Lung glial cells and in-vitro culture method and application thereof

Abstract

The invention discloses lung glial cells as well as an in-vitro culture method and application thereof. The in-vitro culture method comprises the steps that in-vitro mouse lung tissue is subjected to mechanical separation and enzymolysis digestion, a single-cell suspension is prepared, enzymolysis digestion is conducted by adding a mixed digestive enzyme solution, and the mixed digestive enzyme solution comprises type I collagenase and deoxyribonuclease I; the single cells are placed in a DMEM complete medium for subculture, the pulmonary glial cells are obtained, and the DMEM complete medium contains the heparin-binding epidermal growth factors. According to the in-vitro culture method, the lung glial cells can be successfully obtained, and the lung glial cells are high in purity, good in cell morphology and state, normal in function and suitable for follow-up cell experiments.

Description

Technical Field

[0001] The present application relates to the field of cell culture technology, and in particular to a pulmonary glial cell and an in vitro culture method and application thereof. Background Art

[0002] Pulmonary glial cells are non-myelinating peripheral glial cells that primarily surround the proximal large airways and are densely distributed around the mainstem bronchi. Studies have shown that the pulmonary autonomic nervous system influences lung inflammation and function, and that pulmonary glial cells are closely associated with the sympathetic nervous system. However, the function of pulmonary glial cells remains poorly understood, and their involvement in normal lung function remains inconclusive. Furthermore, while pulmonary neuro-immune interactions have been extensively studied during lung infection and chronic disease, the role of glial cells in lung inflammation remains unclear. Primary pulmonary glial cells retain the biological properties of the original tissue, including genetic characteristics and tissue specificity. This allows them to better reflect the growth and function of cells in vivo, thereby obtaining data that more closely resembles in vivo physiological functions and provides a valuable tool for studying the growth, metabolism, and proliferation of living cells. Furthermore, primary cells can be used to establish disease models, conduct a series of in vitro experiments to further elucidate the molecular mechanisms of disease development, and conduct drug screening and toxicity testing, avoiding the bias associated with long-term cell line passages and enhancing the reliability of experimental results.

[0003] However, to date, there is a lack of research in the field on the isolation methods and culture conditions of primary pulmonary glial cells. Summary of the Invention

[0004] Based on this, it is necessary to provide a pulmonary glial cell and an in vitro culture method and application thereof.

[0005] The first aspect of the present application provides an in vitro culture method for pulmonary glial cells, comprising the following steps:

[0006] Mechanically separating and enzymatically digesting isolated mouse lung tissue to prepare a single-cell suspension, wherein the enzymatic digestion is performed by adding a mixed digestive enzyme solution comprising type I collagenase and deoxyribonuclease I;

[0007] The single cell is placed in DMEM complete medium for subculture to obtain the pulmonary glial cell.

[0008] In some embodiments, the DMEM complete medium further comprises one or more of 8% to 12% (v / v) fetal bovine serum, 80 KU / L to 120 KU / L penicillin, and 80 mg / L to 120 mg / L streptomycin.

[0009] In some embodiments, the concentration of heparin-binding epidermal growth factor in the DMEM complete medium is 0.020 ng / µL to 0.025 ng / µL.

[0010] In some embodiments, the mixed digestive enzyme solution comprises 3 mg / mL to 5 mg / mL of type I collagenase and 0.03 mg / mL to 0.05 mg / mL of deoxyribonuclease I.

[0011] In some embodiments, the enzymatic digestion satisfies one or more of the following conditions:

[0012] The temperature of the enzymatic digestion is 35°C to 40°C;

[0013] The enzymatic digestion time is 15 min to 20 min, and shaking is performed every 4 min to 6 min.

[0014] In some embodiments, the step of mechanically separating the lung tissue includes shearing the lung tissue into lung tissue fragments, and using a tissue dissociation instrument to mix and break the lung tissue fragments into fragments less than or equal to 0.1 mm in length.

[0015] In some embodiments, the enzyme used for digestion during subculture is a pancreatic enzyme solution, and the digestion satisfies one or more of the following conditions:

[0016] The concentration of pancreatic enzyme in the pancreatic enzyme solution is 0.25% (w / v);

[0017] The volume of pancreatic enzyme solution added is 4mL~6mL;

[0018] The digestion temperature during the subculture is 35°C to 40°C;

[0019] The digestion time during the subculture is 3 min to 10 min.

