044] In another preferred embodiment, the concentration of epidermal growth factor, based on the total volume of the culture medium, is 0.1-100 ng / mL, more preferably 0.5-80 ng / mL, even more preferably 0.7-50 ng / mL, even more preferably 0.8-30 ng / mL, even more preferably 1-20 ng / mL, and even more preferably 2-10 ng / mL.

[0045] In another preferred embodiment, the concentration of hydrocortisone is 0.1-100 μg / mL, more preferably 0.3-50 μg / mL, even more preferably 0.8-30 μg / mL, even more preferably 1-10 μg / mL, and even more preferably 1-6 μg / mL, based on the total volume of the culture medium.

[0046] In another preferred embodiment, the concentration of Y-27632 is 0.1-100 μM, more preferably 0.3-50 μM, more preferably 0.8-30 μM, more preferably 1-10 μM, and even more preferably 1-6 μM, based on the total volume of the culture medium.

[0047] In another preferred embodiment, the concentration of SB431542 is 1-100 μM, more preferably 5-60 μM, more preferably 8-20 μM, and even more preferably 8-15 μM, based on the total volume of the culture medium.

[0048] In another preferred embodiment, the concentration of Noggin is 1-500 ng / mL, more preferably 5-300 ng / mL, more preferably 8-200 ng / mL, more preferably 10-100 ng / mL, and even more preferably 20-50 ng / mL, based on the total volume of the culture medium.

[0049] In another preferred embodiment, the concentration of R-spondin 1 is 1-500 ng / mL, more preferably 5-300 ng / mL, more preferably 8-200 ng / mL, more preferably 10-100 ng / mL, and even more preferably 30-80 ng / mL, based on the total volume of the culture medium.

[0050] In another preferred embodiment, the concentration of Fibronectin is 0.1-100 μg / mL, more preferably 0.5-50 μg / mL, even more preferably 0.8-30 μg / mL, even more preferably 1-10 μg / mL, and even more preferably 1-6 μg / mL, based on the total volume of the culture medium.

[0051] In another preferred embodiment, the concentration of E-Cadherin is 0.01-10 μg / mL, more preferably 0.05-5 μg / mL, more preferably 0.08-3 μg / mL, even more preferably 0.1-2 μg / mL, and even more preferably 0.6-2 μg / mL, based on the total volume of the culture medium.

[0052] In another preferred embodiment, the bronchial epithelial sample is a normal bronchial epithelial sample or a pathological bronchial epithelial sample.

[0053] In another preferred embodiment, the bronchial epithelial sample is derived from a bronchiolar, preferably a bronchiolar of grade 5 or higher.

[0054] In another preferred embodiment, the bronchial epithelial sample is selected from the group consisting of: tissue samples, non-invasive fluid samples, tissue biopsy forceps samples, biopsy brush samples, surgical excision samples, or combinations thereof.

[0055] In another preferred embodiment, the centrifugation conditions in step (b) are: 300-1000g.

[0056] In another preferred embodiment, in step (b), the sample is cleaned using a sample preservation solution.

[0057] In another preferred embodiment, the sample preservation solution comprises: a basal culture medium containing 5-50 mg / mL (preferably 6-30 mg / mL, more preferably 8-20 mg / mL) BSA, and penicillin (20-800 units / mL, more preferably 50-500 units / mL, even more preferably 80-200 units / mL) and streptomycin (20-800 μg / mL, more preferably 50-500 μg / mL, even more preferably 80-200 μg / mL), based on the total volume of the sample preservation solution.

[0058] In another preferred embodiment, the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof.

[0059] In another preferred embodiment, in step (b), digestion is performed using the digestive enzyme TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013).

[0060] In another preferred embodiment, in step (a), the irradiation dose is 20-80 Gy, preferably 30-60 Gy.

[0061] In another preferred embodiment, in step (c), the inoculation density of the cell suspension containing bronchial epithelial stem / progenitor cells is 8,000-50,000 cells / cm³. 2 Preferably, 10,000-20,000 cells / cm³ 2 .

[0062] In another preferred embodiment, step (c) further includes the step of: culturing at 37°C and with a CO2 concentration of 5%-10%.

[0063] In another preferred embodiment, in step (c), the culture medium is changed 2-3 times per week.

[0064] In another preferred embodiment, step (c) further includes the step of detecting endotoxins and mycoplasma in the cells.

[0065] In another preferred embodiment, step (c) further includes the step:

[0066] (ci) The cell suspension containing bronchial epithelial stem / progenitor cells is inoculated onto the trophoblast cells and primary cultured for 5-10 days in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells.

[0067] (c-ii) Digest the primary cultured cell clones, and seed the resulting cell suspension onto the feeder cells for passage;

[0068] (c-iii) In the presence of a culture medium for culturing bronchial epithelial stem / progenitor cells, the cells are passaged 2-3 times to obtain the bronchial epithelial stem / progenitor cells.

[0069] In another preferred embodiment, in step (c-iii), the number of bronchial epithelial stem / progenitor cells obtained through culture and amplification is ≥1 x 102 6 1 x 10 cells, preferably 1 x 10 6 -10x10 6 1 cell, or better yet, 2 x 10 6 -8x10 6 1 cell, or better yet, 4 x 10 6 -6x10 6 Each cell.

