Culture method of bronchial epithelial stem / progenitor cells

Abstract

The invention provides a culture method of bronchial epithelial stem / progenitor cells, and particularly provides a culture medium for culturing the bronchial epithelial stem / progenitor cells, which comprises a basic culture medium and additives, wherein the basic culture medium is selected from DMEM (Dulbecco's Modified Eagle Medium), Ham's F-12, DMEM / F-12 or a combination thereof, the additives comprise fetal calf serum, glucose, insulin, epidermal growth factors, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin and E-Cadherin, the invention further provides a method for culturing the bronchial epithelial stem / progenitor cells, and the bronchial epithelial stem / progenitor cells amplified by the method are relatively high in cloning rate and relatively high in survival rate. According to the method, epithelial stem cell clones can be separated from a very small amount of tissues, more than 50 times of subculture amplification can be realized, the cell stemness is maintained normal, the normal differentiation potential is maintained, enough bronchial epithelial stem / progenitor cells which can be infused clinically can be obtained through culture, the bronchial epithelial stem / progenitor cells are used for autologous infusion, the repair of small airway injury of the lung is clinically realized, and the clinical application prospect is wide. The lung function is obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for culturing bronchial epithelial stem / progenitor cells. Background Art

[0002] Small airways refer to airways with a diameter of less than 2 mm. It is currently believed that in the early stages of respiratory diseases, small airway obstruction has little effect on changes in airway resistance, so it is also called the silent zone of lung disease. However, in the progressive stage of the disease, small airway obstruction will significantly affect lung function.

[0003] If two of the maximum 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) are lower than 65% of the expected value, it can be judged as small airway dysfunction. (https: / / doi.org / 10.1016 / S2213-2600(20)30155-7)

[0004] According to the research results, among people aged 20 and above in my country, 43.5% have small airway dysfunction, a total of about 426 million people. Among people with normal forced expiratory volume in one second (FEV1) and forced expiratory volume in one second to forced vital capacity ratio (FEV1 / FVC) before inhaling bronchodilators, 25.5% have small airway dysfunction, a total of about 253 million people; among people with normal FEV1 and FEV1 / FVC before and after inhaling bronchodilators, 11.3% have small airway dysfunction, a total of about 111 million people. In addition, among the study population, almost all patients with chronic obstructive pulmonary disease (96.15%) have small airway dysfunction, and 68.97% of asthma patients have small airway dysfunction.

[0005] Even among people who do not suffer from COPD or asthma, 41.3% have small airway dysfunction. These figures suggest that small airway dysfunction may exist before COPD and asthma appear, which needs to attract more attention from health workers and clinicians.

[0006] Research in the past decade has shown that pathological changes such as thickening of the small airway walls, inflammatory cell infiltration characterized by epithelial changes, mucus blocking the lumen, airway smooth muscle hyperplasia, and peribronchial fibrosis are common in patients with chronic obstructive pulmonary disease (COPD). Not only in COPD, but also in asthma, small airway wall thickening is common and is related to the frequency and severity of dyspnea and asthma exacerbations. Another prospective study believes that small airway dysfunction may precede the development of asthma, so small airway dysfunction is also considered a precursor to 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 high secretion of mucus, massive expansion and proliferation of goblet cells, metaplasia of squamous epithelial cells, thickening and fibrosis of airway walls, hyperplasia and hypertrophy of smooth muscles, etc. 2. Airway stenosis: Small airway inflammation can lead to thickening of 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 limited exhalation of small airway airflow, but the pathophysiological state that does not meet the diagnostic criteria for obstructive ventilation dysfunction is considered to be an early signal of chronic airway diseases such as asthma and COPD. 4. Increased mucus secretion: Long-term and repeated inflammation may lead to increased mucus secretion, which is a characteristic of small airway dysfunction. 5. Alveolar loss: Long-term inflammation may also lead to alveolar loss, which is another feature of small airway dysfunction. 6. Bronchiolar stenosis and alveolar destruction: In patients with COPD, the small airways often have pathological changes such as excess mucus, bronchiolar stenosis and alveolar destruction. 7. Small airway dilatation: In patients with idiopathic pulmonary fibrosis (IPF), small airway dilatation is more obvious, which may be related to the tractional small airway dilatation caused by the severe degree of lung parenchymal fibrosis. 8. Imaging manifestations: High-resolution CT (HRCT) can show signs of small airway lesions, including direct features and indirect imaging features. Direct symptoms are mainly centrilobular nodules and tree-bud signs, and indirect symptoms include mosaic attenuation, wedge-shaped ground-glass areas, cylindrical bronchiolar dilatation, etc. These pathological characteristics indicate that small airway damage is closely related to the development of a variety of chronic airway diseases and has a significant impact on patients' respiratory function and clinical outcomes.

