CN114736867A - Lung and lung cancer tissue culture method and method for constructing lung cancer mouse animal model by using same - Google Patents
Lung and lung cancer tissue culture method and method for constructing lung cancer mouse animal model by using same Download PDFInfo
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Abstract
The invention belongs to the technical field of lung and lung cancer tissue culture, and discloses a lung and lung cancer tissue culture method and a method for constructing a lung cancer mouse animal model by using the same, wherein the lung and lung cancer tissue culture method comprises the following steps: acquiring and preprocessing human lung cancer tissues; constructing a lung cancer tissue organoid model; cloning and culturing human lung cancer tissue single cells; and constructing a lung cancer cell organoid library. According to the invention, a large number of in vitro culture cells which are highly consistent with the genetic angle of the source organ can be successfully cultured by the lung and lung cancer tissue culture method, and can be stored for a long time, the culture effect is good, a favorable tool is provided for the research on the development of lung tissue organs and lung cancer, the development of human can be deeply known, and a new visual angle is provided for establishing disease modeling, drug discovery and regenerative medicine; meanwhile, the compound can be differentiated to form adult and in-situ lung tumors after in-situ injection, is very close to the occurrence situation of lung cancer in a human body, and can be better applied to lung cancer research work.
Description
Technical Field
The invention belongs to the technical field of lung and lung cancer tissue culture, and particularly relates to a lung and lung cancer tissue culture method and a method for constructing a lung cancer mouse animal model by using the same.
Background
At present, the clinical manifestations of lung cancer are complex, and the presence, severity and early and late appearance of symptoms and signs depend on the site of tumorigenesis, the type of pathology, the presence or absence of metastasis and complications, and the differences in the degree of response and tolerance of the patients. Early lung cancer symptoms are often mild and may even be without any discomfort. Central lung cancer symptoms appear early and severe, while peripheral lung cancer symptoms appear late and mild, even asymptomatic, and are often found during physical examination. The symptoms of lung cancer are roughly classified into: local symptoms, systemic symptoms, extrapulmonary symptoms, infiltrates and metastases. However, the existing lung and lung cancer tissue culture methods and methods for constructing a lung cancer mouse animal model by using the lung and lung cancer tissue culture methods have poor in-vitro culture effects on the obtained lung tissues and lung cancer tissues; meanwhile, the distance of the research result of the mouse tumor genome is larger for directly guiding the human tumor genomics research; the cell line transplantation tumor model is limited to the difference between the cell line and the human tumor cells in terms of gene and biological functions; the PDX model (human-Derived tumor xenograft model) is established by directly transplanting tumor tissues of patients to immunodeficient mice, retains the characteristics of most primary tumors on the aspects of histopathology, molecular biology and gene level, and has better clinical efficacy predictability. Although the defects of the two models are partially compensated, and the two models are also the current popular main methods for researching biological characteristics of the human tumor or screening and identifying drugs, the traditional PDX model still has limitations, and due to subcutaneous transplantation, the traditional PDX model cannot provide a lung tissue in-situ microenvironment, so that the relevant biological characteristics of the human tumor are possibly lost in the experimental process, and the condition in a human body cannot be simulated.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) the existing lung and lung cancer tissue culture method and the method for constructing a lung cancer mouse animal model by using the lung and lung cancer tissue culture method have poor in-vitro culture effect on the obtained lung tissue and lung cancer tissue; meanwhile, the distance of the research result of the mouse tumor genome is larger for directly guiding the human tumor genomics research; the cell line transplantation tumor model is limited to the difference between the cell line and the human tumor cell in terms of gene and biological functions.
(2) Although the PDX model partially remedies the defects of the two models, and is a current popular main method for researching biological characteristics of the human tumor or screening and identifying drugs, the PDX model still has limitations, and due to subcutaneous transplantation, the conventional PDX model cannot provide a lung tissue in-situ microenvironment, so that the relevant biological characteristics of the human tumor are possibly lost in the experimental process, and the condition in the human body cannot be simulated.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a lung and lung cancer tissue culture method and a method for constructing a lung cancer mouse animal model by using the same.
The invention is realized in such a way, and the lung and lung cancer tissue culture method comprises the following steps:
step one, acquiring and preprocessing human lung cancer tissues: obtaining a fresh human lung cancer tissue; shearing on ice or in the same low-temperature environment or mechanically crushing in the same low-temperature environment to obtain human lung cancer tissue blocks; utilizing a protease dissolving solution to crack the human lung cancer tissue block into human lung cancer tissue single cells;
step two, constructing a lung cancer tissue organoid model: uniformly mixing the human lung cancer tissue single cells with matrigel, placing the mixture in an incubator for solidification and three-dimensional culture, and establishing a lung cancer tissue organoid model;
step three, carrying out clone culture of human lung cancer tissue single cells: carrying out clone culture on the human lung cancer tissue single cell by adopting a clone-promoting culture medium and matrigel to obtain a clone culture product; taking out the clonal cells in the clonal culture product to perform amplification culture, and establishing a lung cancer information database;
step four, constructing a lung cancer cell organoid library: freezing and storing the lung cancer cell organoid, establishing a lung cancer cell organoid library, recovering the lung cancer cell organoid and re-culturing.
