Method for culturing bronchial epithelial stem / progenitor cells

Abstract

This invention provides a method for culturing bronchial epithelial stem / progenitor cells. Specifically, this invention provides a culture medium for culturing 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: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, and R-spondin. 1. Fibronectin and E-Cadherin are provided, along with a method for culturing bronchial epithelial stem / progenitor cells. The bronchial epithelial stem / progenitor cells amplified using the method of this invention have a high cloning rate, can be isolated from trace amounts of tissue, and can be passaged more than 50 times. The cells maintain normal stemness and differentiation potential, and can be cultured to obtain a sufficient number of clinically infused bronchial epithelial stem / progenitor cells for autologous infusion. Clinically, this has achieved the repair of small airway damage in the lungs and significantly improved lung function.

Description

Technical Field

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

[0002] Small airways refer to airways with a diameter of less than 2 mm. Currently, it is believed that in the early stages of respiratory diseases, small airway obstruction has little impact on changes in airway resistance, hence it is also known as the silent zone of lung disease. However, in the progression of the disease, small airway obstruction can significantly affect lung function.

[0003] Small airway dysfunction can be diagnosed if two of the following parameters are below 65% of the predicted values: mid-expiratory flow rate (MMEF), maximum expiratory flow rate at 50% of vital capacity (FEF50), and maximum expiratory flow rate at 75% of vital capacity (FEF75). (https: / / doi.org / 10.1016 / S2213-2600(20)30155-7)

[0004] According to the research results, 43.5% of people aged 20 and above in my country have small airway dysfunction, totaling approximately 426 million. Among those with normal forced expiratory volume in one second (FEV1) and FEV1 / FVC ratio before bronchodilator inhalation, 25.5% have small airway dysfunction, totaling approximately 253 million; among those with normal FEV1 and FEV1 / FVC before and after bronchodilator inhalation, 11.3% have small airway dysfunction, totaling approximately 111 million. Furthermore, in the study population, almost all patients with chronic obstructive pulmonary disease (COPD) (96.15%) had small airway dysfunction, while 68.97% of asthma patients had small airway dysfunction.

[0005] Even among people without chronic obstructive pulmonary disease (COPD) or asthma, 41.3% have small airway dysfunction. These figures suggest that small airway dysfunction may exist before the onset of COPD or asthma, requiring greater attention from healthcare workers and clinicians.

[0006] Research over the past decade has demonstrated that chronic obstructive pulmonary disease (COPD) patients commonly exhibit pathological changes characterized by small airway wall thickening, epithelial changes, inflammatory cell infiltration, mucus obstruction of the lumen, airway smooth muscle hyperplasia, and peribronchial fibrosis. Small airway wall thickening is not only common in COPD but also in asthma, and is associated with the frequency and severity of dyspnea and asthma exacerbations. Another prospective study suggests that small airway dysfunction may precede the development of asthma; therefore, small airway dysfunction is also considered a precursor to both COPD and asthma.

[0007] Small airway lesions and related diseases mainly include the following: 1. Small airway inflammation: In chronic obstructive pulmonary disease (COPD), small airway inflammation is one of the important pathophysiological changes, including hypersecretion of mucus, extensive dilation and proliferation of goblet cells, squamous metaplasia, thickening and fibrosis of the airway walls, and proliferation and hypertrophy of smooth muscle. 2. Airway narrowing: Small airway inflammation leads to thickening of the airway walls and narrowing of the lumen. According to Poiseuille's law, airway resistance is inversely proportional to the fourth power of the airway diameter; when the airway diameter is narrowed, airway resistance will increase significantly. 3. Small airway dysfunction (SAD): Small airway lesions or decreased lung elasticity lead to restricted airflow in the small airways, but do not meet the diagnostic criteria for obstructive ventilatory dysfunction. This pathophysiological state is considered an early sign of chronic airway diseases such as asthma and COPD. 4. Increased mucus secretion: Long-term, repeated inflammation may lead to increased mucus secretion, which is a characteristic of small airway dysfunction. 5. Alveolar loss: Long-term inflammation can also lead to alveolar loss, another characteristic of small airway dysfunction. 6. Bronchiolar stenosis and alveolar destruction: In patients with COPD, pathological changes such as excessive mucus, bronchiolar stenosis, and alveolar destruction are common in the small airways. 7. Small airway dilatation: Small airway dilatation is more pronounced in patients with idiopathic pulmonary fibrosis (IPF), possibly related to the severe degree of pulmonary parenchymal fibrosis causing tractional dilatation of the small airways. 8. Imaging findings: High-resolution CT (HRCT) reveals signs of small airway lesions, including direct and indirect imaging features. Direct symptoms mainly include centrilobular nodules and tree-in-bud sign, while indirect symptoms include mosaic attenuation, wedge-shaped ground-glass opacities, and cylindrical bronchiolar dilatation. These pathological features indicate that small airway damage is closely related to the development of various chronic airway diseases and has a significant impact on patients' respiratory function and clinical outcomes.

