CN112080471B - Cisplatin-resistant lung cancer organoid and culture method, culture medium and application thereof - Google Patents
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Abstract
The invention relates to a method for culturing cisplatin-resistant lung cancer organoids, which comprises the steps of mixing cisplatin-resistant lung cancer histiocyte with a culture medium and matrigel to obtain a to-be-cultured substance, wherein the culture medium contains matrix metalloproteinase-10, and the concentration of the matrix metalloproteinase-10 is 50-200nM by taking the culture medium as a reference; and culturing and amplifying the culture to be cultured to obtain the cisplatin-resistant lung cancer organoid. In the culture method of the cisplatin-resistant lung cancer organoid provided by the disclosure, the adopted culture medium contains the matrix metalloproteinase-10 with the specific concentration, and the matrix metalloproteinase-10 with the specific concentration can promote the in-vitro growth of the cisplatin-resistant lung cancer organoid, so that the culture success rate is higher when the method provided by the disclosure is used for culturing the cisplatin-resistant lung cancer organoid.
Description
Technical Field
The disclosure relates to the technical field of biomedicine, in particular to a method for culturing cisplatin-resistant lung cancer organoids, a culture medium for culturing the cisplatin-resistant lung cancer organoids, the cisplatin-resistant lung cancer organoids and application of the cisplatin-resistant lung cancer organoids in drug screening.
Background
Cisplatin, also known as cis-bis-chloro-diammineplatinum, is an anticancer chemotherapeutic drug widely used in clinic, has a broad spectrum of antitumor properties, and has a good treatment effect in the treatment of tumor diseases such as lung cancer, testicular cancer, ovarian cancer, breast cancer, gastric cancer, bladder cancer and the like since the advent. However, in the process of chemotherapy by using cisplatin, cisplatin resistance phenomenon is generated, which easily causes chemotherapy failure, so that the use of cisplatin in tumor chemotherapy is greatly limited.
Lung cancer is a common malignant tumor worldwide, and the death rate of the lung cancer accounts for about 18.4% of all tumor deaths. The molecular mechanism of cisplatin resistance of lung cancer is complex, and is mainly related to drug resistance related gene change, cell detoxification and DNA damage repair gene change, chromosome change, apoptosis related gene change, cytoskeleton, angiogenesis and extracellular matrix density abnormality. Establishing cisplatin-resistant lung cancer biological model in vitro is the basis and precondition for researching lung cancer cisplatin-resistant mechanism, developing targeted drugs and searching chemosensitizer.
In the related art, cisplatin-resistant lung cancer cell lines are generally adopted as in-vitro cisplatin-resistant lung cancer biological models. At present, the common methods for establishing tumor drug-resistant cell strains mainly comprise a drug concentration increasing continuous action method and a large-dose drug intermittent induction method. The continuous action method with increasing drug concentration usually needs 6 months or more to induce drug resistance, takes longer time, and has obvious difference between the action mode of the chemotherapeutic drug and the chemotherapeutic principle of large dose and short course of treatment of the clinical chemotherapeutic drug. The large-dose drug intermittent induction method has high drug concentration during drug resistance induction, cells are difficult to tolerate the culture environment with sudden change, the induction success rate is low, the cell state is difficult to control, and the drug resistance performance is unstable. In addition, the drug-resistant cell strains induced by the two methods have obvious difference in cell morphology and biological characteristics and belong to different cell sublines, so that data obtained by drug screening in the two drug-resistant cell strains are different.
The establishment of a cisplatin-resistant lung cancer biological model which is more similar to the lung cancer primary tissue in a cisplatin-resistant lung cancer patient has important clinical application value for searching and developing a cisplatin-resistant sensitizer or a cisplatin-resistant reversal agent.
Disclosure of Invention
The invention aims to provide a method for culturing cisplatin-resistant lung cancer organoids, a culture medium for culturing the cisplatin-resistant lung cancer organoids, the cisplatin-resistant lung cancer organoids and application of the cisplatin-resistant lung cancer organoids in drug screening.