Further, the human lung cancer tissues in the first step are transported in a low-temperature storage mode.
Further, the step one of lysing the human lung cancer tissue block into the human lung cancer tissue single cells by using a protease lysis solution comprises:
(1) cleaning the human lung cancer tissue;
(2) cutting up the human lung cancer tissue, mixing the cut-up human lung cancer tissue with the protease dissolving solution, culturing the cut-up human lung cancer tissue in an incubator, and filtering the cut-up human lung cancer tissue through a 70 mu m cell filter to obtain a single cell suspension;
(3) centrifuging the single cell suspension to remove supernatant, and collecting single cell mixed solution; wherein the single cell comprises a normal human lung cell and a human lung cancer cell.
Further, the centrifugation temperature is 2-8 ℃, and 200-400 g of single cell suspension is centrifuged for 10-15 min.
Further, the construction of the lung cancer tissue organoid model in the second step includes:
(1) uniformly mixing the single cell mixed solution with matrigel to obtain mixed cell sap;
(2) pouring the mixed cell sap into a culture plate hole, and placing the culture plate hole in an incubator to be solidified to obtain a three-dimensional structure;
(3) and adding the culture plate hole into a lung tissue organ condition culture medium, and putting the culture plate hole into the carbon dioxide incubator again for culture to construct and obtain the lung cancer tissue organoid model.
Further, the temperature for culturing in the carbon dioxide incubator is 35-37 ℃, and the culturing time is 24-36 h.
Further, the establishment of the lung cancer information database in the third step comprises:
(1) performing first amplification culture on the cloned cells by using an amplification culture medium and matrigel to obtain an amplification culture product;
(2) carrying out first separation treatment on the amplification culture product to obtain an amplification cell;
(3) and performing second amplification culture on the amplified cells by adopting the amplification culture medium, and establishing a lung cancer information database.
Further, the cryopreserving the lung cancer cell organoids and establishing a lung cancer cell organoid bank in step four comprises:
removing the lung tissue organ culture medium and flushing the single cells, centrifuging to remove supernatant, and collecting cell frozen stock solution; pouring the cell frozen stock solution into a freezing tube after resuspension; and (4) placing the freezing tube into a program cooling box for storage for a period of time, and transferring the freezing tube into liquid nitrogen for long-term storage.
Further, the freezing storage tube is placed into a program cooling box to be stored for 24 hours at the temperature of minus 80 ℃.
The invention also aims to provide a method for constructing a lung cancer mouse animal model by applying the lung and lung cancer tissue culture method, wherein the method for constructing the lung cancer mouse animal model comprises the following steps:
(1) obtaining a fresh human lung cancer tissue, digesting collagenase into single cells, mixing the single cells with matrigel, and culturing in a 48-pore plate by using a conditioned medium to obtain a human lung cancer tissue organoid;
(2) injecting human lung cancer tissue organoid into immunodeficient mouse lung tissue in situ to obtain humanized mouse animal model of in situ lung cancer;
(3) the animal model was constructed by injecting human lung cancer tissue into immunodeficient mouse lung tissue.
By combining all the technical schemes, the invention has the advantages and positive effects that: according to the invention, a large number of in vitro culture cells with genetic angle highly consistent with that of the source organ can be successfully cultured by the lung and lung cancer tissue culture method, and can be stored for a long time, the culture effect is good, a favorable tool is provided for research on development of lung tissue organs and lung cancer, the development of human can be deeply known, and a new visual angle is provided for establishing disease modeling, drug discovery and regenerative medicine; meanwhile, a large number of lung cancer organoids with high consistency cultured by the method for constructing the lung cancer mouse animal model based on the in-vitro 3D culture technology are injected in situ, the lung cancer tissue cells cultured by the in-vitro 3D culture technology have high activity, can be differentiated to form adult and in-situ lung tumors after in-situ injection, are very close to the occurrence situation of the lung cancer in a human body, and can be better applied to the lung cancer research work.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a flow chart of a lung and lung cancer tissue culture method according to the present invention.
FIG. 2 is a flowchart of a method for lysing human lung cancer tissue blocks into human lung cancer tissue single cells by using a protease lysis solution according to an embodiment of the present invention.
FIG. 3 is a flow chart of a method for constructing a lung cancer tissue organoid model according to an embodiment of the present invention.