[0008] Small airway disease has a complex etiology, and currently there are no effective drugs or therapies specifically targeting it. Clinical trials are currently exploring stem cell therapy, primarily using mesenchymal stem cells. However, mesenchymal stem cells cannot differentiate into lung cells and therefore cannot repair small airway damage, resulting in unsatisfactory outcomes. Additionally, there are clinical trials attempting to use lung stem cells to treat COPD; however, published data indicate limited improvement in lung function.

[0009] Current bronchial epithelial stem / progenitor cell technology has limitations in culture passages, high costs, and low clone formation rates, resulting in limited effectiveness in improving lung function in clinical trials.

[0010] Therefore, there is an urgent need in this field to develop a method that can improve the cloning rate of bronchial epithelial stem / progenitor cells, achieve multiple passages for expansion, maintain normal cell stemness, and preserve normal differentiation potential. Summary of the Invention

[0011] This invention discloses a method that can improve the cloning rate of bronchial epithelial stem / progenitor cells, achieve multiple passage expansions of bronchial epithelial stem / progenitor cells, and achieve more than 50 passage expansions while maintaining normal stemness and normal differentiation potential of bronchial epithelial stem / progenitor cells.

[0012] In a first aspect of the invention, a culture medium for culturing bronchial epithelial stem / progenitor cells is provided, the culture medium comprising 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.

[0013] In another preferred embodiment, the basal culture medium comprises DMEM and Ham's F-12.

[0014] In another preferred embodiment, the concentration (volume percentage) of the basal medium is 80-90% based on the total volume of the culture medium.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] A second aspect of the present invention provides a culture medium kit for culturing bronchial epithelial stem / progenitor cells, comprising:

[0028] (a) A first container and additives located within the first container, said additives including: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

[0029] In another preferred embodiment, the culture medium kit further includes:

[0030] (b) The second container and the basal culture medium located within the second container.

[0031] In another preferred embodiment, the culture medium kit further includes:

[0032] (c) A third container and trophoblast cells located within the third container, the trophoblast cells being obtained by irradiating fibroblasts.

[0033] In another preferred embodiment, the irradiation dose is 20-80 Gy, preferably 30-60 Gy.

[0034] In another preferred embodiment, the culture medium kit further includes:

[0035] (d) The fourth container and the bronchial epithelial stem / progenitor cells located within the fourth container.

[0036] In another preferred embodiment, the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F12, or combinations thereof.

[0037] In another preferred embodiment, the culture medium kit is used to prepare bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the invention.

[0038] 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 bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the present invention.

[0039] In another preferred embodiment, the specification also describes the recommended concentrations of each component in the culture medium kit when preparing the bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the invention.

[0040] In another preferred embodiment, the first container, the second container, the third container, and the fourth container may be the same or different containers.

[0041] A third aspect of the present invention provides a method for culturing bronchial epithelial stem / progenitor cells, the method comprising the steps of:

[0042] (S1) Provide an isolated bronchial epithelial sample and trophoblast cells, the trophoblast cells being obtained by irradiating fibroblasts;

[0043] (S2) The sample is centrifuged, washed, and digested to obtain a cell suspension containing bronchial epithelial stem / progenitor cells.

[0044] (S3) The cell suspension containing bronchial epithelial stem / progenitor cells is inoculated onto the trophoblast cells and cultured in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells as described in the first aspect of the present invention, thereby obtaining the bronchial epithelial stem / progenitor cells.

[0045] In another preferred embodiment, the bronchial epithelial sample is a normal bronchial epithelial sample or a pathological bronchial epithelial sample.

[0046] In another preferred embodiment, the bronchial epithelial sample is derived from a bronchiole, preferably a bronchiole of grade 5 or higher.

[0047] 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.

[0048] In another preferred embodiment, the centrifugation conditions in step (S2) are: 300-1000g.

[0049] In another preferred embodiment, in step (S2), tissue cleaning solution is used for cleaning.

[0050] In another preferred embodiment, the tissue washing 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 tissue washing solution.

[0051] 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.

[0052] In another preferred embodiment, in step (S2), digestion is performed using the digestive enzyme TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013).

[0053] In another preferred embodiment, in step (S1), the irradiation dose is 20-80 Gy, preferably 30-60 Gy.

[0054] In another preferred embodiment, in step (S3), the inoculation amount 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 .

[0055] In another preferred embodiment, step (S3) further includes the step of: culturing at 37°C and with a CO2 concentration of 5%-10%.

[0056] In another preferred embodiment, in step (S3), the culture medium is changed 2-3 times per week.

[0057] In another preferred embodiment, step (S3) further includes: a step of detecting endotoxins and mycoplasma in the cells.

[0058] In another preferred embodiment, step (S3) further includes the step:

[0059] (S3a) 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 as described in the first aspect of the present invention.

[0060] (S3b) The cell clones that have been cultured in the primary culture are digested, and the cell suspension obtained after digestion is seeded onto the feeder cells for passage.

[0061] (S3c) In the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells as described in the first aspect of the present invention, the cells are passaged multiple times (preferably 3 or more times, more preferably 3-6 times, more preferably 3-5 times, such as 4 times) to obtain the bronchial epithelial stem / progenitor cells.