[0070] In another preferred embodiment, step (c-iii) further includes the step of analyzing and / or identifying the bronchial epithelial stem / progenitor cells.

[0071] In another preferred embodiment, the analysis and / or identification includes: identification of P63 and / or Krt5 markers.

[0072] In another preferred embodiment, in step (S1), the concentration of bronchial epithelial stem / progenitor cells in the bronchial epithelial stem / progenitor cell suspension is ≥0.5 x 10⁻⁶. 6 Cells / mL, preferably 0.8 x 10⁻⁶. 6 -5x10 6 cells / mL, preferably 1x10⁻¹ 6 -2x10 6 Cells / mL.

[0073] In another preferred embodiment, in step (S2), the cell culture chamber includes an upper chamber and a lower chamber, a culture medium for bronchial epithelial stem / progenitor cells is added to the lower chamber, and a suspension of the bronchial epithelial stem / progenitor cells is added to the upper chamber.

[0074] In another preferred embodiment, in step (S2), the amount of culture medium added to the bronchial epithelial stem / progenitor cells is ≥700 μL / well, preferably 700-800 μL / well.

[0075] In another preferred embodiment, in step (S2), the number of bronchial epithelial stem / progenitor cells in the bronchial epithelial stem / progenitor cell suspension is 1 x 102 5 -5x10 5 Cells, preferably 2x10 5 -4x10 5 Each cell.

[0076] In another preferred embodiment, step (S2) further includes the step of: culturing at 37°C and with a CO2 concentration of 5%-8%.

[0077] In another preferred embodiment, step (S2) further includes changing the culture medium for culturing bronchial epithelial stem / progenitor cells every 1-2 days (preferably 2 days).

[0078] In another preferred embodiment, in step (S3), the culture medium described in the first aspect of the present invention is added to the lower chamber of the cell culture chamber for differentiation culture.

[0079] In another preferred embodiment, step (S3) further includes replacing the culture medium described in the first aspect of the invention every 1-2 days (preferably 2 days).

[0080] In another preferred embodiment, in step (S3), the differentiation culture time is 10-20 days.

[0081] In another preferred embodiment, step (S3) further includes washing, membrane separation, and membrane fixation of the bronchial epithelial organoids obtained through differentiation culture.

[0082] In another preferred embodiment, step (S3) further includes washing the bronchial epithelial organoids obtained from the differentiation culture with buffer solution during culture days 14-24 (preferably day 19).

[0083] In another preferred embodiment, the buffer solution comprises PBS.

[0084] In another preferred embodiment, the method is an in vitro method.

[0085] In another preferred embodiment, the method is for non-diagnostic and non-therapeutic purposes.

[0086] A fourth aspect of the present invention provides a bronchial epithelial organoid, which is obtained by means of the method described in the third aspect of the present invention.

[0087] The fifth aspect of the present invention provides the use of a culture medium as described in the first aspect of the present invention, or a culture medium kit as described in the second aspect of the present invention, the use comprising:

[0088] (a) Used as or for the preparation of culture media for culturing bronchial epithelial organoids; and / or

[0089] (b) as an additive used in or for the preparation of bronchial epithelial organoid culture media; and / or

[0090] (c) Used for culturing bronchial epithelial organoids;

[0091] In another preferred embodiment, the use is for non-disease diagnosis or non-disease treatment purposes.

[0092] A sixth aspect of the present invention provides a composition comprising: DMEM, Ham's F-12, DMEM / F-12, or a combination thereof, wherein the additives comprise: glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum.

[0093] A seventh aspect of the present invention provides a use of the composition as described in the sixth aspect of the present invention, the use comprising:

[0094] (a) Culture media used to prepare bronchial epithelial organoids; and / or

[0095] (b) For the cultivation of bronchial epithelial organoids; and / or

[0096] (c) Used as an additive in the culture medium for bronchial epithelial organoids.

[0097] In another preferred embodiment, the use is for non-disease diagnosis or non-disease treatment purposes.

[0098] The eighth aspect of the present invention provides a use of the bronchial epithelial organoid described in the fourth aspect of the present invention for preparing drug screening models, lung toxicity detection models, or microbial infection models.

[0099] In another preferred embodiment, the drug screening model is an in vitro drug screening model, an in vitro lung toxicity detection model, or an in vitro microbial infection model.

[0100] In another preferred embodiment, the drug screening model includes an antifibrotic drug screening model and an antiviral drug screening model.

[0101] In another preferred embodiment, the fibrosis includes pulmonary fibrosis.

[0102] In another preferred embodiment, the lung toxicity detection model includes lung toxicity detection of formaldehyde, tobacco, e-cigarettes, radiation, drugs, and cosmetics.

[0103] In another preferred embodiment, the microbial infection model includes infection models of viruses, bacteria, mycoplasma, and chlamydia.

[0104] In another preferred embodiment, the virus includes SARS-CoV-2, influenza virus, respiratory syncytial virus, adenovirus, rhinovirus, and human metapneumovirus.