Fig. 4 is a flowchart of a method for creating a lung cancer information database according to an embodiment of the present invention.
FIG. 5 is a flow chart of a method for constructing a lung cancer mouse animal model according to embodiments of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a lung and lung cancer tissue culture method and a method for constructing a lung cancer mouse animal model by using the same, and the invention is described in detail by combining the attached drawings.
As shown in fig. 1, the lung and lung cancer tissue culture method provided by the embodiment of the present invention includes the following steps:
s101, acquiring and preprocessing human lung cancer tissues: obtaining a fresh human lung cancer tissue; shearing on ice or in the same low-temperature environment or mechanically crushing in the same low-temperature environment to obtain human lung cancer tissue blocks; utilizing a protease dissolving solution to crack the human lung cancer tissue block into human lung cancer tissue single cells;
s102, constructing a lung cancer tissue organoid model: uniformly mixing the human lung cancer tissue single cells with matrigel, placing the mixture in an incubator for solidification and three-dimensional culture, and establishing a lung cancer tissue organoid model;
s103, carrying out cloning culture on the human lung cancer tissue single cells: carrying out clone culture on the human lung cancer tissue single cell by adopting a clone-promoting culture medium and matrigel to obtain a clone culture product; taking out the clone cells in the clone culture product for amplification culture, and establishing a lung cancer information database;
s104, constructing a lung cancer cell organoid bank: freezing the lung cancer cell organoids, establishing a lung cancer cell organoid library, recovering the lung cancer cell organoids and re-culturing.
In the step S101 provided by the embodiment of the present invention, the human lung cancer tissue is transported by low-temperature storage.
As shown in fig. 2, the step S101 of cleaving the human lung cancer tissue block into the human lung cancer tissue single cell by using the protease dissolving solution according to the embodiment of the present invention includes:
s201, cleaning the human lung cancer tissue;
s202, cutting up the human lung cancer tissues, mixing the cut-up human lung cancer tissues with the protease dissolving solution, culturing the mixture in an incubator, and filtering the mixture through a 70-micron cell filter to obtain a single-cell suspension;
s203, centrifuging the single cell suspension to remove supernatant, and collecting single cell mixed liquor; wherein the single cell comprises a normal human lung cell and a human lung cancer cell.
The centrifugation temperature provided by the embodiment of the invention is 2-8 ℃, and 200-400 g of single cell suspension is centrifuged for 10-15 min.
As shown in fig. 3, the constructing of the lung cancer tissue organoid model in step S102 according to the embodiment of the present invention includes:
s301, uniformly mixing the single cell mixed solution with matrigel to obtain mixed cell sap;
s302, pouring the mixed cell sap into a culture plate hole, placing the mixed cell sap into an incubator, and solidifying the mixed cell sap to obtain a three-dimensional structure;
s303, adding the culture plate hole into a lung tissue organ conditioned medium, and putting the culture plate hole into the carbon dioxide incubator again for culture to construct and obtain the lung cancer tissue organoid model.
The temperature for culturing in the carbon dioxide incubator provided by the embodiment of the invention is 35-37 ℃, and the culture time is 24-36 h.
As shown in fig. 4, the establishment of the lung cancer information database of step S103 according to the embodiment of the present invention includes:
s401, performing first amplification culture on the cloned cells by adopting an amplification culture medium and matrigel to obtain an amplification culture product;
s402, carrying out first separation treatment on the amplification culture product to obtain an amplification cell;
and S403, performing second amplification culture on the amplified cells by using the amplification culture medium, and establishing a lung cancer information database.
The freezing and storing the lung cancer cell organoid and establishing the lung cancer cell organoid library in step S104 provided by the embodiment of the present invention includes:
removing the lung tissue organ culture medium and flushing the single cells, centrifuging to remove supernatant, and collecting cell frozen stock solution; pouring the cell frozen stock solution into a freezing tube after resuspension; and (4) placing the freezing tube into a program cooling box for storage for a period of time, and transferring the freezing tube into liquid nitrogen for long-term storage.
The freezing storage tube provided by the embodiment of the invention is placed into a program cooling box to be stored for 24 hours at the temperature of minus 80 ℃.