In order to achieve the above object, in a first aspect, the present disclosure provides a method for culturing cisplatin-resistant lung cancer organoids, comprising:
mixing the cisplatin-resistant lung cancer tissue cells with a culture medium and matrigel to obtain a to-be-cultured substance, wherein the culture medium contains matrix metalloproteinase-10, and the concentration of the matrix metalloproteinase-10 is 50-200nM on the basis of the culture medium;
and culturing and amplifying the culture to be cultured to obtain the cisplatin-resistant lung cancer organoid.
Optionally, the concentration of the cisplatin-resistant lung cancer tissue cells in the culture is 1 × 104～5×104Per mL, the matrixThe content of the gum is 1-20 vol%.
Optionally, the culture medium further comprises at least one of Advanced DMEM/F12 medium, FBS, streptomycin, Noggin, FGF7, B27, Nicotinamide, and EGF.
Optionally, based on the culture medium, the FBS content is 5-10 vol%, the streptomycin concentration is 50-100 μ g/ml, the Noggin concentration is 50-150 ng/ml, the FGF7 concentration is 1-10 ng/ml, the B27 content is 1-5 vol%, the Nicotinamide concentration is 1-10 mM, the EGF concentration is 1-10 ng/ml, and the balance is the Advanced DMEM/F12 culture medium.
Optionally, the cisplatin-resistant lung cancer tissue cells are obtained by performing enzymolysis on cisplatin-resistant lung cancer primary tissues, wherein the enzymolysis includes:
mixing the cisplatin-resistant lung cancer primary tissue with collagenase I to obtain a first premix;
cutting the cisplatin-resistant lung cancer primary tissues in the first premix into minced shapes to obtain a second premix;
mixing the second premix with an enzymolysis liquid to obtain a to-be-enzymolyzed substance, wherein the enzymolysis liquid contains cisplatin, and the concentration of the cisplatin is 1-100 mu M by taking the enzymolysis liquid as a reference;
culturing and carrying out enzymolysis on the to-be-enzymolyzed substance for 1-2 hours to obtain an enzymolysis product;
and separating the enzymolysis product to obtain the cisplatin-resistant lung cancer tissue cell.
Optionally, the enzymolysis solution further contains collagenase type i, wherein the concentration of the collagenase type i is 1mg/mL to 5mg/mL based on the enzymolysis solution.
In a second aspect, the present disclosure provides a culture medium for culturing cisplatin-resistant lung cancer organoids, wherein the culture medium contains matrix metalloproteinase-10, and the concentration of the matrix metalloproteinase-10 is 50nM to 200nM based on the culture medium;
preferably, the culture medium further comprises at least one of Advanced DMEM/F12 culture medium, FBS, streptomycin, Noggin, FGF7, B27, Nicotinamide and EGF; the medium is taken as a reference, the FBS content is 5-10 vol%, the streptomycin concentration is 50-100 mu g/ml, the Noggin concentration is 50-150 ng/ml, the FGF7 concentration is 1-10 ng/ml, the B27 content is 1-5 vol%, the Nicotinamide concentration is 1-10 mM, the EGF concentration is 1-10 ng/ml, and the balance is the Advanced DMEM/F12 medium.
In a third aspect, the present disclosure provides a cisplatin-resistant lung cancer organoid cultured by the method of any of the first aspect.
Optionally, in the cisplatin-resistant lung cancer organoid, the content of P-glycoprotein accounts for 10-80% of the total content of membrane protein, and the content of multidrug-resistant associated protein 1 accounts for 10-40% of the total content of membrane protein.
In a fourth aspect, the present disclosure provides a use of the cisplatin-resistant lung cancer organoid of the third aspect in drug screening.
According to the technical scheme, in the culture method of the cisplatin-resistant lung cancer organoid provided by the disclosure, as the adopted culture medium contains the matrix metalloproteinase-10 with the specific concentration, and the matrix metalloproteinase-10 with the specific concentration can promote the in-vitro growth of the cisplatin-resistant lung cancer organoid, the culture success rate is high when the method provided by the disclosure is used for culturing the cisplatin-resistant lung cancer organoid.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
A first aspect of the disclosure provides a method of culturing a cisplatin-resistant lung cancer organoid, the method comprising: mixing the cisplatin-resistant lung cancer tissue cells with a culture medium and matrigel to obtain a to-be-cultured substance, wherein the culture medium contains matrix metalloproteinase-10, and the concentration of the matrix metalloproteinase-10 is 50-200nM on the basis of the culture medium; and culturing and amplifying the culture to be cultured to obtain the cisplatin-resistant lung cancer organoid.