[0105] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0106] Figure 1 Clones of bronchial epithelial stem / progenitor cells were displayed.

[0107] Figure 2 The images show HE staining of bronchial epithelial stem / progenitor cells differentiating into bronchial epithelial organoids (top) and morphological structure of human bronchial epithelial tissue (bottom). The cell layers are intact and clearly defined, with ciliated cells and goblet cells arranged in a regular pattern.

[0108] Figure 3 HE staining image of a co-culture model constructed by adding fibroblasts after bronchial epithelial stem / progenitor cells differentiated into bronchial epithelial organoids.

[0109] Figure 4 The study demonstrated the use of a co-culture model constructed from differentiated bronchial epithelial organoids and fibroblasts for screening anti-pulmonary fibrosis drugs, and found that the candidate compound CPD1 had anti-fibrotic activity.

[0110] Figure 5 HE staining image of bronchial epithelial organoids differentiated and cultured using differentiation medium under current technological conditions. The cell layers are incomplete, the cell hierarchy is unclear, and the morphology of ciliated cells and goblet cells is irregular.

[0111] Figure 6 This study demonstrates SARS-CoV-2 infection in bronchial epithelial organoids.

[0112] Figure 7 The study showed that different concentrations of the antiviral drug Paxlovid could inhibit viral replication in bronchial epithelial organoids.

[0113] Figure 8 The bronchial epithelial organoids were used to evaluate the pulmonary toxicity of bleomycin. Detailed Implementation

[0114] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly discovered for the first time that a bronchial epithelial stem / progenitor cell differentiation culture medium, specially formulated with the addition of glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum, can differentiate bronchial epithelial stem / progenitor cells into bronchial epithelial organoids with morphological structures consistent with human bronchial epithelial tissue. Furthermore, the obtained bronchial epithelial organoids, when co-cultured with fibroblasts and induced with bleomycin, can be used as a drug screening model for anti-fibrosis. Based on this, the present invention was completed.

[0115] the term

[0116] bronchial epithelial stem / progenitor cells

[0117] Bronchial epithelial stem / progenitor cells are a type of cell in the bronchial epithelium with self-renewal and differentiation capabilities. They play an important role in maintaining bronchial epithelial homeostasis and in the repair process after injury. Commonly used biomarkers are P63 and Krt5.

[0118] basal culture medium

[0119] Basic cell culture media are the fundamental nutrient media used for cell culture, and they typically need to be selected and supplemented according to different cell types and experimental requirements. DMEM medium is a widely used basic medium suitable for culturing various mammalian cell types. Ham's F12 medium is a classic cell culture medium developed by Ham in 1965. It contains similar nutrients to DMEM, but its formulation differs slightly, particularly in that it contains a richer amount of trace elements and growth factors. DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F-12 medium, combining the advantages of both media to provide more comprehensive nutrients suitable for culturing various cell types.

[0120] fetal bovine serum

[0121] Fetal bovine serum (FBS) is a pale yellow, clear, slightly viscous liquid without hemolysis or foreign matter. FBS should be obtained from fetuses born via cesarean section. Serum is a complex mixture formed by removing fibrinogen from plasma. It contains various plasma proteins, polypeptides, fats, carbohydrates, growth factors, hormones, and inorganic substances.

[0122] glucose

[0123] Glucose is a colorless monosaccharide that is easily soluble in water, has a sweet taste, and is widely distributed in nature. Its molecular formula is C6H12H2O. 12 O6 has a wide range of uses. In the human body and cells, glucose can quickly replenish energy.

[0124] insulin

[0125] Insulin is a protein hormone secreted by the pancreatic β cells in the pancreas in response to stimulation by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, etc.

[0126] Epidermal growth factor

[0127] Epidermal growth factor (EGF) is a heat-resistant single-chain low-molecular-weight polypeptide composed of 53 amino acid residues. After EGF specifically recognizes and binds to EGF receptors on target cells, a series of biochemical reactions occur, ultimately promoting DNA synthesis and mitosis in the target cells.

[0128] Hydrocortisone

[0129] Hydrocortisone, also known as cortisol, is an organic compound with the chemical formula C63-320. 21 H 30 O5 is an adrenocortical hormone extracted from the adrenal cortex that has the strongest effect on carbohydrate metabolism; it belongs to the class of glucocorticoids.

[0130] Y-27632

[0131] Y-27632 is a small molecule inhibitor of Rho-associated protein kinase p160ROCK.

[0132] The structural formula of Y-27632 (CAS.NO:146986-50-7) is shown below:

[0133]

[0134] SB431542

[0135] SB431542 is a potent and selective inhibitor of the TGF-β signaling pathway.

[0136] Structural formula:

[0137]

[0138] Noggin

[0139] Noggin is a secreted homodimeric glycoprotein that is an antagonist of bone morphogenetic proteins (BMPs).