[0008] The cause of small airway disease is relatively complex, and there are currently no effective drugs or therapies for small airway disease. There are currently attempts to use stem cells for clinical trials, mainly mesenchymal stem cells. Mesenchymal stem cells cannot differentiate into lung cells and cannot repair small airway damage, so the effect is unsatisfactory. In addition, there are attempts to use lung stem cells in clinical trials to treat COPD. Judging from the published data, there is no obvious improvement in lung function.

[0009] The current bronchial epithelial stem / progenitor cell technology has limited culture generations, high costs, and low cloning rates, and has not been effective in improving lung function in clinical trials.

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

[0011] The present invention discloses a method for improving the cloning rate of bronchial epithelial stem / progenitor cells, achieving multiple passage expansion of bronchial epithelial stem / progenitor cells, and achieving more than 50 passage expansions, while maintaining normal stemness of bronchial epithelial stem / progenitor cells and normal differentiation potential.

[0012] In the first aspect of the present invention, a culture medium for culturing bronchial epithelial stem / progenitor cells is provided, the culture medium comprising a basal culture medium and additives; wherein the basal culture medium is selected from the following group: DMEM, Ham's F-12, DMEM / F-12, or a combination thereof, and the additives comprise: 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 includes DMEM and Ham's F-12.

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

[0015] In another preferred embodiment, based on the total volume of the culture medium, the volume ratio of the DMEM to Ham's F-12 is 0.5-2:0.5-2, preferably 1:1.

[0016] In another preferred embodiment, based on the total volume of the culture medium, the concentration (volume percentage) of the fetal bovine serum is 10-20%.

[0017] In another preferred embodiment, based on the total volume of the culture medium, the concentration of glucose is 1000-5000 mg / L, preferably 1500-4500 mg / L, more preferably 2000-4000 mg / L, more preferably 2500-3500 mg / L.

[0018] In another preferred embodiment, the concentration of insulin based on the total volume of the culture medium is 0.1-100 μg / mL, preferably 0.3-80 μg / mL, more preferably 0.5-50 μg / mL, more preferably 0.8-30 μg / mL, more preferably 1-10 μg / mL, more preferably 3-10 μg / mL.

[0019] In another preferred embodiment, based on the total volume of the culture medium, the concentration of epidermal growth factor is 0.1-100 ng / mL, preferably 0.5-80 ng / mL, more preferably 0.7-50 ng / mL, more preferably 0.8-30 ng / mL, more preferably 1-20 ng / mL, more preferably 2-10 ng / ml.

[0020] In another preferred embodiment, based on the total volume of the culture medium, the concentration of hydrocortisone is 0.1-100 μg / mL, preferably 0.3-50 μg / mL, more preferably 0.8-30 μg / mL, more preferably 1-10 μg / mL, more preferably 1-6 μg / mL.

[0021] In another preferred embodiment, based on the total volume of the culture medium, the concentration of Y-27632 is 0.1-100 μM, preferably, 0.3-50 μM, more preferably, 0.8-30 μM, more preferably, 1-10 μM, more preferably, 1-6 μM.

[0022] In another preferred embodiment, based on the total volume of the culture medium, the concentration of SB431542 is 1-100 μM, preferably 5-60 μM, more preferably 8-20 μM, and more preferably 8-15 μM.

[0023] In another preferred embodiment, based on the total volume of the culture medium, the concentration of Noggin is 1-500 ng / mL, preferably 5-300 ng / mL, more preferably 8-200 ng / mL, more preferably 10-100 ng / mL, more preferably 20-50 ng / mL.

[0024] In another preferred embodiment, based on the total volume of the culture medium, the concentration of R-spondin 1 is 1-500 ng / mL, preferably 5-300 ng / mL, more preferably 8-200 ng / mL, more preferably 10-100 ng / mL, more preferably 30-80 ng / mL.

[0025] In another preferred embodiment, based on the total volume of the culture medium, the concentration of Fibronectin is 0.1-100 μg / mL, preferably 0.5-50 μg / mL, more preferably 0.8-30 μg / mL, more preferably 1-10 μg / mL, more preferably 1-6 μg / mL.

[0026] In another preferred embodiment, based on the total volume of the culture medium, the concentration of E-Cadherin is 0.01-10 μg / mL, preferably 0.05-5 μg / mL, more preferably 0.08-3 μg / mL, more preferably 0.1-2 μg / mL, more preferably 0.6-2 μg / mL.

[0027] The 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 in the first container, wherein the additives include: 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 comprises:

[0030] (b) a second container and a basal culture medium in the second container.

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

[0032] (c) a third container and trophoblast cells in the third container, wherein the trophoblast cells are 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 comprises:

[0035] (d) A fourth vessel and bronchial epithelial stem / progenitor cells located in the fourth vessel.

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

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

[0038] In another preferred embodiment, the culture medium kit further comprises: an instruction manual, wherein the instruction manual records the concentration of each component in the culture medium kit and the 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 instructions further record the guided usage concentrations of the components in the culture medium kit when preparing the bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the present invention.