In the disclosed embodiments, the matrigel may be, for example, a low growth factor matrigel and the matrix metalloproteinase-10 (MMP-10) may be recombinant human matrix metalloproteinase-10, in particular. When the cisplatin-resistant lung cancer tissue cells are mixed with the culture medium and the matrigel, the cisplatin-resistant lung cancer tissue cells can be mixed with the culture medium firstly, and then the matrigel is added.
When a culture to be cultured is subjected to culture amplification to obtain the cisplatin-resistant lung cancer organoid, the culture to be cultured can be placed in culture holes of a culture plate for culture, and 200-300 μ L of the culture to be cultured is added into each culture hole. In the culture process, the culture plate can be placed in an incubator at 7 ℃ and 5% carbon dioxide for culture for 0.5-2 h, then 200-300 mu L of culture medium is added into each culture hole, and then the culture medium is replaced every 3-5 days until the diameter of the culture in the culture hole is larger than 0.2mm, so that the primary cisplatin-resistant lung cancer organs are obtained. In addition, the primary cisplatin-resistant lung cancer organoids can be subcultured until the cisplatin-resistant lung cancer organoids with the diameter larger than 0.2mm are obtained.
The inventor of the present disclosure finds that matrix metalloproteinase-10 with a concentration of 50-200nM can promote the in vitro growth of cisplatin-resistant lung cancer organoids, which can effectively improve the success rate of in vitro culture of cisplatin-resistant lung cancer organoids, and based on the discovery, the present disclosure is provided.
According to the technical scheme, in the culture method of the cisplatin-resistant lung cancer organoid provided by the disclosure, as the adopted culture medium contains the matrix metalloproteinase-10 with the specific concentration, and the matrix metalloproteinase-10 with the specific concentration can promote the in-vitro growth of the cisplatin-resistant lung cancer organoid, the culture success rate is high when the method provided by the disclosure is used for culturing the cisplatin-resistant lung cancer organoid.
According to the disclosure, the concentration of the cisplatin-resistant lung cancer tissue cells in the cultureMay be 1 × 104～5×104The content of the matrigel in the gel is 1-20 vol%. The cell concentration in the culture can be detected by the prior art and will not be described herein.
In light of the present disclosure, other components of the culture medium can be selected within a wide range, and components that favor the growth of cisplatin-resistant lung cancer tissue cells can all be used in the present disclosure. Illustratively, the medium may further comprise at least one of Advanced DMEM/F12 medium, FBS, streptomycin, Noggin, FGF7, B27, Nicotinamide, and EGF.
Optionally, based on the culture medium, the FBS can be 5-10 vol%, the streptomycin can be 50-100 μ g/ml, Noggin can be 50-150 ng/ml, FGF7 can be 1-10 ng/ml, B27 can be 1-5 vol%, Nicotinamide can be 1-10 mM, EGF can be 1-10 ng/ml, and the balance is the Advanced DMEM/F12 culture medium.
The penicillin streptomycin is a mixed solution of penicillin and streptomycin, wherein the content of the penicillin and the streptomycin in the penicillin streptomycin can be selected within a certain range, for example, the content of the penicillin can be 100-150U/ml, and the content of the streptomycin can be 0.1-0.5 mg/ml.
According to the present disclosure, the cisplatin-resistant lung cancer tissue cells are obtained by subjecting cisplatin-resistant lung cancer primary tissues to enzymatic hydrolysis, wherein the enzymatic hydrolysis includes: mixing the cisplatin-resistant lung cancer primary tissue with collagenase I to obtain a first premix; cutting the cisplatin-resistant lung cancer primary tissues in the first premix into minced shapes to obtain a second premix; mixing the second premix with an enzymolysis liquid to obtain a to-be-enzymolyzed substance, wherein the enzymolysis liquid contains cisplatin, and the concentration of the cisplatin can be 1-100 mu M by taking the enzymolysis liquid as a reference; culturing and carrying out enzymolysis on the to-be-enzymolyzed substance for 1-2 hours to obtain an enzymolysis product; and separating the enzymolysis product to obtain the cisplatin-resistant lung cancer tissue cell.