[0140] During skeletal development, Noggin inhibits chondrocyte proliferation, thereby regulating normal joint formation. In adult central nervous system and peripheral tissue (such as the lungs), when human embryonic stem cells (hESCs) or neural stem cells are cultured under certain conditions, the addition of Noggin to antagonize BMP activity can induce stem cell proliferation while maintaining their undifferentiated state, or differentiate them into dopaminergic neurons. In Noggin-deficient mice, enhanced BMP activity causes a series of developmental abnormalities, including neural tube failure, delayed hair follicle development, axial skeletal deformities, and joint lesions.

[0141] Noggin also plays a crucial role in organoid culture. It is widely used in the long-term culture of various organoids, such as liver, small intestine, and fallopian tube organoids. Noggin binds to bone morphogenetic proteins, coordinating Wnt signaling to activate stem cells and promote their proliferation. During organoid culture, Noggin maintains the self-renewal capacity of stem cells and prevents premature differentiation by inhibiting the BMP signaling pathway. For example, in intestinal organoid culture, Noggin is added to the culture medium to inhibit the BMP signaling pathway and maintain the self-renewal capacity of intestinal stem cells. In brain organoid culture, Noggin promotes the self-renewal of neural stem cells and the generation of neurons by inhibiting BMP signaling.

[0142] R-spondin 1

[0143] R-spondin 1 (RSPO1) is a secreted activating protein belonging to the R-spondin protein family, which includes four members: R-spondin 1-4. RSPO1 possesses two cysteine-rich furin-like domains (FU-like CRs) and a platelet-reactive protein type 1 domain (TSR). It is a pluripotent signaling ligand, best known for activating the Wnt / β-catenin signaling pathway. RSPO1 enhances Wnt / β-catenin signaling by acting on its homologous receptors LGR4 / 5 / 6 expressed in stem cells and progenitor cells, particularly in proliferating stem cells. Therefore, RSPO1 plays a crucial role in stem cell regulation across multiple organs and is a key factor in the in vitro expansion and culture of various adult stem cells, including those from the intestine, stomach, and liver.

[0144] Fibronectin

[0145] Fibronectin is a large extracellular membrane protein present on the surface of various animal cells. It is a major non-collagenous glycoprotein in the extracellular matrix and basement membrane. It plays a central role in cell adhesion and regulates cell polarity, differentiation, and growth.

[0146] E-Cadherin

[0147] E-cadherin, also known as intercellular adhesion molecule 1 (CDH1), plays a crucial role in intercellular connections, maintaining cell adhesion and structural integrity, and participating in cell polarity and migration. Primarily expressed in epithelial tissues, E-cadherin plays a key role in cell recognition and adhesion. Due to its important functions, E-cadherin is involved in physiological and pathological processes such as embryonic development, tissue repair, and tumor metastasis.

[0148] RA

[0149] RA (Retinoic acid), also known as retinoic acid, is a metabolic intermediate of vitamin A. It can induce cell differentiation and apoptosis and plays a key role in cell growth, differentiation and organ formation.

[0150] Structural formula:

[0151]

[0152] Differentiation culture medium for bronchial epithelial stem / progenitor cells

[0153] As used in this article, "differentiation culture medium for bronchial epithelial stem / progenitor cells" and "differentiation culture medium for bronchial epithelial organoids" are interchangeable and both refer to the differentiation culture medium for differentiating bronchial epithelial stem / progenitor cells into bronchial epithelial organoids.

[0154] This invention provides a differentiation culture medium for bronchial epithelial stem / progenitor cells, comprising a basal culture medium and additives; wherein the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof, and the additives include glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum.

[0155] The bronchial epithelial organoids obtained by differentiation and culture using the specially added components have the same morphology and structure as human bronchial epithelial tissue. Furthermore, the obtained bronchial epithelial organoids can be co-cultured with fibroblasts and induced with bleomycin, serving as a drug screening model for anti-fibrosis. Preferably, the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F12, or combinations thereof; more preferably, the basal culture medium is DMEM, Ham's F-12, or combinations thereof.

[0156] Preferably, the differentiation culture medium for bronchial epithelial stem / progenitor cells further includes the following components: glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum.

[0157] The preferred concentrations of each component in the culture medium are as described above, and the most preferred concentrations are those of each component in the embodiments of the present invention.

[0158] Using the culture medium of the present invention and the culture method of the present invention, the bronchial epithelial organoids differentiated and cultured by the present invention have the same morphology and structure as human bronchial epithelial tissue, and can be co-cultured with fibroblasts for screening anti-fibrotic drug candidates.

[0159] Differentiation culture methods for bronchial epithelial stem / progenitor cells to differentiate into bronchial epithelial organoids

[0160] This invention also provides a method for differentiating bronchial epithelial stem / progenitor cells into bronchial epithelial organoids, the method comprising the following steps:

[0161] (S1) Provide a bronchial epithelial stem / progenitor cell, and mix the bronchial epithelial stem / progenitor cell with a culture medium for culturing bronchial epithelial stem / progenitor cells to obtain a bronchial epithelial stem / progenitor cell suspension.

