CN112522184A - Kit and method for separating and obtaining lung stem cells - Google Patents
Kit and method for separating and obtaining lung stem cells Download PDFInfo
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Abstract
The invention discloses a kit and a method for separating and obtaining lung stem cells, wherein the kit for separating the lung stem cells comprises: tissue digestive fluid; irradiating the embryonic fibroblasts; and a lung stem cell separation culture solution. The kit can be used for effectively obtaining a large amount of amplified lung stem cells, and the obtained lung stem cells have good activity, high purity and no bacterial and mycoplasma pollution.
Description
Case information
Parent application number of the present invention: 201610216939.0, filing date: 2016, 4 and 8 months, invention name: a kit and a method for separating and obtaining lung stem cells.
Technical Field
The invention relates to the technical field of separation and transplantation of lung stem cells, in particular to a kit and a method for separating and obtaining lung stem cells.
Background
With the increase of air pollution and other factors such as virus infection, some diseases of the lung, such as pulmonary fibrosis, chronic obstructive pulmonary disease, etc., increasingly affect the health and even survival of human beings. For the treatment of these diseases, the most effective treatment is currently lung transplantation. However, the number of patients who can receive lung transplant therapy is very rare due to the limited number of donors available, immunological rejection between allogenic patients, and the like. No effective treatment is currently available to remove lung transplants.
The lung stem cells belong to adult stem cells, are widely distributed in a body, have organ targeting property in cell transplantation treatment, strong regeneration capacity and no tumorigenicity, can be used for the autologous stem cell transplantation treatment, have small risk of immunological rejection and have strong operability. Further, it has been found that when lung stem cells in the distal trachea are isolated and then transplanted into an immunodeficient mouse with damaged lung after being amplified in vitro in a large amount, the transplanted lung stem cells can be integrated into the lung of the mouse and form a alveolar structure at the damaged site. Therefore, the stem cell transplantation has very wide prospect in the aspect of treating lung injury diseases. However, it has long been difficult to isolate and obtain lung stem cells that can be expanded in large quantities from tissues and organs of adults due to technical limitations.
Therefore, the current method for separating and obtaining lung stem cells still needs to be improved.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art. Therefore, an object of the present invention is to provide a means for efficiently isolating and obtaining lung stem cells that can be expanded in large quantities from tissues and organs of an adult.
In a first aspect of the invention, the invention provides a kit for isolating lung stem cells. According to an embodiment of the invention, the kit comprises: tissue digestive fluid; irradiating the embryonic fibroblasts; and a culture solution for separating the lung stem cells,
wherein the tissue digest comprises:
99% by volume PBS;
0.1-2U/ml DNase;
0.1-0.2mg/ml collagenase type I;
0.1-0.2mg/ml collagenase type IV;
0.04-0.1mg/ml dispase;
0.1-0.5mg/ml protease XIV;
0.1-3mg/ml streptothricin protease E; and
0.2-0.5mg/ml pancreatin,
the lung stem cell separation culture solution comprises:
90% by volume of RPMI1640 medium;
10% by volume fetal bovine serum;
1-3 mmol/LL-glutamine;
0.05-0.15mmol/L beta-mercaptoethanol;
100-1000U/ml leukemia inhibitory factor; and
2-5ng/ml EGF growth factor.
The inventor surprisingly finds that the kit can be effectively used for separating the lung stem cells, and particularly, the tissue digestive juice of the kit can be used for effectively carrying out digestion treatment on a biological sample containing the lung stem cells to obtain single cells; the lung stem cell separation culture solution is mixed with the single cells and placed on the embryo fibroblasts after irradiation treatment for incubation culture, so that the lung stem cells capable of being greatly expanded can be effectively obtained, and the obtained lung stem cells have good activity, high purity and no bacterial and mycoplasma pollution. In addition, the kit has low cost, convenient use and easy mass production and utilization, and is particularly suitable for separating and obtaining the lung stem cells from a trace biological sample.
According to the embodiment of the invention, the embryonic fibroblasts subjected to proliferation inhibition treatment are prepared by any one of the following methods:
(1) irradiating the embryonic fibroblasts for 1 to 5 hours with the irradiation dose of 30 to 80 Gy;
(2) the embryonic fibroblasts were treated with mitomycin C at 1-30ug/ml for 1-5 hours. Therefore, the embryonic fibroblasts subjected to proliferation inhibition treatment lose cell growth activity, and are favorable for incubation culture of the lung stem cells.