In the embodiment of the disclosure, specifically, the cisplatin-resistant lung cancer tissue cells are obtained by performing enzymolysis on cisplatin-resistant lung cancer primary tissues, wherein the cisplatin-resistant lung cancer tissue cells contain mesenchymal cells such as adult tissue stem cells and pluripotent stem cells in addition to cisplatin-resistant lung cancer cells/stem cells, so that the cisplatin-resistant lung cancer organs obtained by culturing the cisplatin-resistant lung cancer tissue cells can better retain heterogeneity, tissue characteristics and gene mutation information of the cisplatin-resistant lung cancer tissues, can simulate the spatial morphological structure of the cisplatin-resistant lung cancer tissues in vitro, have biological characteristics similar to those of the cisplatin-resistant lung cancer primary tissues, can be used for constructing an individual precise disease model of a cisplatin-resistant lung cancer patient, and can be used for researching and screening disease treatment schemes and anticancer drugs.
Optionally, the enzymolysis solution may further contain collagenase type i, wherein the concentration of the collagenase type i is 1mg/mL to 5mg/mL based on the enzymolysis solution.
The second aspect of the disclosure provides a culture medium for culturing cisplatin-resistant lung cancer organoids, wherein the culture medium contains matrix metalloproteinase-10, and the concentration of the matrix metalloproteinase-10 is 50-200nM based on the culture medium; preferably, the culture medium further comprises at least one of Advanced DMEM/F12 culture medium, FBS, streptomycin, Noggin, FGF7, B27, Nicotinamide and EGF; based on the culture medium, the FBS content can be 5-10 vol%, the streptomycin concentration can be 50-100 mu g/ml, Noggin concentration can be 50-150 ng/ml, FGF7 concentration can be 1-10 ng/ml, B27 content can be 1-5 vol%, Nicotinamide concentration can be 1-10 mM, EGF concentration can be 1-10 ng/ml, and the balance is the Advanced DMEM/F12 culture medium.
By utilizing the culture medium provided by the disclosure, the success rate of in-vitro culture of cisplatin-resistant lung cancer organoids can be effectively improved.
In a third aspect of the present disclosure, there is provided a cisplatin-resistant lung cancer organoid cultured by the method of any of the first aspect.
Optionally, in the cisplatin-resistant lung cancer organoid, the content of P-glycoprotein accounts for 10-80% of the total content of membrane protein, and the content of multidrug-resistant associated protein 1 accounts for 10-40% of the total content of membrane protein.
In a fourth aspect of the present disclosure, there is provided a use of the cisplatin-resistant lung cancer organoid of the third aspect in drug screening.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The starting materials, reagents, instruments and equipment referred to in the examples of the present disclosure may be obtained by purchase, unless otherwise specified.
Where specific experimental temperatures are not noted in the examples of the present disclosure, the experimental temperatures are all room temperature (20-25 ℃).
The culture medium used in the embodiment of the disclosure is a culture medium for culturing cisplatin-resistant lung cancer organoids, and comprises: advanced DMEM/F12 medium, 10 vol% FBS, 100 mug/mL streptomycin (penicillin content can be 100U/mL, streptomycin content can be 0.1mg/mL), 100ng/mL Noggin, 5ng/mL FGF7, 2% B27, 5mM Nicotinamide, 5ng/mL EGF, and 50-200nM recombinant human matrix metalloproteinase-10 (MMP-10).
The sources of reagents used in the examples of the disclosure are as follows:
Example 1
This example illustrates the culture of cisplatin-resistant lung cancer organoids.