[0162] (S2) Under suitable culture conditions, culture medium for bronchial epithelial stem / progenitor cells and the suspension of the bronchial epithelial stem / progenitor cells are added to the cell culture chamber for culture;

[0163] (S3) On days 3-5 (preferably day 4) the culture medium for culturing bronchial epithelial stem / progenitor cells is removed, and the culture medium described in the first aspect of the present invention is added for differentiation culture to obtain differentiated bronchial epithelial organoids.

[0164] In this invention, the sample preservation solution contains the following components: DMEM culture medium containing 10 mg / mL BSA and Penicillin (100 units / ml)-Streptomycin (100 μg / ml).

[0165] In this invention, the bronchial epithelial stem / progenitor cell culture medium comprises the following components: 45% (v / v) DMEM medium, 45% (v / v) Ham's F-12 medium, 10% (v / v) fetal bovine serum, 3000 mg / L glucose, 8 μg / mL insulin, 5 ng / mL epidermal growth factor, 3 μg / mL hydrocortisone, 2 μM Y-27632, 10 μM SB431542, 30 ng / mL Noggin, 60 ng / mL R-spondin, 1 ug / mL Fibronectin, and 1 ug / mL E-Cadherin.

[0166] The differentiation medium for bronchial epithelial stem / progenitor cells to differentiate into bronchial epithelial organoids contained the following components: 50% (v / v) DMEM medium, 49% (v / v) Ham's F-12 medium, glucose (3000 mg / L), insulin (3 μg / mL), EGF (400 ng / mL), RA (2 μM), hydrocortisone (3 μg / mL), and 1% fetal bovine serum (v / v).

[0167] The main advantages of this invention include:

[0168] (1) This invention is the first to discover that the bronchial epithelial stem / progenitor cell differentiation culture medium, which is specially formulated with added glucose, insulin, EGF, RA, hydrocortisone and fetal bovine serum, can provide a specific culture medium formula with bronchial stem / progenitor cells as the cell source during the differentiation culture of bronchial epithelial organoids. With the control of differentiation conditions, bronchial epithelial organoids can be stably obtained, which are consistent with the morphology and structure of human bronchial epithelial tissue. They can be co-cultured with fibroblasts for the screening of anti-fibrotic drug candidates.

[0169] (2) This invention is the first to develop a culture medium for the differentiation of bronchial epithelial stem / progenitor cells. The formulation is simple, the cost is low, and it can be applied on a large scale.

[0170] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0171] Unless otherwise stated, all reagents and materials used in the embodiments of this invention are commercially available products.

[0172] The samples were collected in accordance with the Declaration of Helsinki and with the approval of the Ethics Committee of Zhejiang Xinda Hospital, and were obtained using the method of this invention with the informed consent of the volunteers.

[0173] Example 1: Differentiation culture medium for bronchial epithelial organoids 1

[0174] In this embodiment, a differentiation culture medium for bronchial epithelial organoids is provided, which includes a basal culture medium (such as 50% (v / v) DMEM medium, 49% (v / v) Ham's F-12 medium) and various components added according to the formulations in Table 1 below (based on the total volume of the culture medium).

[0175] Table 1

[0176]

[0177] Example 2: Bronchial epithelial stem / progenitor cells cultured and differentiated to form bronchial epithelial organoids

[0178] (1) A small amount of bronchial epithelial cells were collected by bronchoscopic cell brushing. The cell brush was placed in the sample preservation solution and transported to the production workshop at a temperature of 2-8℃.

[0179] (2) Rinse the bronchial epithelial cells off the cell brush, centrifuge at 300g-1000g, wash three times with sample preservation solution, and then digest in TrypLE digestion solution (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10-60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells.

[0180] (3) Resuscitate 10 frozen trophoblast cells from liquid nitrogen 24 hours in advance. 6 One cell (the feeder cells were fibroblast line 3T3-J2 cells (purchased from Kerafast, product number: EF3003) irradiated with 20-80 Gy and then frozen in liquid nitrogen) was seeded onto two wells of a 12-well cell culture plate. The cell suspension obtained in step (2) was then seeded onto the feeder layer.

[0181] (4) Primary culture was performed using bronchial epithelial stem / progenitor cell culture medium at 37°C and CO2 concentration of 7.5%. After the cells adhered and grew, the medium was changed 2-3 times a week. During the culture process, samples were taken for endotoxin and mycoplasma testing to ensure that the results were negative.

[0182] (Bronchial epithelial stem / progenitor cell culture medium formulation: 45% (v / v) DMEM medium, 45% (v / v) Ham's F-12 medium, 10% (v / v) fetal bovine serum, 3000 mg / L glucose, 8 μg / mL insulin, 5 ng / mL epidermal growth factor, 3 μg / mL hydrocortisone, 2 μM Y-27632, 10 μM SB431542, 30 ng / mL Noggin, 60 ng / mL R-spondin, 1 ug / mL Fibronectin, 1 ug / mL E-Cadherin.)

[0183] (5) Clones will appear after 5-10 days of culture. When the cell clones are close to confluence, they will be digested. The resulting cell suspension will be prepared at a concentration of 10,000-20,000 cells / cm³. 2 The cells were seeded onto feeder cells and passaged. After 5 days of culture, cell clones appeared. The clones are as follows... Figure 1.