[0062] In another preferred embodiment, in step (S3c), the number of bronchial epithelial stem / progenitor cells obtained through culture and amplification is ≥1x10. 8 1 x 10 cells, preferably 1 x 10 8 -10x10 8 1 cell, or better yet, 2 x 10 8 -5x10 8 Each cell.

[0063] In another preferred embodiment, the method further includes step (S4): mixing the cultured and amplified bronchial epithelial stem / progenitor cells with a pharmaceutically acceptable carrier to prepare a biological agent.

[0064] In another preferred embodiment, the biological agent (i.e., cell preparation) is an infusion preparation.

[0065] In another preferred embodiment, step (S4) further includes an endotoxin detection step.

[0066] In another preferred embodiment, step (S4) further includes the step of analyzing and / or identifying the bronchial epithelial stem / progenitor cells.

[0067] In another preferred embodiment, the analysis and / or identification includes: identification of P63 and / or Krt5 markers.

[0068] In another preferred embodiment, the method is an in vitro method.

[0069] In another preferred embodiment, the method is for non-diagnostic and non-therapeutic purposes.

[0070] The fourth aspect of the present invention provides a bronchial epithelial stem / progenitor cell, which is obtained by means of the method described in the third aspect of the present invention.

[0071] 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:

[0072] (a) Used as or for the preparation of culture media for culturing bronchial epithelial stem / progenitor cells; and / or

[0073] (b) As an additive used or in the preparation of bronchial epithelial stem / progenitor cell culture media; and / or

[0074] (c) Used for culturing bronchial epithelial stem / progenitor cells;

[0075] In another preferred embodiment, the use is for non-disease diagnosis or non-disease treatment purposes.

[0076] A sixth aspect of the present invention provides a composition comprising: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin1, Fibronectin, and E-Cadherin.

[0077] 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:

[0078] (a) Culture medium for preparing bronchial epithelial stem / progenitor cells; and / or

[0079] (b) For culturing bronchial epithelial stem / progenitor cells; and / or

[0080] (c) Used as an additive in the culture medium for bronchial epithelial stem / progenitor cells.

[0081] In another preferred embodiment, the use is for non-disease diagnosis or non-disease treatment purposes.

[0082] 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

[0083] Figure 1 The cloning rate of stem cells on trophoblast cells with different irradiation doses is better at a dose of 30-60 Gy.

[0084] Figure 2 The bronchial epithelial stem / progenitor cells are well-cloned, with regular and smooth clonal outlines, clear clonal boundaries, and uniform cell size and tight arrangement within the clone.

[0085] Figure 3 The results showed that the bronchial epithelial stem / progenitor cell clones were of uniform density, with regular clonal outlines, clear boundaries, and uniform cell size and tight arrangement within the clones.

[0086] Figure 4 The results showed that after 50 passages, the bronchial epithelial stem / progenitor cells had uniform cell clonal density, regular clonal outlines, clear boundaries, and uniform cell size and tight arrangement within the clones.

[0087] Figure 5 The results showed that bronchial epithelial stem / progenitor cells could still maintain normal differentiation potential after 10 and 50 passages, and could differentiate into ciliated cells, goblet cells, and rod cells, which are the cells that make up the normal bronchial epithelium, consistent with the structure of normal human bronchial epithelium.

[0088] Figure 6 The clone morphology of the clones cultured in culture medium 1 in Comparative Example 1 is shown. The clone morphology is poor, the clone outline is irregular, the clone boundary is unclear, the cell size varies greatly, and there are gaps between cells within the clone.

[0089] Figure 7 The results show the morphology of clones cultured in culture medium 1 for 10 passages in Comparative Example 1. The cell clone morphology deteriorated, the cell size varied greatly, and mast cells were present in the clones.

[0090] Figure 8 The results show the morphology of clones cultured in culture medium 2 in Comparative Example 2. The cell clone morphology is poor, the clone boundaries are unclear, the cell size within the clone is uneven, and mast cells are present within the clone.

[0091] Figure 9The clone morphology of the cultured cells in culture medium 2 after 10 passages in Comparative Example 2 is shown. The cells within the clones are of uneven size, loosely arranged, and have many gaps between them. Detailed Implementation

[0092] Through extensive and in-depth research and numerous screenings, the inventors unexpectedly discovered for the first time that a bronchial epithelial stem / progenitor cell culture medium formulated with the special addition of fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin can improve the formation rate of good clones during bronchial epithelial stem / progenitor cell culture. This allows for the acquisition of good clones from minute amounts of tissue, improving the accessibility of cell therapy, reducing the cost of cell therapy, and maintaining good stemness and high differentiation potential, making the cells more effective in repairing small airway lesions and treating small airway-related diseases.