[0020] In some embodiments, the subculture satisfies one or more of the following conditions:

[0021] The time for cells to crawl out of the tissue block is 1d~7d;

[0022] The first passage time of cells is 5d~10d;

[0023] The time for the cells to be passaged again is 3d~4d.

[0024] In some embodiments, the in vitro culture method comprises the following steps:

[0025] In the presence of mixed digestive enzymes, the lung tissue fragments were evenly broken up for tissue digestion to prepare a single cell suspension;

[0026] Centrifuging the single cell suspension to collect the cell pellet;

[0027] The cell pellet was resuspended in DMEM complete medium and then subcultured to collect the 3rd to 4th generation pulmonary glial cells;

[0028] The mixed digestive enzymes include type I collagenase and deoxyribonuclease I, and the DMEM complete culture medium contains heparin-binding epidermal growth factor.

[0029] The second aspect of the present application provides a pulmonary glial cell, which is cultured by the in vitro culture method described in the first aspect of the present application.

[0030] The third aspect of the present application provides the use of the pulmonary glial cells described in the second aspect of the present application in screening drugs for diseases caused by abnormal pulmonary glial cells.

[0031] The in vitro culture method of pulmonary glial cells provided in the embodiments of the present application can successfully obtain pulmonary glial cells with high purity, good cell morphology and state, and normal function, which is suitable for subsequent cell experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments and examples of this application and to provide a more complete understanding of the application and its beneficial effects, the following briefly introduces the drawings required for use in the description of the embodiments or examples. Obviously, the drawings described below are only some embodiments of this application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0033] Figure 1 This is a diagram of primary lung glial cells observed under a light microscope in one embodiment of the present application;

[0034] Figure 2 This is an immunofluorescence staining image of GFAP and SOX10 proteins in primary lung glial cells in one embodiment of the present application;

[0035] Figure 3 This is an immunofluorescence staining image of S100b protein in primary lung glial cells in one embodiment of the present application;

[0036] Figure 4 This is a calcium imaging image of primary lung glial cells in one embodiment of the present application;

[0037] Figure 5 This is a graph showing the fluorescence intensity-time curve of calcium ions in pulmonary glial cells in one embodiment of the present application. DETAILED DESCRIPTION

[0038] To facilitate understanding of the present application, a more comprehensive description of the present application will be provided below with reference to the accompanying drawings. The accompanying drawings illustrate preferred embodiments of the present application. However, the present application may be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and comprehensive understanding of the disclosure of the present application.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this application pertains. The terms used herein in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0040] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

[0041] In this application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute a limitation on the scope of protection of this application.

[0042] As used herein, the terms "having," "containing," "including," and "comprising" are synonymous and are inclusive or open-ended, not excluding additional, unrecited members or features. Examples of members or features include materials or components, structures, elements, and instruments. Non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, and states.

[0043] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0044] In this application, when referring to the unit of a data range, if the unit is only after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0045] In this application, if a method flow involves multiple steps, unless otherwise specified herein, there is no strict order restriction on the execution of these steps, and they can be executed in an order other than the order described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages do not necessarily need to be completed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating, or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0046] In this application, exemplary descriptions such as "in some embodiments (or examples)" and "in one embodiment (or example)" may include but are not limited to the following meanings: these solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0047] In this application, the terms "first," "second," and "third," etc., in "the first aspect," "the second aspect," "the third aspect," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," and "third," etc., are only used for non-exhaustive enumeration and description purposes and should be understood not to constitute closed-ended limitations on quantity.

[0048] In this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​within the numerical interval is deemed to be continuous and includes the two numerical endpoints of the numerical interval (i.e., the minimum and maximum values), as well as every numerical value between these two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints, which is equivalent to directly listing every integer. When multiple numerical ranges are provided to describe a feature or characteristic, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all subranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, percentage, ratio, etc. "Numerical interval" allows for a broad range of numerical interval types including percentage intervals, ratio intervals, and ratio intervals.

[0049] The function of pulmonary glial cells is currently poorly understood, and their involvement in normal lung physiological function remains unresolved. Furthermore, while the interplay between the lung's neuro-immune system during lung infection and chronic disease has been extensively studied, the role of glial cells in lung inflammation remains unclear. Currently, there is a lack of methods for isolating primary pulmonary glial cells and culture conditions for related research.