According to an embodiment of the invention, the embryonic fibroblasts are derived from a mouse, preferably an E13.5 mouse,
according to an embodiment of the present invention, the irradiation treatment is performed using a cell irradiator. Therefore, the irradiation effect is good, and the embryonic fibroblasts after irradiation treatment lose the cell growth activity, but can still be used for incubation culture of the lung stem cells.
According to an embodiment of the invention, the tissue digest comprises:
99% by volume PBS;
1U/ml DNase;
0.15mg/ml collagenase type I;
0.15mg/ml collagenase type IV;
0.07mg/ml dispase;
0.3mg/ml protease XIV;
2mg/ml streptothricin E; and
0.35mg/ml of pancreatin,
the lung stem cell separation culture solution comprises:
90% by volume of RPMI1640 medium;
10% by volume fetal bovine serum;
2 mmol/LL-glutamine;
0.1mmol/L beta-mercaptoethanol;
500U/ml leukemia inhibitory factor; and
3.5ng/ml EGF growth factor.
Therefore, the tissue digestive juice has good effect when used for carrying out digestion treatment on the biological sample containing the lung stem cells, can effectively obtain single cells, and has no damage to the cell activity; the lung stem cell separation culture solution can be effectively used for incubation culture of the lung stem cells, so that the lung stem cells which can be greatly expanded can be effectively obtained, and the obtained lung stem cells have high purity and are free from bacterial and mycoplasma pollution.
In a second aspect of the present invention, the present invention also provides a method for obtaining lung stem cells by isolation. According to an embodiment of the invention, the method comprises the steps of:
digesting a biological sample containing lung stem cells by using tissue digestive juice so as to obtain single cells; and
mixing the single cell with a lung stem cell separation culture solution, placing the mixture on an embryo fibroblast subjected to irradiation treatment for incubation culture so as to obtain a lung stem cell,
wherein the tissue digest comprises:
99% by volume PBS;
0.1-2U/ml DNase;
0.1-0.2mg/ml collagenase type I;
0.1-0.2mg/ml collagenase type IV;
0.04-0.1mg/ml dispase;
0.1-0.5mg/ml protease XIV;
0.1-3mg/ml streptothricin protease E; and
0.2-0.5mg/ml pancreatin,
the lung stem cell separation culture solution comprises:
90% by volume of RPMI1640 medium;
10% by volume fetal bovine serum;
1-3 mmol/LL-glutamine;
0.05-0.15mmol/L beta-mercaptoethanol;
100-1000U/ml leukemia inhibitory factor; and
2-5ng/ml EGF growth factor.
The inventor surprisingly finds that the method can effectively obtain the lung stem cells which can be greatly expanded, the obtained lung stem cells have good activity and high purity, are free from bacteria and mycoplasma pollution, are particularly suitable for separating and obtaining the lung stem cells from a trace biological sample, and is simple to operate, low in implementation cost and easy to popularize.
According to an embodiment of the invention, the tissue digest comprises:
99% by volume PBS;
1U/ml DNase;
0.15mg/ml collagenase type I;
0.15mg/ml collagenase type IV;
0.07mg/ml dispase;
0.3mg/ml protease XIV;
2mg/ml streptothricin E; and
0.35mg/ml pancreatin. Therefore, the biological sample containing the lung stem cells is well digested, single cells can be effectively obtained, and the activity of the cells is not damaged.
According to an embodiment of the present invention, the lung stem cell isolation medium comprises:
90% by volume of RPMI1640 medium;
10% by volume fetal bovine serum;
2 mmol/LL-glutamine;
0.1mmol/L beta-mercaptoethanol;
500U/ml leukemia inhibitory factor; and
3.5ng/ml EGF growth factor.
Therefore, the isolated culture solution for the lung stem cells can be effectively used for incubation culture of the lung stem cells, so that the lung stem cells capable of being greatly expanded can be effectively obtained, and the obtained lung stem cells have good activity, high purity and no bacterial or mycoplasma pollution.
According to the embodiment of the invention, the embryonic fibroblasts subjected to proliferation inhibition treatment are prepared by any one of the following methods:
(1) irradiating the embryonic fibroblasts for 1 to 5 hours with the irradiation dose of 30 to 80 Gy;
(2) the embryonic fibroblasts were treated with mitomycin C at 1-30ug/ml for 1-5 hours.
Therefore, the embryonic fibroblasts subjected to proliferation inhibition treatment lose cell growth activity, and are favorable for incubation culture of the lung stem cells.
