CN114214282A - Method for culturing lung tumor organoid - Google Patents
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- CN114214282A CN114214282A CN202111678848.6A CN202111678848A CN114214282A CN 114214282 A CN114214282 A CN 114214282A CN 202111678848 A CN202111678848 A CN 202111678848A CN 114214282 A CN114214282 A CN 114214282A
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Abstract
The invention provides a method for culturing lung tumor organoids, and belongs to the field of organoid culture. The invention firstly provides a lung tumor organoid culture container, which is a cell culture container with a lung extracellular matrix imprinted block array on the inner bottom surface; the regions of the basal surface not covered by the lung extracellular matrix are blocked by a blocking agent; the lung extracellular matrix imprinted block array is composed of circular array points with the diameter of 50-200 mu m, and the distance between every two circular array points is 25-300 mu m. The invention also provides a culture method of the lung tumor organoid, which shortens the culture time, simplifies the culture mode, effectively controls the particle size and arrangement of the lung tumor organoid, and improves the repeatability and stability of drug screening. The cultured lung tumor organoids are used for drug screening, so that the method has higher efficiency and safety, and can be used for personalized treatment and guiding clinical medication.
Description
Technical Field
The invention belongs to the field of organoid culture, and particularly relates to a method for culturing lung tumor organoids.
Background
Lung cancer is the most common malignancy with the highest morbidity and mortality worldwide. According to data statistics of the world cancer research Institute (IARC), in 2012, about 182.5 ten thousands of new lung cancer cases, about 159.0 ten thousands of death cases, about 65.3 thousands of new lung cancer cases and about 59.7 thousands of death cases are the most main malignant tumors harmful to the life health of residents. With the further acceleration of industrialization and urbanization in China, air pollution and high smoking rate are caused, and the harm of lung cancer is further aggravated. With the continuous development of scientific technology, the precise treatment of tumors has entered an individualized age: firstly, the high-throughput gene sequencing is utilized to find out tumor-related mutation and search a drug target; secondly, the tumor model is used for testing the sensitivity of the targeted drug and verifying the drug target. However, the diagnosis of lung cancer is often late, and how to efficiently and briefly establish an in vitro model reflecting the heterogeneity of patients is particularly important. In order to solve the above problems, researchers have focused on organoid techniques.
The organoid field in stem cell research has evolved over the last decade. Organoids belong to three-dimensional (3D) cell cultures, contain some key characteristics of their representative organs, can be used as in vitro models of various diseases, and have wide application prospects in stem cells and in various aspects of development, regenerative medicine, etiology and pathology, drug development, and the like. Hans Clevers firstly put forward a concept of tumor organoids, systematically expounds a method for culturing breast cancer organoids in vitro, completes a large-scale breast cancer organoid project in 2017, and establishes a first breast cancer organoid library in the world. In the George Vlachogiannis team of London cancer research institute, 2018, 71 patients with metastatic digestive tract cancer were sampled for tumor tissues, and tumor organoids were cultured in vitro for chemotherapy susceptibility test for clinical medication guidance. Compared with the actual curative effect of a patient, the organoid drug sensitivity result obtains flamboyant data with 100 percent of sensitivity and 93 percent of specificity. The successfully cultured tumor organoid is highly similar to the original tumor tissue in tissue structure, genome, transcriptome and function, has self-assembly characteristic under microscopic condition, can well retain the characteristics of histology and mutation of the original tumor, has the advantages of quick aging, strong proliferation capability, good maneuverability and the like, and has higher in vitro survival rate of about 50-80 percent. Because of such advantages, organoids can be used to accurately predict drug sensitivity of tumors in patients with solid tumors, and can be used to clinically guide accurate treatment of tumors in patients and accurate treatment regimen selection in the overall management of lung cancer. Meanwhile, the lung cancer organoids can also construct a tumorigenesis model, study the etiology mechanism and metastasis clues of the lung cancer, study the microenvironment of the tumor by co-culturing with other cells, and finally supplement the personalized accurate treatment of the lung cancer and supplement the existing multiomic accurate medical decision system. In addition, the tumor organoids also have the advantages of higher culture efficiency, passability, frozen preservation, avoidance of ethical conflict, capability of further gene operation and the like, and are a high-efficiency transformation research means before clinic, such as tumor biological research, new drug screening, accurate individualized treatment and the like.
The tumor organoids can be passaged for a long time without losing or changing genetic information during the subculture process. Intratumoral and extratumoral heterogeneity was preserved. Colorectal cancer, non-small cell lung cancer, breast cancer and pancreatic cancer tumor organoid models are widely applied to drug evaluation, biomarker identification, biological research and personalized treatment at present. However, the current culture method of tumor organoids mainly comprises the 3D encapsulated culture of tumor organoids mixed with matrigel. The culture method has the defects of complex operation, higher cost, small scale, different organoid particle sizes, non-coplanar, difficult analysis and observation and the like, and can not be used for large-scale batch production. Meanwhile, the existing method for culturing tumor organoids has long culture period, and can cause delay of the illness state of patients.