[0050] Based on this, the embodiments of the present application at least provide a pulmonary glial cell and its in vitro culture method and application.

[0051] In a first aspect of the present application, a method for culturing pulmonary glial cells in vitro is provided, comprising the following steps:

[0052] S10: mechanically dissociating and enzymatically digesting isolated mouse lung tissue to prepare a single-cell suspension, wherein the enzymatic digestion is performed by adding a mixed digestion enzyme solution comprising type I collagenase and deoxyribonuclease I;

[0053] S20: The single cell is placed in a DMEM complete medium for subculture to obtain lung glial cells, wherein the DMEM complete medium (Dule's modified Eagle's medium) contains heparin-binding epidermal growth factor (HBEGF).

[0054] In this application, unless otherwise specified, "mechanical separation" refers to a technical method that uses mechanical force to separate tissues, cell clusters or other biological materials into single cells or smaller components.

[0055] It should be noted that, usually when isolating and culturing astrocytes, low concentrations of trypsin (such as 0.125%) can be used, or even without enzyme digestion, and a single-cell dissociation solution of astrocytes can be obtained by gently blowing with a pipette; when isolating and culturing intestinal glial cells, type II collagenase is usually used for dissociation and digestion. However, compared with lung tissue, because it has more connective tissue and lung tissue has higher toughness, it is necessary to use enzymes suitable for lung tissue for digestion, and simply blowing with a pipette cannot achieve the effect of separating the tissues. In addition, if type II collagenase is used for digestion, problems such as cell damage to the lung tissue and insufficient digestion will occur. Therefore, in order to better dissociate the lung tissue and make it into a single-cell suspension, a digestion and dissociation scheme suitable for lung tissue separation is selected in the embodiment of the present application, and a single-cell suspension is obtained by mixing digestive enzymes and mechanical separation.

[0056] In some embodiments, because the cytoplasmic matrix contains a large amount of collagen, collagenase is used to degrade and separate cells in most primary cell isolation procedures. During primary cell isolation, DNase I is sometimes added to digest DNA leaked from damaged cells, thereby reducing viscosity and removing membrane-bound DNA fragments. This reduces cell aggregation and facilitates the formation of a single-cell suspension.

[0057] It should be noted that in the embodiments of this application, lung tissue was dissociated using a specific digestion and dissociation method, and the dissociated single cells were subcultured in DMEM medium containing heparin-binding epidermal growth factor. This successfully cultured lung glial cells with good morphology and high purity for the first time. However, when the dissociated single cells were subcultured in DMEM medium without heparin-binding epidermal growth factor, the lung glial cells could not be well isolated and cultured, and the purity was low, and the cell morphology was heterogeneous.

[0058] In some embodiments, the DMEM complete medium further comprises one or more of 8% to 12% (v / v) fetal bovine serum, 80 KU / L to 120 KU / L penicillin, and 80 mg / L to 120 mg / L streptomycin.

[0059] In some embodiments, "KU / L" means that the activity of penicillin contained in each liter of solution is 1 thousand units, and 1 unit is defined as the amount of penicillin that inhibits the growth of a certain number of standard strains under specific conditions.

[0060] In some embodiments, the volume fraction of fetal bovine serum in DMEM complete medium can be, but is not limited to, 8%, 9%, 10%, 11%, 12%, or a value or range between any two of the above values.

[0061] In some embodiments, the concentration of penicillin in DMEM complete medium can be, but is not limited to, 80 KU / L, 90 KU / L, 100 KU / L, 110 KU / L, 120 KU / L, or a value or range between any two of the above values.

[0062] In some embodiments, the concentration of streptomycin in DMEM complete medium can be, but is not limited to, 80 mg / L, 90 mg / L, 100 mg / L, 110 mg / L, 120 mg / L, or a value or range between any two of the above values.

[0063] In some embodiments, the concentration of heparin-binding epidermal growth factor in DMEM complete medium can be, but is not limited to, 0.020 ng / µL, 0.021 ng / µL, 0.022 ng / µL, 0.023 ng / µL, 0.024 ng / µL, 0.025 ng / µL, or a value or range between any two of the above values.

[0064] In some embodiments, the concentration of type I collagenase in the mixed digestive enzyme solution can be 3 mg / mL to 5 mg / mL. Without limitation, the concentration of type I collagenase in the mixed digestive enzyme solution can be, but is not limited to, 3 mg / mL, 4 mg / mL, 5 mg / mL, or a value or range between any two of the foregoing values.