According to an embodiment of the invention, the embryonic fibroblasts are derived from a mouse, preferably an E13.5 mouse.
According to an embodiment of the present invention, the irradiation treatment is performed using a cell irradiator. Therefore, the embryonic fibroblasts after irradiation treatment lose cell growth activity, and are beneficial to incubation culture of the lung stem cells.
According to an embodiment of the invention, the digestion treatment is performed at 36-38 degrees celsius, preferably 37 degrees celsius, for 1-5 hours. Therefore, the digestion treatment effect is good, single cells can be effectively obtained, and the activity of the cells is not damaged.
According to an embodiment of the present invention, in performing the digestion treatment, 1ml of tissue digestive fluid is added per biological sample for less than 0.1mg of the biological sample containing the lung stem cells; for not less than 0.1mg of the biological sample containing the lung stem cells, 2.5ml of tissue digestive juice is added per cubic centimeter of the biological sample. Therefore, the digestion treatment effect is good, and the activity of the obtained single cell is not damaged.
According to the embodiment of the present invention, the mixing ratio of the single cells to the isolated culture medium of the lung stem cells is 300000 cells/2 ml of the isolated culture medium, wherein the number of the single cells is less than 300000 calculated according to 300000. Therefore, the effect of incubation culture is good by utilizing the growth and amplification of the lung stem cells, and the activity of the obtained lung stem cells is good.
According to an embodiment of the invention, 4% -6.5% CO at 36-38 deg.C2The incubation is carried out for 2 to 5 days. Therefore, the effect of incubation culture is good, and the activity of the obtained lung stem cell is good.
According to an embodiment of the present invention, further comprising: and carrying out cell identification and pollution detection on the lung stem cells. Thus, the purity and quality of the obtained lung stem cells can be further determined.
According to an embodiment of the invention, the contamination detection comprises a bacterial contamination detection and a mycoplasma contamination detection.
According to an embodiment of the present invention, the lung stem cells were detected for the expression of Krt5 and P63 markers using an immunofluorescence staining method, so as to achieve the cell identification. This enables further determination of the purity of the obtained lung stem cells.
According to some specific examples of the present invention, the method for separating and obtaining lung stem cells of the present invention may include the following steps:
digesting the biopsy tissue with a tissue digestion solution at 37 ℃ for 1-5 hours in a sterile laboratory, collecting the digested cells, adding a lung stem cell separation culture solution, and culturing on REGEND-FEEDER for 2-5 days; then carrying out expression detection on markers Krt5 and P63 on the cells obtained by culture by an immunofluorescence staining method so as to carry out cell identification, determining the obtained cells as lung stem cells, and separating the purity of the obtained lung stem cells; and (4) carrying out quality detection on bacterial contamination and mycoplasma contamination on the separated lung stem cells.
In the case of using the kit or the method of the present invention for the isolation of lung stem cells, the source of the "biological sample containing lung stem cells" is not particularly limited, and the lung tissue containing lung stem cells of a general animal is suitable. Moreover, the invention is particularly suitable for separating and obtaining the lung stem cells from a trace biological sample.
It should be noted that, when the present invention is applied to clinical isolated culture and transplantation of lung stem cells, since the biological sample containing the lung stem cells is derived from a human, it is necessary to avoid damage to the human body during sampling. Aiming at patients who can not be repaired due to serious lung laceration, patients with lung malignant tumor without long-distance metastasis, partial tuberculosis patients, patients with bronchiectasis, patients with lung abscess, patients with interstitial lung disease and the like, normal tissues far away from focuses can be selected as the biological sample containing the lung stem cells for separating the lung stem cells while the lung resection is performed in clinical treatment. However, the method has the disadvantages of great damage to patients, high risk of operation, great difficulty and high risk. Is not suitable for normal healthy people or most patients with chronic lung diseases. Therefore, for a patient or a healthy person who needs to store lung stem cells, the patient or the healthy person can be sent to a bronchoscopy room for bronchoscopy, a disposable cell brush is inserted into a biopsy hole of a bronchoscope, the cell brush is sent to a far-end bronchus under bronchoscope monitoring, after clearly observing the condition of a tracheal cavity around the cell brush, a position far away from a focus is selected, the brush is lightly brushed on the tracheal wall for one to five times, then the bronchoscope and the cell brush are taken out of the body together, and a cell brush head part with a small amount of biopsy tissue is cut off by a sterile scissors, so that a 'biological sample containing lung stem cells' can be obtained. The method for obtaining a small amount of biopsy tissue by brushing inspection has the advantages of low risk, very small damage to a human body and very strong operability. Meanwhile, the method is suitable for a wide range of people, and is suitable for both patients and normal people.