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

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

[0042] (S1) providing an ex vivo bronchial epithelial sample and trophoblast cells, wherein the trophoblast cells are obtained by irradiating fibroblasts;

[0043] (S2) centrifuging, washing, and digesting the sample to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0044] (S3) inoculating the cell suspension containing bronchial epithelial stem / progenitor cells onto the trophoblast cells, and culturing them in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells 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 from bronchioles, preferably bronchioles of level 5 and above.

[0047] In another preferred embodiment, the bronchial epithelial sample is selected from the following group: tissue sample, non-invasive liquid sample, tissue biopsy forceps sample, biopsy brush sample, surgical excision sample, or a combination thereof.

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

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

[0050] In another preferred embodiment, the tissue cleaning solution comprises: based on the total volume of the tissue cleaning solution, a basal medium containing 5-50 mg / mL (preferably 6-30 mg / mL, more preferably 8-20 mg / mL) BSA, Penicillin (20-800 units / mL, preferably 50-500 units / mL, more preferably 80-200 units / mL) -Streptomycin (20-800 μg / mL, preferably 50-500 μg / mL, more preferably 80-200 μg / mL)

[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 a combination thereof.

[0052] In another preferred embodiment, in step (S2), digestion is performed using 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 8000-50000 cells / cm 2 , preferably, 10000-20000 cells / cm 2 .

[0055] In another preferred embodiment, step (S3) further comprises the step of: heating the mixture at 37°C and 5%-10% CO 2 cultured under the conditions of .

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

[0057] In another preferred embodiment, the step (S3) further comprises: a step of performing endotoxin and mycoplasma detection on the cells.

[0058] In another preferred embodiment, the step (S3) further comprises the steps of:

[0059] (S3a) inoculating the cell suspension containing bronchial epithelial stem / progenitor cells onto the trophoblast cells, and performing primary culture for 5-10 days in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells described in the first aspect of the present invention;

[0060] (S3b) digesting the primary cultured cell clones, and inoculating the cell suspension obtained after digestion onto the trophoblast cells for passaging;

[0061] (S3c) performing multiple passaging (preferably more than 3 times, more preferably 3-6 times, more preferably 3-5 times, for example 4 times) 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.

[0062] In another preferred embodiment, in the step (S3c), the number of the bronchial epithelial stem / progenitor cells obtained by culture and expansion is ≥1x10 8 cells, preferably 1x10 8 -10x10 8 cells, preferably 2x10 8 -5x10 8 cells.

[0063] In another preferred embodiment, the method further comprises the step (S4): mixing the bronchial epithelial stem / progenitor cells obtained by culture and expansion with a pharmaceutically acceptable carrier to prepare a biological preparation.

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

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

[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, wherein the bronchial epithelial stem / progenitor cell is obtained by the method described in the third aspect of the present invention.

[0071] The fifth aspect of the present invention provides a use of the culture medium according to the first aspect of the present invention, or the culture medium kit according to the second aspect of the present invention, the use comprising:

[0072] (a) for use as or for preparing a culture medium for culturing bronchial epithelial stem / progenitor cells; and / or

[0073] (b) for use as or for preparing a supplement for a bronchial epithelial stem / progenitor cell culture medium; and / or

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

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

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

[0077] The seventh aspect of the present invention provides a use of the composition according to the sixth aspect of the present invention, the use comprising:

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

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

[0080] (c) Used as a supplement for the culture medium of bronchial epithelial stem / progenitor cells.

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

[0082] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0084] Figure 2 The bronchial epithelial stem / progenitor cells were well cloned, with regular and smooth clone outlines, clear clone boundaries, and cells within the clones of uniform size and compact arrangement.

[0085] Figure 3 The results showed that the density of bronchial epithelial stem / progenitor cell clones was uniform, the clone outlines were regular, the boundaries were clear, and the cells within the clones were uniform in size and compactly arranged.

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

[0087] Figure 5 It showed that bronchial epithelial stem / progenitor cells could maintain normal differentiation potential after 10 and 50 passages, and could differentiate into ciliated cells, goblet cells, club cells and other cells that constitute normal bronchial epithelium, which is consistent with the structure of normal human bronchial epithelium.

[0088] Figure 6 The clone morphology cultured in 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 difference is large, and there are gaps between cells in the clone.

[0089] Figure 7 The clone morphology of the culture medium 1 in comparative example 1 after 10 passages is shown. The clone morphology of the cells deteriorates, the cell sizes vary greatly, and mast cells are present in the clones.

[0090] Figure 8 The clone morphology of the cells cultured in medium 2 in comparative example 2 is shown. The clone morphology of the cells is deteriorated, the clone boundary is unclear, the cell sizes within the clone are not uniform, and mast cells are present in the clone.