[0065] In some embodiments, the concentration of DNase I in the mixed digestive enzyme solution can be 0.03 mg / mL to 0.05 mg / mL. Without limitation, the concentration of DNase I in the mixed digestive enzyme solution can be, but is not limited to, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, or a value or range between any two of the foregoing values.

[0066] In some embodiments, the temperature of the enzymatic digestion can be 35° C. to 40° C. Without limitation, the temperature of the enzymatic digestion can be, but is not limited to, 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., or a value or range between any two of the above values.

[0067] In some embodiments, the enzymatic digestion time can be 15 min to 20 min, and the shaking is performed every 4 min to 6 min. In a non-limiting manner, the enzymatic digestion time can be, but is not limited to, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, or a value or range between any two of the above values.

[0068] In some embodiments, the step of mechanically separating the mouse lung tissue comprises shearing the mouse lung tissue into lung tissue fragments, and using a tissue dissociation instrument to mix and break the lung tissue fragments into fragments less than or equal to 0.1 mm in length.

[0069] In some embodiments, the volume of the mixed and broken pieces is less than or equal to 0.001 mm 3 .

[0070] It should be noted that the mouse lung tissue can be selected from but not limited to the lung tissue of adult mice, suckling mice or other transgenic mice, and the in vitro culture method provided in the embodiments of the present application can be used to culture lung glial cells with good morphology and high purity.

[0071] In some embodiments, the enzyme used for digestion during subculture in step S20 is a trypsin solution, and the concentration of trypsin in the trypsin solution is 0.25% (w / v).

[0072] In some embodiments, the volume of the pancreatic enzyme solution added can be, but is not limited to, 4 mL, 5 mL, 6 mL, or a value or range between any two of the above values. It should be noted that the pancreatic enzyme solution is added to initiate digestion when the cells have grown to cover 80% to 90% of the area of ​​the culture flask.

[0073] In some embodiments, the digestion temperature during subculture is 35° C. to 40° C. Without limitation, the digestion temperature during subculture can be, but is not limited to, 35° C., 36° C., 37° C., 38° C., 39° C., 40° C., or a value or range between any two of the above values.

[0074] In some embodiments, the digestion time during subculture is 3 min to 10 min. In a non-limiting manner, the digestion time during subculture can be, but is not limited to, 3 min, 5 min, 7 min, 8 min, 10 min, or a value or range between any two of the above values.

[0075] In some embodiments, during the subculture process, the time it takes for cells to crawl out of the tissue block is 1 to 7 days. In a non-limiting manner, the time it takes for cells to crawl out of the tissue block can be, but is not limited to, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or a value or range between any two of the above values.

[0076] It should be noted that the time when cells crawl out of the tissue block is the time when single cells are placed in the incubator and subcultured. Regular observation under a microscope is performed and the time when cells begin to migrate and stretch outward from the edge of the tissue block is recorded, which is the time when cells crawl out of the tissue block.

[0077] In some embodiments, during the subculture process, the time of the first cell passage is 5 to 10 days. In a non-limiting manner, the time of the first cell passage can be, but is not limited to, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or a value or range between any two of the above values.

[0078] In some embodiments, "d" means "day".

[0079] It should be noted that the time of the first cell passage refers to the time from when the cells are first inoculated into the culture vessel and culture begins until the cells grow to an appropriate confluence and are in good condition.

[0080] In some embodiments, during the subculture process, the cells are subcultured again for 3 to 4 days.

[0081] It should be noted that the time for the second cell passage refers to the time from when the cells that have completed the last passage are re-seeded into the culture container and culture begins to when the cells are cultured again.

[0082] In some embodiments, the in vitro culture method of pulmonary glial cells comprises the following steps:

[0083] S100: Evenly break up the lung tissue fragments in the presence of mixed digestive enzymes for tissue digestion to prepare a single-cell suspension;

[0084] S200: centrifuge the single cell suspension and collect the cell pellet;

[0085] S300: The cell pellet was resuspended in DMEM complete medium and placed in a 37°C 5% CO2 incubator for subculture, and the 3rd to 4th generation pulmonary glial cells were collected;

[0086] The digestive enzyme mixture includes type I collagenase and deoxyribonuclease I, and the DMEM complete medium contains heparin-binding epidermal growth factor.