By the kit or the method for separating and culturing the lung stem cells, cells which can be greatly expanded can be obtained from a very small amount of biopsy tissues, and the possibility of transplantation treatment of the lung stem cells is provided. For patients, the chances of recovering health and prolonging life are provided without spending a great deal of time and resources to obtain an abnormally rare matching successful lung for organ transplantation. Thus, more patients have the opportunity for treatment than organ transplantation. For normal people, the healthy stem cells can be stored for the future needs when the body is healthy, and meanwhile, the health-care medicine has better repairing effect and curative effect when unforeseen diseases occur, and a very favorable insurance is put into the future body health. That is, the kit and method of the present invention can be effectively used for isolated culture of lung stem cells for normal healthy people with a requirement for storage of lung stem cells or for most patients with chronic lung diseases, as well as for patients with severe lung diseases with a requirement for transplantation therapy of lung stem cells.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:
FIG. 1 shows morphological observations of isolated lung stem cells-growing clonally (10-fold objective, 10-fold eyepiece), according to one embodiment of the invention;
FIG. 2 shows immunofluorescence staining assay results of p63 and Krt5 markers isolated from obtained lung stem cells, according to one embodiment of the present invention; and
FIGS. 3-5 show immunofluorescent staining assay results of human lung stem cells transplanted into immunodeficient mice according to one embodiment of the present invention (FIG. 3 was observed with a 0.8-fold objective lens and a 10-fold eyepiece; FIGS. 4 and 5 were observed with a 40-fold objective lens and a 10-fold eyepiece).
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
Obtaining lung stem cells by separating the biological sample containing the lung stem cells, which comprises the following steps:
1. sample preparation: the sample was from a patient with pulmonary fibrosis who had signed an informed consent.
And (3) a sample collection process: the patient is scheduled for distal tracheal examination in an endoscopy room and, after a clear view of his distal bronchi (roughly positioned at 3-8 levels) through the endoscope, inserting a disposable cell brush into a biopsy hole of a bronchoscope, selecting a normal part, keeping away from a focus as far as possible, extending a protective brush out of the tail end of the bronchoscope, then pushing out an inner sleeve, pushing off a protective plug at the tail end of the brush, extending the inner sleeve out of the tail end of the outer sleeve, then pushing out the brush, after gently brushing the trachea wall for one to five times, the cytobrush is drawn back into the protective tube, the bronchoscope and the cytobrush are taken out of the body together, the front part of the hairbrush is cut off by sterile scissors, the hairbrush is extended out, the cell brush head part with a small amount of biopsy tissue is cut off by 3-6cm by sterile scissors and is placed into a sample collection tube, so that a biological sample containing the lung stem cells is obtained.
2. Reagent:
a tissue digest comprising:
99% by volume PBS;
0.1-2U/ml DNase;
0.1-0.2mg/ml collagenase type I;
0.1-0.2mg/ml collagenase type IV;
0.04-0.1mg/ml dispase;
0.1-0.5mg/ml protease XIV;
0.1-3mg/ml streptothricin protease E; and
0.2-0.5mg/ml pancreatin,
a lung stem cell isolation medium comprising:
90% by volume of RPMI1640 medium;
10% by volume fetal bovine serum;
1-3 mmol/LL-glutamine;
0.05-0.15mmol/L beta-mercaptoethanol;
100-1000U/ml leukemia inhibitory factor; and
2-5ng/ml EGF growth factor.
Irradiation-treated embryonic fibroblasts: obtained by irradiating E13.5 mouse embryo fibroblast MEF with 30-80Gy radiation for 1-5 hr by using cell irradiator.