[0184] (6) The above clonal passages were repeated using bronchial epithelial stem / progenitor cell culture medium (passed twice, with cells sequentially passaged into 3.5cm and 6cm culture dishes, expanding the epithelial stem cells to a quantity of 5 x 10⁻⁶). 6 Each cell.

[0185] (7) Collect the bronchial epithelial stem / progenitor cells expanded in (6), add an appropriate amount of preheated bronchial epithelial stem / progenitor cell culture medium, and gently pipette several times to form a single-cell suspension (concentration = 1.5 x 10⁻⁶). 6 / mL).

[0186] (8) Add 700 μL of bronchial epithelial stem / progenitor cell culture medium per well (24-well plate) to the lower chamber of the cell culture chamber.

[0187] (9) Vertically add 200 μL of a solution containing 3*10 to the upper chamber of the cell culture chamber. 5 After suspending the cells in a cell suspension, the well plate was placed in a 37°C, 7.5% CO2 incubator for incubation.

[0188] (10) Change the bronchial epithelial stem / progenitor cell culture medium in the lower chamber every two days, 700 μL each time.

[0189] (11) After the fourth day of culture, remove the bronchial epithelial stem / progenitor cell culture medium from the upper and lower chambers. Add 700 μL of the bronchial epithelial stem / progenitor cell differentiation medium from Example 1 to the lower chamber, and then replace it with fresh 700 μL of bronchial epithelial stem / progenitor cell differentiation medium every two days.

[0190] (12) After culturing for 19 days, differentiation was completed, and differentiated bronchial epithelial organoids were obtained. 700 μL of PBS buffer was added to the lower chamber of the microchamber for washing.

[0191] (13) After discarding PBS, the membrane covering the bronchial epithelial organoid was separated using a sterile blade and transferred to 5 mL of 4% PFA and fixed at room temperature for 30 min. After fixation, the membrane was washed with PBS and transferred to a 1.5 mL EP tube for subsequent paraffin embedding.

[0192] (14) Section the embedded paraffin sections to a thickness of 5-10 μm. Stain the sections with hematoxylin and eosin (HE) and eosin (H&E), and then scan a panoramic image. The bronchial epithelial organoids show intact cell layers with clear hierarchies, and regular distribution and arrangement of ciliated cells and goblet cells. A local image is shown below. Figure 2 Above. Its morphology and structure are consistent with those of human bronchial epithelial tissue. Figure 2 Down).

[0193] Furthermore, immunofluorescence staining was used to detect the expression of recognized goblet cells and ciliated cells biomarkers in organoids.

[0194] The results showed that the bronchial epithelial organoids expressed Muc5AC protein (a marker of goblet cells) and FoxJ1 or acetylated α-tubulin (a marker of ciliated cells), indicating that the bronchial epithelial organoids expressed markers similar to those in human bronchial epithelial tissue.

[0195] Example 3: Bronchial epithelial stem / progenitor cells were cultured and differentiated to form bronchial epithelial organoids for screening antifibrotic drugs.

[0196] (1) Similar to Example 2, the bronchial epithelial organoids obtained in Example 2 (12) and MRC-5 fibroblasts (purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences) were co-cultured. The co-culture method was as follows: 100,000 to 1,000,000 MRC-5 fibroblasts were seeded into the bottom outer side of the chamber. Cultured for 1-2 days. A co-culture model was constructed, and a portion of the model chamber was stained with sections as in Example 2. HE staining image is shown below. Figure 3 .

[0197] (2) Add drugs to the chambers in the co-culture model. When adding drugs, add 200 μL of differentiation culture medium for bronchial epithelial stem / progenitor cells to the upper chamber of each chamber.

[0198] The dosing regimen is as follows:

[0199] The blank control group (con) only added DMSO;

[0200] Model group (BLM): 75 μg / mL of bleomycin (purchased from MedChemExpress, catalog number: HY-17565) was added to the upper layer, using DMSO as the solvent.

[0201] Candidate compound group (BLM+CPD1): 75 μg / mL bleomycin was added to the upper layer, and 100 μM of candidate compound CPD1 (purchased from MedChemExpress) was added to both the upper and lower layers simultaneously, using DMSO as the solvent.

[0202] (3) After 24 hours, wash the chamber once with 700ul PBS, cut off the membrane at the bottom of the chamber with a blade, lyse it with Trizol, and extract RNA using the standard method.

[0203] (4) After reverse transcription of RNA, the expression levels of fibrosis-related genes Collagen I, Fibronectin and α-SMA were detected by qPCR.

[0204] (5) Analyze and compare the expression of these three genes in each group. The results are as follows: Figure 4 *** indicates p<0.001, * indicates p<0.05. The results show that bleomycin significantly induced the expression of fibrosis-related genes Collagen I, Fibronectin, and α-SMA, indicating a successful pulmonary fibrosis model.

[0205] from Figure 4 It can be seen that the CPD1 candidate compound can reduce the expression of fibrosis-related genes Collagen I, Fibronectin and α-SMA, thus the candidate compound CPD1 has an anti-fibrotic effect.