[0091] Fig. 9The morphology of clones cultured in medium 2 for 10 passages in comparative example 2 is shown. The cell sizes in the clones are uneven, the cells in the clones are loosely arranged, and there are many gaps between the cells. DETAILED DESCRIPTION

[0092] After extensive and in-depth research and a large amount of screening, the inventors unexpectedly discovered for the first time that a bronchial epithelial stem / progenitor cell culture medium prepared by specially adding fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin components can improve the formation rate of good clones during the bronchial epithelial stem / progenitor cell culture process, can obtain good clones from extremely small amounts of tissue, improve the accessibility of cell therapy, reduce the cost of cell therapy, and maintain good stemness and high differentiation potential, so that the cells are more effective in repairing small airway lesions and treating diseases related to small airway lesions.

[0093] Furthermore, the present invention has also discovered for the first time that the present invention inoculates bronchial epithelial stem / progenitor cells into trophoblast cells obtained by irradiating fibroblasts, and cultured them in a bronchial epithelial stem / progenitor cell culture medium prepared by adding fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin components, and the clone rate of the amplified bronchial epithelial stem / progenitor cells is high, epithelial stem cell clones can be isolated from a very small amount of tissue, and more than 50 passages can be amplified, the stemness of the cells is maintained normally, the normal differentiation potential is maintained, and a sufficient number of bronchial epithelial stem / progenitor cells that can be infused clinically can be cultured for autologous infusion, and the repair of small airway damage in the lungs is clinically achieved, and lung function is significantly improved. The present invention was completed on this basis.

[0094] the term

[0095] Bronchial epithelial stem / progenitor cells

[0096] Bronchial epithelial stem / progenitor cells are a type of cells that exist in the bronchial epithelium and have the ability to self-renew and differentiate. They play an important role in maintaining the homeostasis of the bronchial epithelium and repairing it after injury. Commonly used markers are P63 and Krt5.

[0097] Small airway disease and small airway dysfunction

[0098] Small airway disease refers to structural or pathological abnormalities in small airways (airways with an inner diameter of less than 2 mm, including terminal bronchioles and respiratory bronchioles), such as inflammation, fibrosis, stenosis or obstruction. It is usually related to chronic inflammation, infection, smoking or other damaging factors. Small airway dysfunction (SAD) refers to functional abnormalities in small airways (airways with an inner diameter of less than 2 mm), resulting in airflow limitation or decreased ventilation function, but has not yet reached the diagnostic criteria for obstructive ventilation dysfunction. It reflects the abnormal performance of small airways in gas exchange and airflow conduction. Small airway lesions may lead to small airway dysfunction, but dysfunction may also occur before the lesions are obvious. Small airway dysfunction may be an early manifestation of small airway lesions, indicating potential pathological changes.

[0099] Basic culture medium

[0100] Cell culture basal medium is the basic nutrient medium used for cell culture, which usually needs to be selected and supplemented according to different cell types and experimental requirements. DMEM medium is a widely used basal medium suitable for the culture of a variety of mammalian cells. Ham's F12 medium is a classic cell culture medium developed by Ham in 1965. It contains similar nutrients to DMEM, but its formula is slightly different, especially it contains richer trace elements and growth factors. DMEM / F12 medium is a mixture of DMEM medium and Ham's F-12 medium in a 1:1 ratio, combining the advantages of the two culture media, providing more comprehensive nutrients, and is suitable for the culture of a variety of cell types.

[0101] Fetal bovine serum

[0102] Fetal bovine serum is a light yellow, clear, non-hemolytic, and slightly viscous liquid. Fetal bovine serum should be obtained from fetal bovines delivered by caesarean section; serum is a very complex mixture formed by removing fibrin from plasma. Serum contains various plasma proteins, polypeptides, fats, carbohydrates, growth factors, hormones, inorganic substances, etc.

[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 C 6 H 12 O 6 , 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 pancreatic beta cells in the pancreas in response to 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 polypeptide composed of 53 amino acid residues. After EGF specifically recognizes and binds to the EGF receptor on the target cell, a series of biochemical reactions occur, which ultimately promotes DNA synthesis and mitosis of the target cell.

[0109] Hydrocortisone

[0110] Hydrocortisone, also known as cortisol, is an organic compound with the chemical formula C 21 H 30 O 5 It is an adrenal cortex hormone extracted from the adrenal cortex that has the strongest effect on carbohydrate metabolism, that is, it is a type of glucocorticoid.

[0111] Y-27632

[0112] Y-27632 is a small molecule inhibitor of the 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 prevents chondrocyte proliferation and regulates the normal formation of joints. When human embryonic stem cells (hESCs) or neural stem cells are cultured under certain conditions in the adult central nervous system and peripheral tissues (such as the lung), the addition of Noggin to antagonize BMP activity can allow stem cells to proliferate while maintaining their undifferentiated state or differentiate into dopaminergic neurons. In Noggin-deficient mice, enhanced BMP activity causes a series of developmental abnormalities, including failure of neural tube formation, delayed hair follicle development, axial skeletal deformities, and joint lesions.