3. The method comprises the following steps:
the lung stem cells were isolated from the above-obtained biological sample containing the lung stem cells (i.e., the cytobrush head with a small amount of biopsy tissue) according to the following procedure:
within 24 hours, the specimen collection tubes are transported to a sterile laboratory in a low temperature environment. Carefully taking out a brush head (which is a biological sample containing lung stem cells and less than 0.1mg) with a small amount of tissue to be brushed from a sample collection tube in a sterile laboratory, adding 1ml of tissue digestion solution, standing at 37 ℃ for 1-5 hours for digestion treatment, adding a stop solution (wherein 1L of the stop solution contains 900ml of RPMI1640 culture medium and 100ml of fetal bovine serum) to stop digestion, centrifuging at 1200rpm for 5 minutes, removing supernatant to remove the digestion solution, and centrifuging at 1200rpm for 5 minutes, and removing the digestion solution by using a reagent containing double-componentAfter washing the cells twice in phosphate buffer against (50U/ml penicillin and 50ug/ml streptomycin), centrifuging at 1200rpm for 5 minutes, removing the supernatant, resuspending the cells in lung stem cell isolation medium (300000 cells/2 ml isolation medium, mixed at 300000 cells when the number of single cells is less than 300000), spreading the cells on irradiated human embryonic fibroblasts, and culturing in a carbon dioxide cell incubator (36-38 ℃, 4% -6.5% CO)2) After 2-5 days, the colonies consisting of lung stem cells were observed under an inverted microscope (see FIG. 1).
Then, the cells obtained by the culture were subjected to expression detection of the markers Krt5 and P63 by immunofluorescence staining for cell identification, the obtained cells were determined to be lung stem cells, and the purity of the obtained lung stem cells was isolated. Immunofluorescent staining results showed that a number of clones expressed the corresponding lung stem cell markers Krt5 (see fig. 2A) and p63 (see fig. 2B). Fig. 2C is DAPI staining, indicating nuclei.
Then, the supernatant of the separated lung stem cells subjected to cell identification is subjected to quality detection of bacterial contamination and mycoplasma contamination.
The detection method of bacterial contamination comprises the following steps: 500ul of the supernatant of the lung stem cell culture medium is added into the stem cell isolation culture solution and cultured for 14 days at the temperature of 37 ℃, the condition of the culture medium is observed every day, if the culture medium is still clear, no bacteria are generated, and if the culture solution is turbid, bacteria pollution is caused. In our assay, the medium was observed daily in a clear state, indicating no bacterial contamination.
The detection method of mycoplasma contamination is as follows: 100ul of the lung stem cell culture supernatant was collected and tested by the Lonza MycoAlert Mycoplasma Detection Kit (cat # LT07-118) manufactured by Lonza, USA according to the procedures described in the specification. The final value of the test result was less than 0.8, indicating no contamination by mycoplasma.
Example 2
The method of example 1, wherein the lung stem cells are isolated from a biological sample comprising lung stem cells, wherein the only difference is that:
the tissue digest comprises:
99% by volume PBS;
1U/ml DNase;
0.15mg/ml collagenase type I;
0.15mg/ml collagenase type IV;
0.07mg/ml dispase;
0.3mg/ml protease XIV;
2mg/ml streptothricin E; and
0.35mg/ml of pancreatin,
the lung stem cell separation culture solution comprises:
90% by volume of RPMI1640 medium;
10% by volume fetal bovine serum;
2 mmol/LL-glutamine;
0.1mmol/L beta-mercaptoethanol;
500U/ml leukemia inhibitory factor; and
3.5ng/ml EGF growth factor.
As a result, the lung stem cells are successfully obtained, and the separated lung stem cells have good cell activity, high purity and no bacterial and mycoplasma pollution.
Example 3
After the lung stem cells obtained in example 1 were continuously expanded and lentiviruses expressing a green fluorescent protein marker were added to the cell culture for 24 hours, the cells were cultured again after washing the viruses out with a phosphate buffer, and after 72 hours, it was observed that almost all the cells expressed green fluorescent protein, indicating that the cells were successfully labeled with green fluorescent protein. Then, the lung stem cells expressing green fluorescent protein are resuspended in 30-70ul of phosphate buffer, and then infused into the immunodeficient mouse with damaged lung through the airway (bleomycin is infused into the respiratory tract of an NOD-SCID mouse through airway perfusion 8 days before cell transplantation to cause lung damage of the mouse, so as to obtain the immunodeficient mouse with damaged lung, wherein the NOD-SCID mouse is purchased from the lung of Shanghai Slek laboratory animals Co., Ltd.), the lung of the mouse is taken out after half a month, after being embedded by OCT, the mouse is sliced by a freezing microtome to carry out immunofluorescence staining, and the expression condition of fluorescence is photographed under a fluorescence microscope.