Therefore, the method is rapid, simple and convenient to operate and low in cost, the lung tumor organoids which are uniform in size, grow on the same plane and are convenient to analyze and observe are cultured, and the method has important significance in achieving rapid large-scale culture of the lung tumor organoids.
Disclosure of Invention
The invention aims to provide a method for culturing lung tumor organoids.
The invention provides a lung tumor organoid culture container, which is a cell culture container with an array of lung extracellular matrix blotting blocks on the inner bottom surface;
the regions of the basal surface not covered by the lung extracellular matrix are blocked by a blocking agent;
the lung extracellular matrix imprinted block array is composed of circular array points with the diameter of 50-200 mu m, and the distance between every two circular array points is 25-300 mu m.
Further, the lung extracellular matrix imprinted block array is composed of circular array points with the diameter of 100 μm, and the spacing between each circular array point is 50 μm.
Furthermore, the area of the lung extracellular matrix imprinted block array is 1-10 cm2；
Preferably, the area of the lung extracellular matrix imprinted block array is 4cm2。
Further, the container is a culture dish, a culture box, a culture plate or a culture bottle;
and/or, the sealant is Pluronic F-127;
preferably, the mass fraction of the Pluronic F-127 is 1-5%.
Further, the lung extracellular matrix is a porcine lung extracellular matrix;
preferably, the preparation method of the porcine lung extracellular matrix comprises the following steps:
(1) preparing a porcine whole lung decellularized scaffold by adopting a Triton-SLES-Triton perfusion method;
(2) the obtained decellularized lung scaffold is obtained by freeze-drying, crushing and enzymolysis by adopting pepsin.
Preferably, the Triton-SLES-Triton perfusion method comprises the steps of taking a new pig lung, cannulating a pulmonary artery, removing blood, freezing and thawing the lung, perfusing for 3 hours by adopting 1% Triton X-100, perfusing for 6 hours by adopting 1% sodium lauryl polyoxyethylene ether sulfate (SLES), perfusing for 3 hours by adopting 1% Triton X-100, and finally perfusing for 2 hours by using PBS to balance the decellularized lung, thus obtaining the decellularized lung scaffold. The flow rate was 100mL/min throughout the perfusion.
Preferably, during the enzymolysis, the concentration of the lung extracellular matrix obtained after crushing is 10 mg/mL; the mass ratio of the pepsin to the extracellular matrix powder is 1: 10. In addition, the pH during enzymolysis was 2, the temperature was room temperature, and the enzymolysis time was 72 hours.
Further, the concentration of the lung extracellular matrix is 0.05-0.5 mg/mL;
preferably, the concentration of the lung extracellular matrix is 0.1-0.5 mg/mL.
The lung tumor organoid culture container is prepared by adopting PDMS micro-pattern printing technology: etching a silicon wafer with a specific pattern by using laser as a template, inverting the template to obtain a micro-array PDMS stamp with an outward convex circular micro pattern with a specific size, coating a pulmonary extracellular matrix adhesive on the surface of the PDMS stamp, stamping the surface of the PDMS stamp on the bottom surface of a culture container to obtain an extracellular matrix micro pattern array, and sealing the rest part of the culture container by using a sealing agent to obtain the lung tumor organoid culture container.
The invention also provides a lung tumor organoid culture method, which comprises the following steps:
and (3) inoculating the lung cancer cells into the culture container, culturing for 4-6 h, washing away the cells which are not attached, and culturing for 3-5 days to obtain the lung tumor organoids.
Further, the seeding density of the lung cancer cells is 104～106Seed/culture container；
And/or, the lung cancer cell is derived from a human;
preferably, the lung cancer cells are seeded at a density of 105And (4) culturing the cells in a container.
The invention also provides a lung tumor organoid prepared by the method.
The invention also provides application of the lung tumor organoid in constructing a lung cancer model or screening medicaments.
The invention provides a method for culturing lung tumor organs, which adopts a lung decellularization ECM pattern microarray chip with specific lattice point shape, size and density to culture lung tumor cells to prepare the lung tumor organs, shortens the in-vitro amplification culture time of the lung tumor organs, simplifies the culture mode of the lung tumor organs, effectively improves the defects of complex operation, high cost, small scale, inconsistent particle size, difficult analysis and observation and the like, effectively controls the particle size and arrangement of the lung tumor organs, and improves the repeatability and stability of drug screening. The lung decellularized ECM provides a tumor microenvironment for lung tumor organoid culture, is beneficial to maintaining tumor heterogeneity and simulates lung cancer tissues in vivo. The lung tumor organoids are used for drug screening, so that the efficiency and the safety are higher, and meanwhile, the PDO can be used for personalized treatment and guiding clinical medication.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 is a culture route of lung tumor organoids according to the present invention.