[0093] Furthermore, this invention is the first to discover that by seeding bronchial epithelial stem / progenitor cells into trophoblast cells obtained through irradiation of fibroblasts, and culturing them in a bronchial epithelial stem / progenitor cell culture medium specially formulated with added fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin, the resulting bronchial epithelial stem / progenitor cell clones exhibit a high clonal rate. Epithelial stem cell clones can be isolated from minute amounts of tissue and expanded through more than 50 passages, maintaining normal cell stemness and differentiation potential. Sufficient numbers of clinically infused bronchial epithelial stem / progenitor cells can be cultured for autologous infusion, clinically achieving the repair of small airway damage and significantly improving lung function. This invention was completed based on these findings.

[0094] the term

[0095] bronchial epithelial stem / progenitor cells

[0096] 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.

[0097] Small airway disease and small airway dysfunction

[0098] Small airway disease refers to structural or pathological abnormalities in the small airways (airways with an internal diameter of less than 2 mm, including terminal bronchioles and respiratory bronchioles), such as inflammation, fibrosis, stenosis, or obstruction. It is usually associated with chronic inflammation, infection, smoking, or other damaging factors. Small airway dysfunction (SAD) refers to functional abnormalities in the small airways (airways with an internal diameter of less than 2 mm), leading to airflow restriction or decreased ventilation, but not yet meeting the diagnostic criteria for obstructive ventilatory dysfunction. It reflects abnormalities in gas exchange and airflow conduction in the small airways. Small airway disease can lead to small airway dysfunction, but dysfunction can also occur before the disease becomes apparent. Small airway dysfunction may be an early manifestation of small airway disease, indicating underlying pathological changes.

[0099] basal culture medium

[0100] 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.

[0101] fetal bovine serum

[0102] 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.

[0103] glucose

[0104] 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.

[0105] insulin

[0106] 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.

[0107] Epidermal growth factor

[0108] 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.

[0109] Hydrocortisone

[0110] 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.

[0111] Y-27632

[0112] Y-27632 is a small molecule inhibitor of Rho-associated protein kinase p160ROCK.

[0113] The structural formula of Y-27632 (CAS.NO:146986-50-7) is shown below:

[0114]

[0115] SB431542

[0116] SB431542 is a potent and selective inhibitor of the TGF-β signaling pathway.

[0117] Structural formula:

[0118]

[0119] Noggin

[0120] Noggin is a secreted homodimeric glycoprotein that is an antagonist of bone morphogenetic proteins (BMPs).

[0121] 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.

[0122] 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.

[0123] R-spondin 1

[0124] 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.

[0125] Fibronectin

[0126] 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.

[0127] E-Cadherin

[0128] 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.

[0129] Culture medium for bronchial epithelial stem / progenitor cells

[0130] This invention provides a culture medium for culturing bronchial epithelial stem / progenitor cells, comprising a basal medium and additives; wherein the basal medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F12, 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.

[0131] The added ingredients amplify bronchial epithelial stem / progenitor cell clones with a high cloning rate. Epithelial stem cell clones can be isolated from trace amounts of tissue and expanded through more than 50 passages. The cells maintain normal stemness and differentiation potential, and can be cultured to obtain a sufficient number of clinically infused bronchial epithelial stem / progenitor cells for autologous infusion. Clinically, this has achieved the repair of small airway damage in the lungs and significantly improved lung function.

[0132] 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.

[0133] Preferably, the culture medium for culturing bronchial epithelial stem / progenitor cells further includes the following components: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

[0134] 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.

[0135] Using the culture medium and culture method of this invention, the bronchial epithelial stem / progenitor cells amplified by this invention have a high cloning rate. Epithelial stem cell clones can be isolated from trace amounts of tissue and amplified through more than 50 passages. The cells maintain normal stemness and differentiation potential, and a sufficient number of clinically infused bronchial epithelial stem / progenitor cells can be cultured for autologous infusion. Clinically, this has achieved the repair of small airway damage in the lungs and significantly improved lung function.

[0136] Bronchial Epithelial Stem / Progenitor Cell Culture Medium Kit

[0137] The present invention also provides a bronchial epithelial stem / progenitor cell culture medium kit, the culture medium kit comprising:

[0138] (a) A first container and additives located within the first container, said additives including: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

[0139] In another preferred embodiment, the culture medium kit further includes:

[0140] (b) The second container and the basal culture medium located within the second container.

[0141] In another preferred embodiment, the culture medium kit further includes:

[0142] (c) A third container and trophoblast cells located within the third container, the trophoblast cells being obtained by irradiating fibroblasts.

[0143] In another preferred embodiment, the irradiation dose is 20-80 Gy, preferably 30-60 Gy.

[0144] In another preferred embodiment, the culture medium kit further includes:

[0145] (d) The fourth container and the bronchial epithelial stem / progenitor cells located within the fourth container.

[0146] In another preferred embodiment, the basal culture medium is selected from the group consisting of DMEM, Ham's F-12, DMEM / F12, or combinations thereof.

[0147] In another preferred embodiment, the culture medium kit is used to prepare bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the invention.

[0148] 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 bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the present invention.

[0149] In another preferred embodiment, the specification also describes the recommended concentrations of each component in the culture medium kit when preparing the bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the invention.