[0206] Example 4: Bronchial epithelial stem / progenitor cells were cultured and differentiated to form bronchial epithelial organoids, which were used as a SARS-CoV-2 infection model.

[0207] (1) As in Example 2, bronchial epithelial organoids (HAM-1, 2, and 3) from three donors (obtained from Zhejiang Xinda Hospital) were differentiated.

[0208] (2) Prior to infection, the HAM model was washed twice with Opti-MEM and differentiation medium was added. SARS-CoV-2 (obtained from Fudan University) (MOI=1) was diluted in Opti-MEM and added to the upper chamber, incubated at 37°C for 1 hour. The control group used the same volume of Opti-MEM. Subsequently, the viral inoculum was removed. At 24, 48, 72, and 96 hours post-inoculation, the upper chamber was flushed with 200 μL of Opti-MEM at 37°C for 10 minutes to assess the amount of virus released.

[0209] (3) Using PureLink TM Viral RNA was extracted using a viral RNA kit, yielding 60 μL of eluted RNA. Viral RNA in the cell culture supernatant was quantified using TaqManFast one-step viral premix. The cycling parameters for the qPCR reaction were: 50℃ for 1 min, 95℃ for 20 s, followed by 45 cycles (95℃ for 3 s, 60℃ for 30 s). Primer and probe sequences targeting SARS-CoV-2 nonstructural protein 14 (nsp14) are shown in Table 2. Relative quantification of viral RNA was performed using the 2-ΔCt method.

[0210] After cell lysis, RNA was extracted using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Vazyme, RC112-01), eluted in 50 μL of nuclease-free water, and diluted 1:10 before gene expression analysis. IIQ RT SuperMix (Vazyme, RC222-01, China) was used to reverse transcribe intracellular RNA, followed by qPCR analysis. The reaction cycling parameters were: 50℃ for 1 min, 95℃ for 5 min, followed by 50 cycles (95℃ for 15 s, 60℃ for 30 s; nsp14 annealing temperature was 53℃). Finally, melting curve analysis was performed at 40℃ for 1 min to confirm product specificity. Primer sequences are shown in Table 2. GAPDH was used as the internal control. Changes in intracellular gene expression were analyzed using 2... -ΔΔCt Method calculation.

[0211] Table 2 Primer sequences

[0212]

[0213] (4) Plot the viral RNA copy number calculated in (3) (see...) Figure 6 After four days of viral infection, the viral RNA copy number in the supernatant approached 10. 9 This indicates that the bronchial epithelial organoid is an excellent model for viral infection.

[0214] Example 5: Bronchial epithelial stem / progenitor cells were cultured and differentiated to form bronchial epithelial organoids, which were used to screen antiviral drugs.

[0215] (1) Same as in Example 4, bronchial epithelial organoids were obtained.

[0216] (2) Same as in Example 4, viral infection was performed. SARS-CoV-2 virus (obtained from Fudan University) (MOI=1) was diluted in Opti-MEM and added to the upper chamber, incubated at 37°C for 1 hour. Subsequently, the viral inoculum was removed. 0.05, 0.5, and 5 μM Paxlovid (purchased from Pfizer) were added to the culture medium in each chamber, respectively; the control group received no drug. At 24, 48, 72, and 96 hours after viral inoculation, the upper chamber was flushed with 200 μL Opti-MEM at 37°C for 10 minutes to assess the amount of virus released.

[0217] (3) Viral RNA was extracted as in Example 4, reverse transcribed, and then analyzed by qPCR to calculate the viral copy number.

[0218] (4) Plot the data of different drug concentration treatment groups into graphs. The results are shown in [the figure]. Figure 7 It can be seen that viral replication is significantly inhibited as the drug concentration increases.

[0219] The above results indicate that bronchial epithelial organoids can be used for antiviral drug screening.

[0220] Example 6: Bronchial epithelial stem / progenitor cells were cultured and differentiated to form bronchial epithelial organoids for lung toxicity assessment.

[0221] (1) Same as in Example 2, bronchial epithelial organoids were obtained.

[0222] (2) Add drugs to the chambers. When adding drugs, add 200 μl of differentiation culture medium for bronchial epithelial stem / progenitor cells to differentiate into bronchial epithelial organoids to the upper chamber of each chamber.

[0223] The dosing regimen is as follows:

[0224] The control group only added DMSO;

[0225] Drug A (penicillin), B (bleomycin), and C (streptomycin) were added to the drug group at a concentration of 50 μM (drugs purchased from MedChemExpress), using DMSO as the solvent.

[0226] (3) After 24 hours, the supernatant of the upper chamber was collected, and the concentration of IL-6 in the supernatant was detected using an IL-6 ELISA kit (purchased from Beyotime, catalog number: PI330).

[0227] (4) Collect bronchial epithelial organoids and use the CCK-8 kit (purchased from Beyotime, catalog number: C0038) to detect the cell viability in the organoids.

[0228] (5) Analyze and plot cell viability and IL-6 expression levels, such as... Figure 8 ** indicates p < 0.01. The results show that the drug B bleomycin significantly reduces cell viability, increases the expression of the inflammatory factor IL-6, and is toxic to bronchial epithelial organoids, indicating that the drug is toxic to the lungs. The bronchial epithelial organoids of this invention can be used to assess the pulmonary toxicity of the drug.