(1) Storage and transportation of cisplatin-resistant lung cancer tissue samples
And (3) obtaining a bronchoscopy lung biopsy of a clinically confirmed cisplatin-resistant lung cancer patient as a cisplatin-resistant lung cancer tissue sample. Cisplatin-resistant lung cancer tissue sample is placed in a sample container
In the preservation solution of RS-I Medium, and is transported to the operation room in a 4-degree cold chain within 48 hours.
(2) Acquisition of cisplatin-resistant lung cancer tissue cells
Placing the cisplatin-resistant lung cancer tissue sample in a sterile culture dish, removing blood clots and necrotic tissues, flushing for 3 times by using sterile PBS, absorbing the PBS, adding 1.5mg/mL type I collagenase, shearing the cisplatin-resistant lung cancer tissue sample into a minced shape by using a sterile instrument, adding an enzymolysis solution containing cisplatin (containing 26 mu M cisplatin and 1.5mg/mL type I collagenase), placing the culture dish in a shaking table with the rotating speed of 60rpm after adding liquid, and performing enzymolysis for 2 hours in a 37 ℃ culture box. Wherein, 0.1mL of enzymolysis solution is correspondingly added into each 1mg of cisplatin-resistant lung cancer tissue sample.
And after enzymolysis is finished, collecting enzymolysis liquid, filtering the collected enzymolysis liquid by using a 100-micron cell sieve, and collecting filtrate to obtain cell suspension. The resulting cell suspension was centrifuged at 1500rpm for 5min and then rinsed 3 times with PBS. And after rinsing, centrifuging for 3min under the condition of centrifugation of 300g, discarding the supernatant, and collecting cell sediment to obtain the cisplatin-resistant lung cancer tissue cells. Wherein, the cisplatin-resistant lung cancer tissue cells can be preserved in an Advanced DMEM/F12 culture medium.
(3) Culture of cisplatin-resistant lung cancer organoids
Mixing the cisplatin-resistant lung cancer tissue cells obtained in the step (2) with a culture medium (MMP-10 concentration: 100nM) so that the concentration of the cisplatin-resistant lung cancer tissue cells is 3X 104Adding 2 Xlow growth factor matrigel with the same volume per mL to obtain the final productAnd (5) nourishing the food. The above-mentioned culture to be cultured was added to the center of the culture well in a 24-well plate in an amount of 250. mu.L per well, and then the plate was incubated at 37 ℃ in an incubator containing 5% carbon dioxide for 1 hour, after which 250. mu.L of a culture medium was added to each well of the 24-well plate and the culture was continued. During the culture, the newly prepared culture medium was replaced every 3 days.
Observing under microscope, when the diameter of culture in single culture well is larger than 0.2mm, absorbing the original culture solution, adding 500 μ L TrypLE Express into each well of 24-well plate, performing enzymolysis for 1min, and collecting the enzymolysis solution. Centrifuging the collected enzymolysis solution for 4min under 400rcf, removing supernatant, collecting cell precipitate, adding culture medium containing 5% low growth factor matrigel into the collected cell precipitate to make the concentration of cisplatin-resistant lung cancer tissue cell be 3 × 104And (4) obtaining the culture to be cultured. The culture to be cultured is inoculated into a 24-well plate and is subjected to subculture expansion, and the condition and operation of subculture are similar to those of the culture process. And when the diameter of the subculture is not less than 0.2mm, removing the culture solution to obtain the cisplatin-resistant lung cancer organoid.
Example 2
This example illustrates the use of cisplatin-resistant lung cancer organoids in drug screening.
The cisplatin-resistant lung cancer organoid obtained in example 1 is subjected to enzymolysis for 1min by TrypLE Express, and then enzymolysis liquid is collected and centrifuged for 4min under the condition of 400 rcf. And after the centrifugation is finished, collecting the precipitate, and adding a culture medium containing 10% of low-growth-factor matrigel into the collected precipitate to obtain the to-be-cultured substance. Sucking the culture at 6X 103The inoculum size per well was inoculated into a low adsorption 96-well plate, and then the 96-well plate was incubated at 37 ℃ in an incubator with 5% carbon dioxide for 1 hour, after which 40. mu.L of the medium for culture was added to each well of the 96-well plate and the incubation was continued.