[0150] In addition, the first container, the second container, the third container and / or the fourth container mentioned above can be the same or different containers.

[0151] This culture medium kit can be used to prepare the bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of this invention. Based on this, the culture medium kit may further include: a specification stating the concentrations of each component in the culture medium kit and a method for preparing the culture medium as described in the first aspect of this invention. The specification may also state the recommended concentrations of each component in the culture medium kit when preparing the culture medium as described in the first aspect of this invention.

[0152] Culture methods for bronchial epithelial stem / progenitor cells

[0153] This invention also provides a method for culturing bronchial epithelial stem / progenitor cells, the method comprising the steps of:

[0154] (S1) Provide a tracheal epithelial sample and trophoblast cells, the trophoblast cells being obtained by irradiating fibroblasts;

[0155] (S2) The sample is centrifuged, washed, and digested to obtain a cell suspension containing bronchial epithelial stem / progenitor cells.

[0156] (S3) The cell suspension containing bronchial epithelial stem / progenitor cells is inoculated onto the trophoblast cells and cultured in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells as described in the first aspect of the present invention, thereby obtaining the bronchial epithelial stem / progenitor cells.

[0157] Preferably, the bronchial epithelial sample is a bronchial epithelial sample or a pathological bronchial epithelial sample.

[0158] Preferably, the method further includes step (S4): mixing the bronchial epithelial stem / progenitor cells obtained through culture and amplification with a pharmaceutically acceptable carrier to prepare a biological agent.

[0159] In another preferred embodiment, step (S4) further includes an endotoxin detection step.

[0160] In another preferred embodiment, step (S4) further includes the step of analyzing and / or identifying the bronchial epithelial stem / progenitor cells.

[0161] In a preferred embodiment, the cultivation method of the present invention includes the following steps:

[0162] 1. Using a bronchoscope and a cell brush, lung bronchial epithelial tissue samples are collected from the airways of COPD patients (samples can also be non-invasive liquid samples, tissue biopsy forceps, biopsy brush, or surgical excision). The tissue samples are flushed off the cell brush, centrifuged at 300g-1000g, washed three times with tissue preservation solution, and then digested with the digestive enzyme TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10-60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells.

[0163] 2.20-80 Gy irradiation of 3T3 mouse fibroblasts was used to prepare trophoblast cells, which were then cryopreserved in liquid nitrogen.

[0164] 3. Resuscitate the frozen cells from step 2 24 hours in advance to cultivate feeder cells, and culture the cell suspension obtained in step 1 at a concentration of 10,000-20,000 cells / cm³. 2 Planted on irradiated 3T3 mouse fibroblast trophoblasts;

[0165] Primary culture was performed using bronchial epithelial stem / progenitor cell culture medium at 37°C and a CO2 concentration of 5%-10%. After the cells adhered and grew, the medium was changed 2-3 times per week. During the culture process, samples were taken for endotoxin and mycoplasma testing to ensure that the results were negative.

[0166] 4. After 5-10 days of primary culture, when the cell clones are nearing confluence, digest the clones. The resulting cell suspension is then incubated at 10,000-20,000 cells / cm³. 2 The cells were seeded on irradiated 3T3 mouse fibroblast trophoblasts and passaged.

[0167] 5. Repeat the above clonal passages using bronchial epithelial stem / progenitor cell culture medium, passage 4 times, and then sequentially passage the cells into 3.5cm, 6cm, 10cm, and 15cm culture dishes to expand the epithelial stem cells to 2x10⁻⁶ cells / cm². 8 One cell;

[0168] 6. Collect the amplified bronchial epithelial stem / progenitor cells from step 5, prepare a cell suspension with PBS, retain the washing buffer for endotoxin detection, and collect cells for flow cytometry analysis. Package the cell suspension into pre-filled syringes and store and transport at 2-8°C.

[0169] 7. Transport the cell suspension prepared in step 6 to the hospital, and inject the cell suspension into different parts of the same patient's airway at multiple points under bronchoscopy to complete the clinical infusion.

[0170] In this invention, the tissue preservation solution comprises the following components: DMEM culture medium containing 10 mg / mL BSA and Penicil lin (100 units / ml)-Streptomycin (100 μg / ml).

[0171] 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.

[0172] The main advantages of this invention include:

[0173] (1) This invention is the first to discover that the bronchial epithelial stem / progenitor cell culture medium, which is specially formulated with the addition of fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin, can improve the formation rate of good clones during the culture of bronchial epithelial stem / progenitor cells, obtain good clones from trace amounts of tissue, improve the accessibility of cell therapy, reduce the cost of cell therapy, and maintain good stemness and high differentiation potential, making the cells more effective in repairing small airway lesions and more effective in treating diseases related to small airway lesions.