[0229] Comparative Example 1: The differentiation culture medium for bronchial epithelial stem / progenitor cells to differentiate into epithelial organoids according to the present invention is superior to currently commercially available differentiation culture media.

[0230] The differentiation culture steps were the same as in Example 2, except that the existing culture medium (purchased from STEMCELL, catalog number: Catalog#100-0620, used for the differentiation culture of primary lung epithelial cells) was used instead of the differentiation culture medium for the differentiation of bronchial epithelial stem / progenitor cells into epithelial organoids in this invention. The results showed that the cell layers of the differentiated bronchial epithelial organoids were incomplete, the cell layers were unclear, and the morphology of ciliated cells and goblet cells was irregular, differing from the morphology and structure of human epithelial tissue. Figure 5 As shown.

[0231] Therefore, the above-mentioned effects of the present invention cannot be obtained using culture media with existing technology.

[0232] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A differentiation culture medium for bronchial epithelial organoids, wherein the differentiation culture medium is used for the differentiation of bronchial epithelial stem / progenitor cells into bronchial epithelial organoids, characterized in that, The culture medium includes a basal medium and additives; wherein the basal medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F-12, or combinations thereof, and the additives include glucose, insulin, EGF, RA, hydrocortisone, and fetal bovine serum. The basal culture medium comprises DMEM and Ham's F-12, with a volume ratio of DMEM to Ham's F-12 of 40-60:45-55. Based on the total volume of the culture medium, the concentration of fetal bovine serum is 0.5-4%, the concentration of glucose is 1500-5000 mg / L, the concentration of insulin is 0.08-8 μg / mL, the concentration of EGF is 0.08-1 μg / mL, the concentration of hydrocortisone is 0.2-10 μg / mL, and the concentration of RA is 0.01-8 μM.

2. The differentiation culture medium as described in claim 1, characterized in that, The concentration of glucose is 3000-4500 mg / L based on the total volume of the culture medium.

3. The differentiation culture medium as described in claim 1, characterized in that, The concentration of RA is 0.08-5 μM based on the total volume of the culture medium.

4. The differentiation culture medium as described in claim 1, characterized in that, The concentration of EGF is 0.1-0.5 μg / mL based on the total volume of the culture medium.

5. The differentiation culture medium as described in claim 1, characterized in that, The concentration of hydrocortisone is 0.5-5 μg / mL based on the total volume of the culture medium.

6. A method for differentiating bronchial epithelial stem / progenitor cells into bronchial epithelial organoids, characterized in that, The method includes the following steps: (S1) Provide bronchial epithelial stem / progenitor cells, and mix the bronchial epithelial stem / progenitor cells with a culture medium for culturing bronchial epithelial stem / progenitor cells to obtain a bronchial epithelial stem / progenitor cell suspension. (S2) Add the culture medium for culturing bronchial epithelial stem / progenitor cells and the suspension of the bronchial epithelial stem / progenitor cells to the cell culture chamber for culture; (S3) On days 3-5 of culture, remove the culture medium for bronchial epithelial stem / progenitor cells and add the differentiation medium described in claim 1 for differentiation culture to obtain differentiated bronchial epithelial organoids; the culture medium for bronchial epithelial stem / progenitor cells includes a basal medium and additives; wherein the basal medium is selected from the group consisting of: DMEM, Ham's F-12, DMEM / F-12, or combinations thereof, and the additives include: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin. Based on the total volume of the culture medium, the concentration of the fetal bovine serum is 10-20%, the concentration of glucose is 2000-4000 mg / L, and the concentration of insulin is 1-10 μg / L. The concentrations of the following drugs were as follows: Epidermal growth factor (1-20 ng / mL), hydrocortisone (1-10 μg / mL), Y-27632 (1-10 μM), SB431542 (8-20 μM), Noggin (20-50 ng / mL), R-spondin 1 (10-100 ng / mL), Fibronectin (1-10 μg / mL), and E-Cadherin (0.1-2 μg / mL). The bronchial epithelial stem / progenitor cells were obtained using the following method: (a) Provide an isolated bronchial epithelial sample and trophoblast cells, the trophoblast cells being obtained by irradiating fibroblasts; (b) The sample is centrifuged, washed, and digested to obtain a cell suspension containing bronchial epithelial stem / progenitor cells; (c) The cell suspension containing bronchial epithelial stem / progenitor cells is inoculated onto the trophoblast cells and cultured in the presence of a culture medium for bronchial epithelial stem / progenitor cells to obtain the bronchial epithelial stem / progenitor cells.

7. The method as described in claim 6, characterized in that, In step (a), the irradiation dose is 20-80 Gy.

8. The method as described in claim 7, characterized in that, The irradiation dose is 30-60 Gy.

9. The use of the differentiation culture medium as described in claim 1, characterized in that, The intended use is to differentiate bronchial epithelial stem / progenitor cells into bronchial epithelial organoids.

Images