[0087] In a second aspect of the present application, a pulmonary glial cell is provided, which is cultured by the in vitro culture method of the first aspect of the present application.

[0088] In some embodiments, the purity of the pulmonary glial cells is greater than or equal to 90%.

[0089] In the third aspect of the present application, a use of the pulmonary glial cells according to the third aspect of the present application is provided.

[0090] In some embodiments, pulmonary glial cells cultured using the provided in vitro culture method can be used for stimulation with LPS (bacteria), poly (I:C) (virus), or β-glucan (fungus) to screen and visualize changes in related factors and genes.

[0091] In some embodiments, the pulmonary glial cells cultured using the provided in vitro culture method can be used to construct a disease model.

[0092] In some embodiments, the pulmonary glial cells cultured using the provided in vitro culture method can be used for drug screening for diseases related to abnormal pulmonary glial cells.

[0093] Some examples are provided below.

[0094] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and may also be based on the experimental manuals or conventional conditions in this area, or on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0095] In the following examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operating accuracy are allowed.

[0096] Example 1

[0097] Healthy C57BL / 6 mice aged 6 to 8 weeks were selected. The lungs of the mice were removed and gently washed in pre-cooled phosphate-buffered saline (PBS) containing 1% double-antibody (100 U / mL penicillin, 100 μg / mL streptomycin), and the connective tissue around the lung tissue was removed. Cut the lung tissue into small pieces as much as possible and place the tissue fragments into a tissue dissociation tube containing 5 mL of mixed digestive enzymes (5 mL of PBS solution, the components and concentrations of which are: 20 mg type I collagenase + 0.04 mg / mL Deoxyribonuclease I). The resulting mixture was mixed and broken into small fragments of approximately 0.1 mm × 0.1 mm using a tissue dissociation instrument (Miltenyi Biotec gentleMACS Dissociator 130-093-235). Digestion was then performed in a 37°C water bath. The mixture was manually shaken every 5 minutes to ensure thorough digestion. Wait for 15 to 20 minutes until no tissue fragments are visible. The tube was then transferred to a 50 mL centrifuge tube and centrifuged at 1000 r.min. -1 Discard the supernatant and resuspend the cells in 1-5 mL of DMEM complete medium (DMEM supplemented with Hyclone 10% fetal bovine serum, 100 kU / L penicillin, 100 mg / L streptomycin, and 0.025 ng / µL HBEGF (heparin-binding epidermal growth factor)). Culture and passage the cells in a 37°C, 5% CO2 incubator. It takes 3-7 days for the cells to crawl out of the tissue block; the first passage is 5-10 days, and the next passage is 3-4 days. The digestion enzyme for passaging is 0.25% trypsin. Transfer the cell suspension to a T75 culture flask, add 4-6 mL of 0.25% trypsin, and digest for 3-10 minutes in a 37°C, 5% CO2 incubator. Continue culturing in DMEM complete medium. Pulmonary glial cells are in optimal condition at passages 3-4 and are ready for subsequent experiments.

[0098] Cell morphology verification

[0099] The isolated and cultured pulmonary glial cells were observed under an optical microscope. Figure 1 As shown, it can be seen that the pulmonary glial cells isolated and cultured in the embodiment of the present application have complete morphology and are in good shape and condition.

[0100] Cell specificity and purity verification

[0101] The isolated and cultured pulmonary glial cells were identified by immunofluorescence staining, that is, anti-GFAP and SOX10 primary antibodies were added for incubation, and then the corresponding fluorescent-labeled secondary antibodies were added for incubation, and the cell nuclei were labeled with DAPI and observed under a fluorescence microscope. Figure 2As shown. Anti-S100b primary antibody was added to the isolated and cultured lung glial cells for incubation, and then the corresponding fluorescent-labeled secondary antibody was added for incubation. The cell nuclei were labeled with DAPI and observed under a fluorescence microscope, as shown. Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the method provided in the examples of the present application was used to successfully isolate and culture pulmonary glial cells with high purity. The ratio of fluorescently labeled cells to the total number of cells was calculated, and the purity was greater than 90%.