When the diameter of the culture in a single culture well is greater than 0.1mm and the degree of fusion of the culture reaches 50%, a test amount of the drug to be screened is added to each well of a 96-well plate. After 48 hours of drug action, the stock culture in each well was replaced with 180. mu.L of culture medium, 10. mu.L of 5mg/ml MTT solution was added to each well, and the culture was continued at 37 ℃ for 4 hours. And then removing the original culture solution, adding 150 mu l of DMSO into each hole, incubating at room temperature for 10min, detecting the absorbance value of the sample under the condition of 570nm by using an enzyme-labeling instrument after the crystal violet is completely dissolved, and judging the effect of the drug to be screened on the cisplatin-resistant lung cancer organoid cells according to the absorbance value.
Example 3
Cisplatin-resistant lung cancer organoids were cultured according to the method of example 1, except that: the concentration of MMP-10 in the culture medium used in this example was 50 nM.
Example 4
Cisplatin-resistant lung cancer organoids were cultured according to the method of example 1, except that: the concentration of MMP-10 in the culture medium used in this example was 200 nM.
Comparative example 1
Cisplatin-resistant lung cancer organoids were cultured according to the method of example 1, except that: the culture medium used in this comparative example did not contain MMP-10.
Comparative example 2
Cisplatin-resistant lung cancer organoids were cultured according to the method of example 1, except that: the culture medium used in this comparative example had a MMP-10 concentration of 10 nM.
Comparative example 3
Cisplatin-resistant lung cancer organoids were cultured according to the method of example 1, except that: the MMP-10 concentration in the culture medium used in this comparative example was 250 nM.
Test examples
Cisplatin-resistant lung cancer organoids were cultured in the same manner as in example 1, examples 3 and 4, and comparative examples 1-3, respectively, in 17 cases each.
(1) Culture success rate detection
After 14 days of culture, performing morphology and immunofluorescence staining detection to determine the success rate of organoid culture, wherein, when the diameter of the culture is not less than 0.2mm, and the cell number is not less than 5 × 107At all times, organoid culture was considered successful. Detection knotAs shown in table 1.
TABLE 1
| Group of | Number of successful cases/total cases of culture | Culture success rate (%) |
| Example 1 | 11/17 | 64.71 |
| Example 3 | 9/17 | 52.94 |
| Example 4 | 10/17 | 58.82 |
| Comparative example 1 | 2/17 | 11.76 |
| Comparative example 2 | 4/17 | 23.53 |
| Comparative example 3 | 5/17 | 29.41 |
As can be seen from Table 1, the culture medium adopted by the method disclosed by the invention contains MMP-10 with a specific concentration, and the success rate of culturing cisplatin-resistant lung cancer organoids can be remarkably improved.
(2) Drug resistance index detection
Treating the organs successfully cultured in the groups by using cisplatin with different concentrations, and detecting and calculating the IC50 value of the cisplatin on the cells of the organs. In addition, normal lung cancer cells were treated with different concentrations of cisplatin, and the IC50 values of cisplatin on normal lung cancer cells were detected and calculated. The drug resistance index of each group of organoids was then calculated according to the following formula:
drug Resistance Index (RI) ═ IC50 for organoid cells/IC 50 for normal lung cancer cells.
When RI >3, organoid cell resistance is considered satisfactory. The success rate of drug resistance of each group of organoids was counted, and the results are shown in table 2.
TABLE 2
| Group of | Number of successful drug resistance cases/total number of cases | Success rate of drug resistance (%) |
| Example 1 | 9/17 | 52.94 |
| Example 3 | 6/17 | 35.29 |
| Example 4 | 8/17 | 47.06 |
| Comparative example 1 | 1/17 | 5.88 |
| Comparative example 2 | 3/17 | 17.65 |
| Comparative example 3 | 2/17 | 11.76 |
As can be seen from Table 2, the cisplatin-resistant lung cancer organoids cultured by the method provided by the present disclosure have high drug-resistant success rate.