[0174] (2) This invention is the first to discover that bronchial epithelial stem / progenitor cells are seeded into trophoblast cells obtained by irradiation of fibroblasts and cultured in a bronchial epithelial stem / progenitor cell culture medium specially prepared with the addition of fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin. The bronchial epithelial stem / progenitor cells amplified have a high cloning rate, can isolate epithelial stem cell clones from trace amounts of tissue, and achieve more than 50 passages of amplification. The cells maintain normal stemness and differentiation potential, and can be cultured to obtain a sufficient number of bronchial epithelial stem / progenitor cells that can be infused clinically for autologous infusion. Clinically, this has achieved the repair of small airway damage in the lungs and significantly improved lung function.

[0175] (3) The culture medium and culture method of the present invention can culture bronchial epithelial cell clones from extremely small samples, requiring a small sample volume, which leads to high accessibility of sampling, high sampling success rate and high culture success rate.

[0176] (4) The culture medium and culture method of the present invention can positively screen out stem cell clones with good morphology.

[0177] (5) The culture medium and culture method of the present invention can improve the clonal morphology, cloning rate and proliferation rate of epithelial stem cells.

[0178] (6) The culture medium and culture method of the present invention can maintain good stemness and preserve the differentiation potential of stem cells after continuous passage and expansion.

[0179] (7) The autologous bronchial epithelial stem / progenitor cells obtained by the method of the present invention have no rejection reaction after being infused into the human body, and have high safety.

[0180] (8) The bronchial epithelial stem / progenitor cell preparation obtained by the method of the present invention can repair small airway lesions and treat lung diseases related to small airway lesions after being infused into the human body.

[0181] (9) This invention improves the current bronchial epithelial stem / progenitor cell culture technology, enabling the large-scale expansion of bronchial epithelial stem / progenitor cells with good stemness from extremely small amounts of tissue samples, thereby reducing culture costs.

[0182] (10) The bronchial epithelial cell preparation of the present invention increases the accessibility of using cell therapy for small airway lesion-related lung diseases and enables precise regenerative treatment of small airway lesion-related lung diseases.

[0183] (11) The lung cell preparation of the present invention maintains high cell stemness and has high differentiation potential. When infused into the human body, it can differentiate into normal airway cells and has a better effect on the treatment of lung diseases related to small airway lesions.

[0184] 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.

[0185] Unless otherwise stated, all reagents and materials used in the embodiments of this invention are commercially available products.

[0186] Example 1: Bronchial Epithelial Stem / Progenitor Cell Culture Medium 1

[0187] In this embodiment, a bronchial epithelial stem / progenitor cell culture medium 1 is provided, which includes a basal culture medium (such as 45% (v / v) DMEM medium, 45% (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).

[0188] Table 1

[0189]

[0190] Example 2: Preparation of autologous bronchial epithelial stem / progenitor cell preparation and maintenance of high passage and normal differentiation of stem cells

[0191] (1) Mr. Li, male, 65 years old, 60 kg, clinically diagnosed with chronic obstructive pulmonary disease. The preparation steps of his autologous bronchial epithelial stem / progenitor cell preparation are as follows:

[0192] (2) In September 2023, Li underwent a bronchoscopy and obtained a small amount of bronchial epithelial tissue (less than 300 cells) through bronchoscopic cell brushing. The cell brush was placed in tissue preservation solution and transported to the production workshop at a temperature of 2-8℃.

[0193] (3) Rinse the bronchial epithelial tissue off the cell brush, centrifuge at 300g-1000g, wash three times with tissue 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.

[0194] (4) Resuscitate 10 frozen trophoblast cells from liquid nitrogen 24 hours in advance. 6 Several cells (trophoblast cells were 3T3-J2 fibroblast cells (purchased from Kerafast, product number: EF3003)) were irradiated with 20-80 Gy and then cryopreserved in liquid nitrogen. Results showed that trophoblast cells irradiated with 20-80 Gy had a high cloning rate, with a dose of 30-60 Gy showing even better results. Figure 1 As shown in the figure, the cells were spread onto two wells of a 12-well cell culture plate. The entire cell suspension obtained in step (2) was then seeded onto the feeder layer.

[0195] (5) Primary culture was performed using the bronchial epithelial stem / progenitor cell culture medium described in Example 1. The cells were cultured at 37°C and CO2 concentration of 5%-10%. After the cells adhered and grew, the medium was changed 2-3 times per week. During the culture process, samples were taken for endotoxin and mycoplasma testing to ensure that the results were negative.

[0196] (6) After 5-10 days of culture, clones appear. More than 80% of the clones have regular and smooth outlines, clear clonal boundaries, and tightly packed, uniformly sized cells within the clones. Figure 2 When the cell clones are nearing confluence, they are digested. The resulting cell suspension is 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, exhibiting uniform density, regular outlines, clear boundaries, and homogeneous cell size within each clone. Figure 3 As shown, the cell cloning rate is consistently above 90%, reaching as high as 96%, as... Figure 1 As shown.

[0197] (7) The bronchial epithelial stem / progenitor cell culture medium described in Example 1 was used to repeat the above clonal passage (passage 4 times, with cells sequentially passaged into 3.5cm, 6cm, 10cm, and 15cm culture dishes, expanding the epithelial stem cells to a quantity of 2.2 x 10⁻⁶). 8 Each cell.