[0122] Noggin also plays an important role in organoid culture. It is widely used in the long-term culture of various organoids, such as liver organoids, small intestinal organoids and fallopian tube organoids. Noggin binds to bone morphogenetic protein to coordinate Wnt signals to activate stem cells and promote their proliferation. During organoid culture, Noggin maintains the self-renewal ability of stem cells and prevents premature differentiation of stem cells 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 ability of intestinal stem cells. In the culture of brain organoids, Noggin promotes the self-renewal of neural stem cells and the generation of neurons by inhibiting BMP signals.

[0123] R-spondin 1

[0124] R-spondin 1 (RSPO1) is a secreted activator protein that belongs to the R-spondin protein family, which includes four members, R-spondin 1-4. RSPO1 has two cysteine-rich furin-like domains (FU-likeCR) and a thrombospondin type 1 domain (TSR). It is a multipotent signaling ligand, and its best-known function is to activate the Wnt / β-catenin signaling pathway. RSPO1 enhances Wnt / β-catenin signaling by acting on the cognate receptors LGR4 / 5 / 6 expressed by stem and progenitor cells, especially in proliferative stem cells. Therefore, RSPO1 plays an important role in stem cell regulation in multiple organs and is a key factor in the in vitro expansion culture of multiple adult stem cells (including intestine, stomach and liver).

[0125] Fibronectin

[0126] Fibronectin is a macromolecular extracellular membrane protein present on the surface of various animal cells. It is the main non-collagenous glycoprotein in the extracellular matrix and basement membrane. It plays a central role in cell adhesion and can regulate cell polarity, differentiation and growth.

[0127] E-Cadherin

[0128] E-Cadherin (E-cadherin) protein is also called intercellular adhesion molecule 1 (CDH1). It plays an important role in intercellular connections, maintaining cell adhesion and structural integrity and participating in cell polarity and cell migration. E-Cadherin is mainly expressed in epithelial tissues and can play a key role in cell-to-cell recognition and adhesion. Due to its important functions, E-Cadherin plays an important role in physiological and pathological processes such as embryonic development, tissue repair and tumor metastasis.

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

[0130] The present invention provides a culture medium for culturing bronchial epithelial stem / progenitor cells, which comprises a basal culture medium and additives; wherein the basal culture medium is selected from the following group: DMEM, Ham's F-12, DMEM / F12, or a combination thereof, and the additives comprise: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

[0131] The cloning rate of bronchial epithelial stem / progenitor cells amplified by the above specially added ingredients is high, and epithelial stem cell clones can be isolated from extremely small amounts of tissue and expanded for more than 50 generations. The cell stemness and differentiation potential are maintained normally, and a sufficient number of clinically infusible bronchial epithelial stem / progenitor cells can be cultured for autologous infusion, which 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 a combination thereof; more preferably, the basal culture medium is DMEM, Ham's F-12, or a combination thereof.

[0133] Preferably, the culture medium for culturing bronchial epithelial stem / progenitor cells further comprises 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 the components in the culture medium are as described above, and the most preferred concentrations may be the concentrations of the components in the embodiments of the present invention.

[0135] By utilizing the culture medium of the present invention and combining it with the culture method of the present invention, the cloning rate of the bronchial epithelial stem / progenitor cells amplified by the present invention is high, epithelial stem cell clones can be isolated from a very small amount of tissue, and more than 50 passage amplifications can be achieved, the stemness of the cells is maintained normal, the normal differentiation potential is maintained, and a sufficient number of clinically infusible bronchial epithelial stem / progenitor cells can be cultured for autologous infusion, which clinically achieves the repair of small airway damage in the lungs and significantly improves lung function.

[0136] Bronchial Epithelial Stem / Progenitor Cell Culture 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 in the first container, wherein the additives include: 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 comprises:

[0140] (b) a second container and a basal culture medium in the second container.

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

[0142] (c) a third container and trophoblast cells in the third container, wherein the trophoblast cells are 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 comprises:

[0145] (d) A fourth vessel and bronchial epithelial stem / progenitor cells located in the fourth vessel.

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

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

[0148] In another preferred embodiment, the culture medium kit further comprises: an instruction manual, wherein the instruction manual records the concentration of each component in the culture medium kit and the 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 instructions further record the guided usage concentrations of the components in the culture medium kit when preparing the bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the present invention.

[0150] In addition, the first container, the second container, the third container and / or the fourth container may be the same (identical) or different containers.

[0151] The culture medium kit can be used to prepare the bronchial epithelial stem / progenitor cell culture medium as described in the first aspect of the present invention. Based on this, the culture medium kit can also include: an instruction manual, which records the concentration of each component in the culture medium kit and the method for preparing the culture medium as described in the first aspect of the present invention. The instruction manual can also record the guiding concentration of each component in the culture medium kit when preparing the culture medium as described in the first aspect of the present invention.