As shown in fig. 5, the method for constructing a mouse animal model of lung cancer provided by the embodiment of the present invention includes:
s501, obtaining a fresh human lung cancer tissue, digesting collagenase into single cells, mixing the single cells with matrigel, and culturing the mixture in a 48-hole plate by using a conditioned medium to obtain a human lung cancer tissue organoid;
s502, injecting human lung cancer tissue organoids into immunodeficient mouse lung tissues in situ to obtain a humanized mouse animal model of in situ lung cancer;
s503, injecting the human lung cancer tissue into the lung tissue of the immunodeficient mouse through injection to construct an animal model.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A lung and lung cancer tissue culture method is characterized by comprising the following steps:
step one, acquiring and preprocessing human lung cancer tissues: obtaining fresh human lung cancer tissue (generally lung puncture or surgical specimen); shearing on ice or in the same low-temperature environment or mechanically crushing in the same low-temperature environment to obtain human lung cancer tissue blocks; utilizing a protease dissolving solution to crack the human lung cancer tissue blocks into human lung cancer tissue single cells;
step two, constructing a lung cancer tissue organoid model: uniformly mixing the human lung cancer tissue single cells with matrigel, placing the mixture in an incubator for solidification and three-dimensional culture, and establishing a lung cancer tissue organoid model;
step three, carrying out clone culture of human lung cancer tissue single cells: carrying out clone culture on the human lung cancer tissue single cell by adopting a clone-promoting culture medium and matrigel to obtain a clone culture product; taking out the clone cells in the clone culture product for amplification culture, and establishing a lung cancer information database;
step four, constructing a lung cancer cell organoid library: freezing and storing the lung cancer cell organoid, establishing a lung cancer cell organoid library, recovering the lung cancer cell organoid and re-culturing.
2. The method for culturing lung and lung cancer tissue according to claim 1, wherein the human lung cancer tissue in the first step is transported by low-temperature storage.
3. The lung and lung cancer tissue culture method according to claim 1, wherein the step one of lysing the human lung cancer tissue mass into single human lung cancer tissue cells using a protease lysis solution comprises:
(1) washing the human lung cancer tissue;
(2) cutting up the human lung cancer tissue, mixing the cut-up human lung cancer tissue with the protease dissolving solution, culturing the cut-up human lung cancer tissue in an incubator, and filtering the cut-up human lung cancer tissue through a 70 mu m cell filter to obtain a single cell suspension;
(3) centrifuging the single cell suspension to remove supernatant and collecting single cell mixed solution; wherein the single cell comprises a normal human lung cell and a human lung cancer cell.
4. The method for tissue culture of lung and lung cancer according to claim 3, wherein the centrifugation temperature is 2-8 ℃ and 200-400 g of single cell suspension is centrifuged for 10-15 min.
5. The lung and lung cancer tissue culture method according to claim 1, wherein the constructing of the lung cancer tissue organoid model in the second step comprises:
(1) uniformly mixing the single cell mixed solution with matrigel to obtain mixed cell sap;
(2) pouring the mixed cell sap into a culture plate hole, placing the culture plate hole in an incubator, and curing to obtain a three-dimensional structure;
(3) and adding the culture plate hole into a lung tissue organ conditioned medium, and putting the culture plate hole into the carbon dioxide incubator again for culture to construct and obtain a lung cancer tissue organoid model.
6. The method for culturing lung and lung cancer tissues according to claim 5, wherein the temperature for culturing in the carbon dioxide incubator is 35-37 ℃, and the culturing time is 24-36 h.
7. The lung and lung cancer tissue culture method according to claim 1, wherein the establishment of the lung cancer information database in the third step comprises:
(1) performing first amplification culture on the cloned cells by using an amplification culture medium and matrigel to obtain an amplification culture product;
(2) carrying out first separation treatment on the amplification culture product to obtain an amplification cell;
(3) and performing second amplification culture on the amplified cells by adopting the amplification culture medium, and establishing a lung cancer information database.
8. The lung and lung cancer tissue culture method of claim 1, wherein the cryopreserving the lung cancer cell organoids and establishing a lung cancer cell organoid bank in step four comprises:
removing the lung tissue organ culture medium and flushing the single cells, centrifuging to remove supernatant, and collecting cell frozen stock solution; pouring the cell freezing solution into a freezing tube after resuspension; and (4) placing the freezing tube into a program cooling box for storage for a period of time, and transferring the freezing tube into liquid nitrogen for long-term storage.
9. The lung and lung cancer tissue culture method according to claim 8, wherein the freezing tube is placed in a programmed cooling box and stored for 24h at-80 ℃.
10. A method for constructing a mouse animal model of lung cancer by applying the lung and lung cancer tissue culture method of any one of claims 1 to 9, wherein the method for constructing the mouse animal model of lung cancer comprises the following steps:
(1) obtaining a fresh human lung cancer tissue, digesting collagenase into single cells, mixing with matrigel, and culturing in a 48-pore plate by using a conditioned medium to obtain a human lung cancer tissue organoid;
(2) injecting human lung cancer tissue organoid into immunodeficient mouse lung tissue in situ to obtain humanized mouse animal model of in situ lung cancer;
(3) the animal model was constructed by injecting human lung cancer tissue into immunodeficient mouse lung tissue.
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