(3) Drug resistance-related protein detection
Collecting organoids obtained by successful culture in each group, adding cell lysate, placing on ice for cracking for 30min, collecting cell protein samples after cracking, and detecting the protein concentration by using a BCA method or a Western blot method, thereby calculating the mass percentage of the drug-resistant related protein content in each organ to the total content of the membrane protein, wherein the drug-resistant related protein is P-glycoprotein and multidrug resistant protein 1. The results are shown in Table 3.
TABLE 3
| Group of | P-glycoprotein (%) | Multidrug resistance protein 1 (%) |
| Example 1 | 70.14 | 35.23 |
| Example 3 | 58.31 | 28.16 |
| Example 4 | 61.47 | 30.28 |
| Comparative example 1 | 8.29 | 5.32 |
| Comparative example 2 | 10.65 | 9.34 |
| Comparative example 3 | 12.19 | 11.25 |
As can be seen from table 3, in the cisplatin-resistant lung cancer organoids cultured by the method provided by the present disclosure, the percentage of the drug-resistant-related protein content in the total content of membrane proteins is higher, which indicates that the organoids cultured by the present disclosure have better drug resistance.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
Claims (5)
1. A method of culturing a cisplatin-resistant lung cancer organoid, comprising:
mixing the cisplatin-resistant lung cancer tissue cells with a culture medium and matrigel to obtain a to-be-cultured substance, wherein the culture medium contains matrix metalloproteinase-10, and the concentration of the matrix metalloproteinase-10 is 50-200nM on the basis of the culture medium; the culture medium also contains Advanced DMEM/F12 culture medium, FBS, streptomycin, Noggin, FGF7, B27, Nicotinamide and EGF;
culturing and amplifying the culture to obtain cisplatin-resistant lung cancer organoids;
wherein the cisplatin-resistant lung cancer tissue cell is obtained by performing enzymolysis treatment on cisplatin-resistant lung cancer primary tissues,
mixing the cisplatin-resistant lung cancer primary tissue with collagenase I to obtain a first premix;
cutting the cisplatin-resistant lung cancer primary tissues in the first premix into minced shapes to obtain a second premix;
mixing the second premix with an enzymolysis liquid to obtain a to-be-enzymolyzed substance;
the enzymolysis liquid contains cisplatin, and the concentration of the cisplatin is 26-100 mu M on the basis of the enzymolysis liquid; the enzymolysis liquid also contains I-type collagenase, wherein the concentration of the I-type collagenase is 1 mg/mL-5 mg/mL by taking the enzymolysis liquid as a reference;
culturing and carrying out enzymolysis on the to-be-enzymolyzed substance for 1-2 hours to obtain an enzymolysis product;
and separating the enzymolysis product to obtain the cisplatin-resistant lung cancer tissue cell.
2. The method according to claim 1, wherein the concentration of the cisplatin-resistant lung cancer tissue cells in the culture is 1 x 104～5×104The gel is coated on the surface of a substrate, wherein the content of the substrate gel is 1-20 vol%.
3. The method according to claim 1, wherein the FBS is 5-10 vol%, the streptomycin is 50-100 μ g/ml, the Noggin is 50-150 ng/ml, the FGF7 is 1-10 ng/ml, the B27 is 1-5 vol%, the Nicotinamide is 1-10 mM, the EGF is 1-10 ng/ml, and the balance is the Advanced DMEM/F12 medium, based on the culture medium.
4. A culture medium for culturing cisplatin-resistant lung cancer organoids is characterized by comprising matrix metalloproteinase-10, wherein the concentration of the matrix metalloproteinase-10 is 50-200nM on the basis of the culture medium;
the culture medium also contains Advanced DMEM/F12 culture medium, FBS, streptomycin, Noggin, FGF7, B27, Nicotinamide and EGF.
5. The culture medium for cisplatin-resistant lung cancer organoid culture as claimed in claim 4, wherein based on the culture medium, the content of FBS is 5-10 vol%, the concentration of streptomycin is 50-100 μ g/ml, the concentration of Noggin is 50-150 ng/ml, the concentration of FGF7 is 1-10 ng/ml, the content of B27 is 1-5 vol%, the concentration of Nicotinamide is 1-10 mM, the concentration of EGF is 1-10 ng/ml, and the balance is the Advanced DMEM/F12 culture medium.
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