[0198] (8) Digest and collect the expanded bronchial epithelial stem / progenitor cells, taking 2 x 102 cells. 8 Each cell was used to prepare a 20ml cell suspension using PBS.

[0199] (9) Take a portion of the cells and continue passage, passageing every 3-5 days. After 50 passages, the cell clone density is uniform, the clone outline is regular, the boundary is clear, and the cell size within the clone is uniform. Figure 4 .

[0200] (10) After 10 and 50 passages, the stem cells still retain the ability to differentiate into normal bronchial epithelial cells, and can differentiate into ciliated cells, goblet cells, and rod cells, etc., consistent with the structure of normal human bronchial epithelium, such as... Figure 5 .

[0201] Example 3: Autologous bronchial epithelial stem / progenitor cell infusion improves lung function in patients with small airway disease.

[0202] Mr. Li, male, 65 years old, 60 kg, clinically diagnosed with chronic obstructive pulmonary disease. An autologous bronchial epithelial stem / progenitor cell preparation was prepared and administered via infusion. The autologous bronchial epithelial stem / progenitor cell preparation was obtained through Example 2, and the infusion steps are as follows:

[0203] (1) The cell preparation from Example 2(8) was bottled into a pre-filled syringe and stored and transported at 2-8°C. The retained cells were tested for endotoxin, mycoplasma, and sterility; all results were negative. Simultaneously, 6x10 cells were retained. 6 The cells were analyzed by ITGA6 flow cytometry, and the cell positivity rate was 99.2%.

[0204] (2) The cell preparation in the pre-filled needle was transported to the hospital, and the cell suspension (concentration of bronchial epithelial stem / progenitor cells 1x10⁻⁶) was extracted via bronchoscopy. 7 The infusion (1 cell / mL) was administered into the bronchi of five lobes of Li's lungs to complete the clinical infusion.

[0205] (3) No adverse reactions related to the preparation occurred after the infusion. Before treatment, the patient walked 260 meters in 6 minutes, and after treatment, the patient walked 550 meters in 6 minutes. The patient reported that before treatment, the patient could only climb 1-2 floors, and after treatment, the patient could climb 6 floors. Symptoms such as shortness of breath and wheezing were significantly improved, and the patient's swimming distance increased from less than 25 meters to 200 meters. Lung function tests were performed at 1, 3, and 6 months after transplantation. The results are shown in Table 2 below. The patient's lung function continued to improve and stabilize. In terms of small airway lesion indicators, after the infusion of autologous bronchial epithelial stem / progenitor cells, the patient's small airway lesion indicators FEF75, FEF50, and MMEF continued to improve. After 3 months, they were normal (>65%), and after 6 months, they remained normal. The patient's lung function recovered, indicating that the infusion of autologous bronchial epithelial stem / progenitor cell preparation had a significant therapeutic effect on the patient's small airway lesions.

[0206] Table 2

[0207] detection indicators Before transplantation 1 month after transplant 3 months after transplant 6 months after transplantation FEF75 (%) 49.7 68.4 79.6 88.4 FEF50 (%) 42.3 66.1 68.2 76.5 MMEF (%) 37.6 60.8 66.7 67.6

[0208] Comparative Example 1

[0209] The culture procedure was the same as in Example 2, using existing culture medium 1 (purchased from STEMCELL, catalog number: Catalog #05040) to culture bronchial epithelial stem / progenitor cells. More than 50% of the clones had poor morphology, with irregular outlines, unclear boundaries, significant differences in cell size, hypertrophic cells within the clones, and gaps between cells. The clonal morphology was as follows. Figure 6 As shown. After passages of well-morphologically sound clones in culture medium 1 more than 10 times, the cell clone morphology deteriorated, cell size varied considerably, and mast cells were present within the clones. The clone morphology is as follows. Figure 7 As shown.

[0210] Comparative Example 2

[0211] The culture procedure was the same as in Example 2, using existing culture medium 2 (formulation as follows: 225 mL DMEM, 225 mL LF12, 50 mL FBS, 1 mM L-glutamine, 5 ng / mL insulin, 0.5 ng / mL epidermal growth factor, 10 μg / mL adenine, 5 μg / mL hydrocortisone) to culture bronchial epithelial stem / progenitor cells. More than 50% of the clones had irregular outlines, indistinct clonal boundaries, and heterogeneous cell size within the clones. Mast cells were present within the clones. The clonal morphology was as follows. Figure 8 As shown, after passages of well-morphologically sound clones in culture medium 2 more than 10 times, the cell morphology deteriorated, the cell size within the clones became uneven, the cells were loosely arranged, and there were many gaps between the cells. Figure 9 As shown.

[0212] Therefore, the above-mentioned effects of the present invention cannot be obtained using existing culture media.