[0152] Method for culturing bronchial epithelial stem / progenitor cells

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

[0154] (S1) providing a bronchial epithelial sample and trophoblast cells, wherein the trophoblast cells are obtained by irradiating fibroblasts;

[0155] (S2) centrifuging, washing, and digesting the sample to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0156] (S3) inoculating the cell suspension containing bronchial epithelial stem / progenitor cells onto the trophoblast cells, and culturing them in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells 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 comprises the step (S4): mixing the bronchial epithelial stem / progenitor cells obtained by culture and expansion with a pharmaceutically acceptable carrier to prepare a biological preparation.

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

[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 culture method of the present invention comprises the following steps:

[0162] 1. Use a cytology brush to brush lung bronchial epithelial tissue samples from the airways of COPD patients through a bronchoscope (the samples can also be non-invasive liquid samples, tissue biopsy forceps, biopsy brushes, or surgical excisions). Flush the tissue samples from the cytology brush, centrifuge at 300g-1000g, wash with tissue preservation solution three times, and then digest with 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] 2.20-80Gy irradiated 3T3 mouse fibroblasts were prepared into trophoblast cells and cryopreserved in liquid nitrogen;

[0164] 3. Resuscitate the frozen cell culture trophoblast cells in step 2 24 hours in advance, and culture the cell suspension obtained in step 1 at 10,000-20,000 cells / cm 2 Seeded on irradiated 3T3 mouse fibroblast feeder layer;

[0165] Primary cultures were cultured in bronchial epithelial stem / progenitor cell medium at 37°C, CO 2 Culture at a concentration of 5%-10%. After the cells adhere to the wall and grow, change the medium 2-3 times a week. During the culture process, take samples for endotoxin and mycoplasma testing to ensure that they are negative.

[0166] 4. After 5-10 days of primary culture, when the cell clones are close to fusion, digest the clones. After digestion, the cell suspension is obtained with a density of 10,000-20,000 cel ls / cm 2 The cells were seeded on irradiated 3T3 mouse fibroblast feeder layers to complete the passage;

[0167] 5. Repeat the above cloning and subculturing using bronchial epithelial stem / progenitor cell culture medium for 4 times, and sequentially subculture the cells onto 3.5 cm, 6 cm, 10 cm, and 15 cm culture dishes to expand the epithelial stem cells to 2 x 10 8 cells;

[0168] 6. Collect the bronchial epithelial stem / progenitor cells amplified in step 5, prepare a cell suspension preparation with PBS, retain the wash solution for endotoxin detection, and retain the cells for flow cytometry. Fill the cell suspension into a pre-filled syringe and store and transport at 2-8°C.

[0169] 7. The cell suspension prepared in step 6 is transported to the hospital, and the cell suspension is injected into different parts of the airway of the same patient at multiple points under bronchoscope to complete clinical infusion.

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

[0171] In the present invention, the bronchial epithelial stem / progenitor cell culture medium contains 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, 2 ug / mL Fibronectin, and 1 ug / mL E-Cadherin.

[0172] The main advantages of the present invention include:

[0173] (1) The present invention first discovered that a bronchial epithelial stem / progenitor cell culture medium prepared by adding fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin components can improve the formation rate of good clones during the bronchial epithelial stem / progenitor cell culture process, and can obtain good clones from extremely small amounts of tissue, thereby improving the accessibility of cell therapy and reducing the cost of cell therapy. In addition, the stemness is well maintained, and the high differentiation potential is maintained, making the cells more effective in repairing small airway lesions and treating diseases related to small airway lesions.

[0174] (2) The present invention first discovered that the present invention inoculates bronchial epithelial stem / progenitor cells into trophoblast cells obtained by irradiating fibroblasts, and cultured them in a bronchial epithelial stem / progenitor cell culture medium prepared by adding fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin. The cloning rate of the amplified bronchial epithelial stem / progenitor cells is high, epithelial stem cell clones can be isolated from extremely small amounts of tissue, and more than 50 passage amplifications can be achieved. The stemness of the cells is maintained normal, and the normal differentiation potential is maintained. In addition, a sufficient number of clinically infusible bronchial epithelial stem / progenitor cells can be cultured for autologous infusion, which clinically achieves the repair of small airway damage in the lungs and significantly improves lung function.

[0175] (3) The culture medium and culture method of the present invention can culture bronchial epithelial cell clones from extremely small amounts of samples, requiring a small amount of sample, resulting in high accessibility for 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 clone morphology, cloning rate and proliferation rate of epithelial stem cells.

[0178] (6) The culture medium and culture method of the present invention can enable stem cells to maintain good stemness and differentiation potential 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 are highly safe.

[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) The present invention improves the current bronchial epithelial stem / progenitor cell culture technology and can amplify a large number of bronchial epithelial stem / progenitor cells with good dryness from extremely small amounts of tissue samples, thereby reducing the culture cost.

[0182] (10) The bronchial epithelial cell preparation of the present invention increases the accessibility of using cells to treat lung diseases associated with small airway lesions, and can achieve precise regenerative treatment of lung diseases associated with small airway lesions.

[0183] (11) The lung cell preparation of the present invention maintains high stemness and high differentiation potential. When infused into the human body, the cells can differentiate into normal airway cells and have better effects in treating lung diseases related to small airway lesions.