[0102] Cell function verification

[0103] Add an appropriate amount of buffer containing a calcium indicator (such as Fluo-4AM) to the isolated and cultured lung glial cells and incubate them. After incubation, wash them with a calcium-free buffer. Observe the treated cells under a fluorescence microscope. After 120 seconds, add 100µM ATP solution through a pipette and observe and record the changes in the intracellular calcium ion fluorescence intensity, such as Figure 4 As shown, from Figure 4 Three cells were selected to analyze the changes of calcium ion fluorescence intensity over time, named 1, 2, and 3 respectively, and the fluorescence intensity-time curve was made accordingly, as shown in the figure. Figure 5 As shown. Figure 4 and Figure 5 It can be seen that after adding 100 μM ATP, the intracellular calcium ion concentration increased significantly, indicating that the pulmonary glial cells isolated and cultured in the embodiment of the present application have good responsiveness to ATP stimulation, and the cell signal transduction function, ion balance regulation function and metabolic function are normal.

[0104] The above verification results show that the method provided in the examples of the present application can successfully obtain lung glial cells with high purity, good cell morphology and state, normal function, and are suitable for subsequent cell experiments.

[0105] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0106] The above-described embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art could make several modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the patent in this application shall be based on the appended claims, and the specification and drawings shall be used to interpret the scope of the claims.

Claims

1. A method for culturing pulmonary glial cells in vitro, characterized in that: The following steps are involved: Mechanically separating and enzymatically digesting isolated mouse lung tissue to prepare a single-cell suspension, wherein the enzymatic digestion is performed by adding a mixed digestive enzyme solution comprising type I collagenase and deoxyribonuclease I; The single cell is placed in a DMEM complete medium for subculture to obtain the pulmonary glial cell, wherein the DMEM complete medium contains heparin-binding epidermal growth factor.

2. The in vitro culture method according to claim 1, wherein The DMEM complete medium meets one or more of the following conditions: The DMEM complete medium further comprises one or more of 8% to 12% (v / v) fetal bovine serum, 80 KU / L to 120 KU / L penicillin, and 80 mg / L to 120 mg / L streptomycin; The concentration of heparin-binding epidermal growth factor in the DMEM complete medium is 0.020 ng / µL to 0.025 ng / µL.

3. The in vitro culture method according to claim 1, wherein The mixed digestive enzyme solution includes 3 mg / mL to 5 mg / mL of type I collagenase and 0.03 mg / mL to 0.05 mg / mL of deoxyribonuclease I.

4. The in vitro culture method according to claim 3, wherein The enzymatic digestion satisfies one or more of the following conditions: The temperature of the enzymatic digestion is 35°C to 40°C; The enzymatic digestion time is 15 min to 20 min, and shaking is performed every 4 min to 6 min.

5. The in vitro culture method according to claim 1, wherein The step of mechanically separating the lung tissue includes shearing the lung tissue into lung tissue fragments, and mixing and crushing the lung tissue fragments into fragments less than or equal to 0.1 mm in length.

6. The in vitro culture method according to claim 1, wherein The enzyme used for digestion during the subculture is a pancreatic enzyme solution, and the digestion meets one or more of the following conditions: The pancreatic enzyme concentration in the pancreatic enzyme solution is 0.25% (w / v); The volume of pancreatic enzyme solution added is 4mL~6mL; The digestion temperature during the subculture is 35°C to 40°C; The digestion time during the subculture is 3 min to 10 min.

7. The in vitro culture method according to claim 6, wherein The subculture satisfies one or more of the following conditions: The time for cells to crawl out of the tissue block is 1d~7d; The first passage time of cells is 5d~10d; The time for the cells to be passaged again is 3d~4d.

8. The in vitro culture method according to any one of claims 1 to 7, wherein The in vitro culture method comprises the following steps: In the presence of mixed digestive enzymes, the lung tissue fragments were evenly broken up for tissue digestion to prepare a single cell suspension; Centrifuging the single cell suspension to collect the cell pellet; The cell pellet was resuspended in DMEM complete medium and then subcultured to collect the 3rd to 4th generation pulmonary glial cells; The mixed digestive enzymes include type I collagenase and deoxyribonuclease I, and the DMEM complete culture medium contains heparin-binding epidermal growth factor.

9. A pulmonary glial cell, characterized in that The product is obtained by culturing the in vitro culture method according to any one of claims 1 to 8.

10. Use of the pulmonary glial cells according to claim 9 in screening drugs for diseases caused by abnormal pulmonary glial cells.

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