[0213] 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 culture medium for culturing bronchial epithelial stem / progenitor cells, characterized by, The culture medium consists of a basal medium and additives; the additives consist of fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin. The basal culture medium consists of DMEM and Ham's F-12, and the volume ratio of DMEM to Ham's F-12 is 1:

1. Based on the total volume of the culture medium, the concentration of fetal bovine serum is 10-20%, the concentration of glucose is 2000-4000 mg / L, the concentration of insulin is 1-10 μg / mL, the concentration of epidermal growth factor is 1-20 ng / mL, the concentration of hydrocortisone is 1-10 μg / mL, the concentration of Y-27632 is 1-10 μM, the concentration of SB431542 is 8-20 μM, the concentration of Noggin is 20-50 ng / mL, the concentration of R-spondin 1 is 10-100 ng / mL, the concentration of Fibronectin is 1-10 μg / mL, and the concentration of E-Cadherin is 0.1-2 μg / mL.

2. The culture medium as described in claim 1, characterized in that, The concentration of glucose is 2500-3500 mg / L based on the total volume of the culture medium.

3. The culture medium as described in claim 1, characterized in that, The insulin concentration is 3-10 μg / mL based on the total volume of the culture medium.

4. The culture medium as described in claim 1, characterized in that, The concentration of epidermal growth factor is 2-10 ng / ml based on the total volume of the culture medium.

5. The culture medium as described in claim 1, characterized in that, The concentration of hydrocortisone is 1-6 μg / mL based on the total volume of the culture medium.

6. The culture medium as described in claim 1, characterized in that, The concentration of Y-27632 is 1-6 μM based on the total volume of the culture medium.

7. The culture medium as described in claim 1, characterized in that, The concentration of SB431542 is 8-15 μM based on the total volume of the culture medium.

8. The culture medium as described in claim 1, characterized in that, The concentration of R-spondin 1 is 30-80 ng / mL based on the total volume of the culture medium.

9. The culture medium as described in claim 1, characterized in that, The concentration of Fibronectin is 1-6 μg / mL based on the total volume of the culture medium.

10. The culture medium according to claim 1, characterized in that, The concentration of E-Cadherin is 0.6-2 μg / mL based on the total volume of the culture medium.

11. A culture medium kit for culturing bronchial epithelial stem / progenitor cells, characterized in that, include: (a) A first container and an additive located within the first container, the additive comprising fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin; The culture medium kit also includes: (b) A second container and a basal culture medium located within the second container, wherein the basal culture medium consists of DMEM and Ham's F-12 in a volume ratio of 1:1; Based on the total volume of the culture medium, the concentration of fetal bovine serum is 10-20%, the concentration of glucose is 2000-4000 mg / L, the concentration of insulin is 1-10 μg / mL, the concentration of epidermal growth factor is 1-20 ng / mL, the concentration of hydrocortisone is 1-10 μg / mL, the concentration of Y-27632 is 1-10 μM, the concentration of SB431542 is 8-20 μM, the concentration of Noggin is 20-50 ng / mL, the concentration of R-spondin 1 is 10-100 ng / mL, the concentration of Fibronectin is 1-10 μg / mL, and the concentration of E-Cadherin is 0.1-2 μg / mL. The culture medium kit also includes: (c) A third container and trophoblast cells located within the third container, the trophoblast cells being obtained by irradiating fibroblasts at an irradiation dose of 20-80 Gy.

12. The culture medium kit according to claim 11, characterized in that, The irradiation dose is 30-60 Gy.

13. The culture medium kit as described in claim 11, characterized in that, The culture medium kit also includes: (d) The fourth container and the bronchial epithelial stem / progenitor cells located within the fourth container.

14. A method for culturing bronchial epithelial stem / progenitor cells, characterized in that, The method includes the following steps: (S1) Provide an isolated bronchial epithelial sample and trophoblast cells, the trophoblast cells being obtained by irradiating fibroblasts; (S2) The sample is centrifuged, washed, and digested to obtain a cell suspension containing bronchial epithelial stem / progenitor cells; (S3) The cell suspension containing bronchial epithelial stem / progenitor cells is inoculated onto the trophoblast cells and cultured in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells as described in claim 1, thereby obtaining the bronchial epithelial stem / progenitor cells. In step (S1), the irradiation dose is 20-80 Gy.

15. The cultivation method as described in claim 14, characterized in that, The bronchial epithelial samples were taken from the bronchioles.

16. The cultivation method as described in claim 15, characterized in that, The bronchial epithelial samples were taken from bronchioles of grade 5 and above.

17. The cultivation method as described in claim 14, characterized in that, In step (S1), the irradiation dose is 30-60 Gy.

18. The cultivation method as described in claim 14, characterized in that, The step (S3) further includes the following steps: (S3a) The cell suspension containing bronchial epithelial stem / progenitor cells is seeded 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 as described in claim 1. (S3b) The cell clones that have been cultured in the primary culture are digested, and the cell suspension obtained after digestion is seeded onto the feeder cells for passage. (S3c) In the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells as described in claim 1, the cells are passaged multiple times to obtain the bronchial epithelial stem / progenitor cells.

19. Use of a culture medium as described in claim 1, or a culture medium kit as described in claim 11, characterized in that, The intended use is for culturing bronchial epithelial stem / progenitor cells.

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