[0184] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually 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 under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0185] Unless otherwise stated, the reagents and materials used in the examples of the present 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 culture medium, 45% (v / v) Ham's F-12 culture medium) and various components added according to the formula shown 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) Li underwent a bronchoscopy in September 2023, and obtained a trace amount of bronchial epithelial tissue (less than 300 cells) through bronchoscopic cell brushing. The cell brush was placed in tissue preservation fluid and transported to the production workshop at a temperature of 2-8°C.

[0193] (3) The bronchial epithelial tissue was flushed from the cell brush, centrifuged at 300 g to 1000 g, washed three times with tissue preservation solution, and then digested in digestion solution TrypLE (purchased from Thermo Fisher Scientific, catalog number: 12604013) for 10 to 60 minutes to obtain a cell suspension containing bronchial epithelial stem / progenitor cells;

[0194] (4) Resuscitate trophoblast cells frozen in liquid nitrogen 24 hours in advance 6 The cells (trophoblast cells are fibroblast cell line 3T3-J2 cells (purchased from Kerafast, product number: EF3003) were irradiated with 20-80Gy and then frozen in liquid nitrogen. The results showed that the trophoblast stem cell cloning rate was high after 20-80Gy irradiation, and the effect of 30-60Gy was better. Figure 1 The cell suspension obtained in step (2) was all seeded on the nutrient layer.

[0195] (5) Primary culture was performed using the bronchial epithelial stem / progenitor cell culture medium described in Example 1 at 37°C, CO 2 Culture at a concentration of 5%-10%. After the cells adhere to the wall and grow, change the medium 2-3 times a week. During the culture process, take samples for endotoxin and mycoplasma testing to ensure that they are negative.

[0196] (6) After 5-10 days of culture, clones appeared. More than 80% of the clones had regular and smooth outlines, clear clone boundaries, and cells in the clones were tightly arranged and uniform in size. Figure 2 When the cell clones are close to fusion, the clones are digested. The cell suspension obtained after digestion is 10000-20000 cells / cm 2 After 5 days of culture, cell clones appeared, with uniform cell clone density, regular clone outline, clear boundaries, and uniform cell size within the clone. Figure 3 As shown. The cell cloning rate is higher than 90%, and the highest can reach 96%. Figure 1 shown.

[0197] (7) The above cloning was repeated using the bronchial epithelial stem / progenitor cell culture medium described in Example 1 (passaged 4 times, and the cells were sequentially passaged to 3.5 cm, 6 cm, 10 cm, and 15 cm culture dishes, and the epithelial stem cells were expanded to a number of 2.2×10 8 cells.

[0198] (8) Digest and collect the amplified bronchial epithelial stem / progenitor cells, and take 2×10 8 Prepare 20 ml of cell suspension with PBS.

[0199] (9) Take a portion of the cells and continue to subculture them every 3-5 days. After 50 subcultures, 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 cells were differentiated and the stem cells still maintained the ability to differentiate into normal bronchial epithelial cells. They could differentiate into ciliated cells, goblet cells, and club cells, which are consistent with the structure of normal human bronchial epithelium, such as Figure 5 .

[0201] Example 3 Infusion of autologous bronchial epithelial stem / progenitor cell preparation 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. Prepare an autologous bronchial epithelial stem / progenitor cell preparation for infusion. The autologous bronchial epithelial stem / progenitor cell preparation is obtained by Example 2. The infusion steps are as follows:

[0203] (1) The cell preparation in Example 2 (8) was bottled into a prefilled syringe and stored at 2-8°C for transportation. The sample cells were tested for endotoxin, mycoplasma, and sterility, and the results were all negative. At the same time, 6x10 6 The cells were tested for ITGA6 by flow cytometry, and the positive rate was 99.2%.

[0204] (2) The cell preparation in the prefilled needle was transported to the hospital, and the cell suspension (the concentration of bronchial epithelial stem / progenitor cells was 1x10 7 cells / mL) were infused into the bronchi of five lobes of Li's lungs to complete the clinical infusion.

[0205] (3) After the infusion, Li did not experience adverse reactions related to the preparation. The 6-minute walking distance before treatment was 260 meters, and the 6-minute walking distance after treatment was 550 meters. He said that he could only climb 1-2 floors before treatment, but could climb to the 6th floor after treatment. Symptoms such as shortness of breath and wheezing were significantly improved, and his swimming ability increased from less than 25 meters to 200 meters. Pulmonary function tests were performed 1, 3, and 6 months after transplantation. The test results are shown in Table 2 below. Li's lung function continued to improve and stabilize. From the perspective of small airway lesion indicators, after the infusion of autologous bronchial epithelial stem / progenitor cells, Li's small airway lesion indicators FEF75, FEF50, and MMEF continued to improve. After 3 months, they were normal (>65%), and continued to remain normal for 6 months. Lung function was restored, indicating that the infusion of autologous bronchial epithelial stem / progenitor cell preparations had a significant effect on the treatment of small airway lesions in this patient.