The results are shown in FIGS. 3-5. Specifically, the stem cells were transplanted into human lungs differentiated after transplantation into mouse lungs (see fig. 3, in which the signal is GFP signal), the transplanted stem cells expressed the bronchial secretory epithelium marker CC10 (see fig. 4, in which panel a is CC10 and panel B is GFP signal of the same region), and the transplanted stem cells expressed the alveolar epithelial marker Ager (see fig. 5, in which panel a is Ager signal and panel B is GFP signal of the same region).
As is clear from fig. 3 to 5, the stem cell characteristics of the lung stem cells were confirmed by differentiation of tissue structures such as human bronchi and alveoli in the lungs of immunodeficient mice after transplantation. Therefore, the stem cells obtained by the method can be used for clinical stem cell transplantation treatment. Wherein, the specific clinical treatment scheme design and ethical demonstration are strictly executed according to the related regulations of the health council.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
While embodiments of the invention have been shown and described, it will be understood by those of ordinary skill in the art that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.
Claims (8)
1. A method for separating and obtaining lung stem cells is characterized by comprising the following steps:
digesting a biological sample containing lung stem cells by using tissue digestive juice so as to obtain single cells; and
mixing the single cell with a lung stem cell separation culture solution, placing the mixture on an embryonic fibroblast subjected to proliferation inhibition treatment for incubation culture so as to obtain an expanded lung stem cell,
wherein the tissue digest comprises: 99% by volume PBS; 0.1-2U/ml DNase; 0.1-0.2mg/ml collagenase type I; 0.1-0.2mg/ml collagenase type IV; 0.04-0.1mg/ml dispase; 0.1-0.5mg/ml protease XIV; 0.1-3mg/ml streptothricin protease E; and 0.2-0.5mg/ml pancreatin,
the lung stem cell separation culture solution comprises: 90% by volume of RPMI1640 medium; 10% by volume fetal bovine serum; 1-3mmol/L L-glutamine; 0.05-0.15mmol/L beta-mercaptoethanol; 100-1000U/ml leukemia inhibitory factor; and 2-5ng/ml EGF growth factor,
the biological sample is lung tissue, including distal bronchial tissue.
2. The lung stem cell of claim 1, wherein the tissue digest comprises: 99% by volume PBS; 1U/ml DNase; 0.15mg/ml collagenase type I; 0.15mg/ml collagenase type IV; 0.07mg/ml dispase; 0.3mg/ml protease XIV; 2mg/ml streptothricin E; and 0.35mg/ml pancreatin.
3. The lung stem cell of claim 1, wherein the isolated culture medium comprises: 90% by volume of RPMI1640 medium; 10% by volume fetal bovine serum; 2mmol/L L-glutamine; 0.1mmol/L beta-mercaptoethanol; 500U/ml leukemia inhibitory factor; and 3.5ng/ml EGF growth factor.
4. The lung stem cell according to claim 1, wherein the proliferation-suppressing embryonic fibroblast is obtained by any one of the following methods:
(1) irradiating the embryonic fibroblasts for 1 to 5 hours with the irradiation dose of 30 to 80 Gy;
(2) treating the embryonic fibroblasts for 1-5 hours by using mitomycin C at a concentration of 1-30ug/ml,
optionally, the embryonic fibroblasts are derived from a mouse, preferably an E13.5 mouse,
optionally, the irradiation treatment is performed using a cell irradiator.
5. The lung stem cells according to claim 1, wherein the digestion treatment is performed at 36-38 degrees Celsius, preferably 37 degrees Celsius, for 1-5 hours,
optionally, in the digesting treatment, 1ml of tissue digest solution is added per biological sample for less than 0.1mg of the biological sample containing the lung stem cells; for not less than 0.1mg of the biological sample containing the lung stem cells, 2.5ml of tissue digestive juice is added per cubic centimeter of the biological sample.
6. The lung stem cell according to claim 1, wherein the mixing ratio of the single cell to the isolated culture medium of the lung stem cell is 300000 cells/2 ml of the isolated culture medium, wherein the number of the single cell is less than 300000 as calculated according to 300000,
optionally, 4% -6.5% CO at 36-38 deg.C2The incubation is carried out for 2 to 5 days.
7. The lung stem cell of claim 1, further comprising:
performing cell identification and contamination detection on the lung stem cells;
optionally, the contamination detection comprises a bacterial contamination detection and a mycoplasma contamination detection;
optionally, detecting the expression of Krt5 and P63 markers of the lung stem cells using immunofluorescence staining to effect the cell identification.
8. Use of the lung stem cells of any one of claims 1-7 in the manufacture of a stem cell medicament for the treatment of an acute or chronic lung disease.
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