[0206] Table 2

[0207] Detection indicators Before transplantation 1 month after transplantation 3 months after transplantation 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 steps were the same as those in Example 2. The bronchial epithelial stem / progenitor cells were cultured using the prior art culture medium 1 (purchased from STEMCELL Company, catalog number: 05040). More than 50% of the clones had poor morphology, irregular clone outlines, unclear clone boundaries, large differences in cell size, hypertrophy of cells within the clones, spaces between cells within the clones, and clone morphology such as Figure 6 After the clones with good morphology were subcultured more than 10 times with medium 1, the morphology of the cell clones became worse, the cell sizes varied greatly, and mast cells appeared in the clones. Figure 7 shown.

[0210] Comparative Example 2

[0211] The culture steps were the same as those in Example 2. The bronchial epithelial stem / progenitor cells were cultured using the prior art culture medium 2 (formulated as follows: 225 mL DMEM, 225 mL F12, 50 mL FBS, 1 mM L-glutamine, 5 ng / mL insulin, 0.5 ng / mL epidermal growth factor, 10 ug / mL adenine, 5 ug / mL hydrocortisone). More than 50% of the clones had irregular outlines, unclear clone boundaries, and uneven cell sizes within the clones. There were mast cells in the clones. The clone morphology was as follows: Figure 8 As shown. After the clones with good morphology were subcultured more than 10 times with medium 2, the morphology of the cell clones became worse, the size of the cells in the clones was uneven, the cells in the clones were loosely arranged, and there were many gaps between the cells. Fig. 9 shown.

[0212] Therefore, the above-mentioned effects of the present invention cannot be obtained by using the culture medium of the prior art.

[0213] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A culture medium for culturing bronchial epithelial stem / progenitor cells, characterized in that: The culture medium comprises a basal culture medium and additives; wherein the basal culture medium is selected from the following group: DMEM, Ham's F-12, DMEM / F-12, or a combination thereof, and the additives comprise: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

2. The culture medium according to claim 1, characterized in that Based on the total volume of the culture medium, the concentration of glucose is 1000-5000 mg / L, preferably 1500-4500 mg / L, more preferably 2000-4000 mg / L, more preferably 2500-3500 mg / L.

3. The culture medium according to claim 1, characterized in that Based on the total volume of the culture medium, the concentration of epidermal growth factor is 0.1-100 ng / mL, preferably 0.5-80 ng / mL, more preferably 0.7-50 ng / mL, more preferably 0.8-30 ng / mL, more preferably 1-20 ng / mL, more preferably 2-10 ng / ml.

4. The culture medium according to claim 1, characterized in that Based on the total volume of the culture medium, the concentration of hydrocortisone is 0.1-100 μg / mL, preferably 0.3-50 μg / mL, more preferably 0.8-30 μg / mL, more preferably 1-10 μg / mL, more preferably 1-6 μg / mL.

5. A culture medium kit for culturing bronchial epithelial stem / progenitor cells, characterized in that: include: (a) A first container and additives in the first container, wherein the additives include: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin 1, Fibronectin, and E-Cadherin.

6. A method for culturing bronchial epithelial stem / progenitor cells, characterized in that: The method comprises the steps of: (S1) providing an ex vivo bronchial epithelial sample and trophoblast cells, wherein the trophoblast cells are obtained by irradiating fibroblasts; (S2) centrifuging, washing, and digesting the sample to obtain a cell suspension containing bronchial epithelial stem / progenitor cells; (S3) inoculating the cell suspension containing bronchial epithelial stem / progenitor cells onto the trophoblast cells, and culturing them in the presence of the culture medium for culturing bronchial epithelial stem / progenitor cells according to claim 1, thereby obtaining the bronchial epithelial stem / progenitor cells.

7. A bronchial epithelial stem / progenitor cell, characterized in that: The bronchial epithelial stem / progenitor cells are obtained by the method according to claim 6.

8. A use of the culture medium according to claim 1 or the culture medium kit according to claim 5, characterized in that: The uses include: (a) for use as or for preparing a culture medium for culturing bronchial epithelial stem / progenitor cells; and / or (b) for use as or in the preparation of a supplement for a bronchial epithelial stem / progenitor cell culture medium; and / or (c) Used to culture bronchial epithelial stem / progenitor cells.

9. A composition, characterized in that The composition comprises: fetal bovine serum, glucose, insulin, epidermal growth factor, hydrocortisone, Y-27632, SB431542, Noggin, R-spondin1, Fibronectin and E-Cadherin.

10. Use of the composition according to claim 9, characterized in that: The uses include: (a) preparing a culture medium for culturing bronchial epithelial stem / progenitor cells; and / or (b) for culturing bronchial epithelial stem / progenitor cells; and / or (c) Used as a supplement for the culture medium of bronchial epithelial stem / progenitor cells.

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