FIG. 2 shows the results after decellularization of the porcine whole lung: a is the general view before and after decellularization; b is electron microscope images before and after decellularization; c is H & E staining before and after decellularization; d is trichromatic collagen staining before and after decellularization; e is elastic fiber dyeing before and after decellularization; f is IV type collagen fiber dyeing before and after decellularization; g is fibronectin staining before and after decellularization; h is laminin staining before and after decellularization; i is a DNA content detection result before and after decellularization; j is the detection result of glycosaminoglycan (GAG) content before and after decellularization; denotes p < 0.05; scale 50 μm; in the figure, Native is not Decellularized, and Decellularized lung scaffold is Decellularized.
Fig. 3 shows lung decellularized ECM powder (a) and lung decellularized ECM gel (B).
FIG. 4 shows the result of the lung tumor organoid dying and alive staining test after the action of each drug.
Fig. 5 shows a decellularized lung extracellular matrix pattern microarray chip prepared by micropattern printing technology and a cultured lung tumor organoid: a is a PDMS stamp; b is an extracellular matrix pattern microarray; c is the result of the adhesion of human lung cancer cells for 6 h; d is a lung tumor organoid formed after 3 days of culture; e is lung tumor organoid dead stain (scale 100 μm).
Detailed Description
Unless otherwise indicated, the starting materials and equipment used in the embodiments of the present invention are known products and obtained by purchasing commercially available products.
The culture route of the lung tumor organoid is shown in figure 1, and comprises the following steps:
(1) preparation of lung decellularized ECM: the porcine whole lung decellularized scaffold is prepared by adopting a Triton-SLES-Triton perfusion method. The obtained decellularized lung scaffold (lung extracellular matrix) is further lyophilized, pulverized by a ball mill, and then subjected to enzymolysis by pepsin to obtain lung decellularized extracellular matrix (ECM) gel.
(2) Preparing a pattern microarray master plate by adopting a PDMS micro pattern printing technology: a silicon wafer with a specific pattern is etched by using laser as a template, the etching precision reaches 1 mu m, the maximum etching area reaches 12 inches, and a Polydimethylsiloxane (PDMS) seal is obtained by reversing the template. And coating extracellular matrix gel (called extracellular matrix gel or matrigel) on the surface of the stamp, then paving the matrigel pattern microarray at the bottom of the cell culture dish subjected to cell non-attachment treatment by utilizing a PDMS micro pattern printing technology, and sealing the space at the bottom of the rest culture dish by Pluronic F-127 so that the added cells can only grow attached to the matrigel pattern. Obtaining the lung decellularization ECM pattern microarray chip.
(3) Culturing lung tumor organoids: the lung tumor organoid is cultured by adopting a lung decellularization ECM pattern microarray chip, the in-vitro amplification culture time is shortened, and the particle size and the arrangement of the lung tumor organoid can be effectively controlled.
Example 1 culture method of lung tumor organoid of the present invention
The culture method of the lung tumor organoid comprises the following steps:
(1) preparing the pig lung decellularized extracellular matrix glue: after taking fresh pig lungs, the pulmonary artery was cannulated and washed with PBS for 10 minutes to remove blood. The lungs were then frozen and thawed, perfused with 1% Triton X-100 for 3 hours, followed by 1% sodium laureth sulfate (SLES) for 6 hours, then perfused with 1% Triton X-100 for 3 hours, and finally perfused with PBS for 2 hours to equilibrate the decellularized lungs to give a decellularized lung scaffold (lung extracellular matrix). The flow rate was 100mL/min throughout the perfusion. The Decellularized Lung Scaffolds (DLSs) were then cut into cubes of 1X 1cm for lyophilization. Further crushing by a ball mill to obtain the lung decellularized extracellular matrix powder. And (3) carrying out enzymolysis on the extracellular matrix powder by adopting pepsin to obtain the lung decellularized extracellular matrix gel (extracellular matrix gel). During enzymolysis, the extracellular matrix powder is dissolved in water, the concentration is 10mg/mL, the mass ratio of pepsin to the extracellular matrix powder is 1:10, the pH value during enzymolysis is 2, the temperature is room temperature, and the enzymolysis time is 72 hours.
The pig lung is white and transparent after decellularization, after immunofluorescence, histochemistry and enzyme-linked immunosorbent assay, extracellular matrix protein is found to be well retained (figure 2), and decellularized lung extracellular matrix gel is obtained by further crushing and enzymolysis (figure 3).
(2) Etching a silicon wafer with a specific pattern by using laser as a template, reversing the template to obtain a PDMS stamp, and preparing a pattern microarray with convex circular array points (the diameter of each circular array point is 100 μm, the interval between each circular array point is 50 μm, and the size of the microarray is 4 cm)2) The PDMS stamp of (1).
(3) Adjusting the concentration of matrigel by PBS, coating 1mL of porcine lung decellularized extracellular matrix gel with the concentration of 0.1mg/mL on the surface of a PDMS stamp, incubating at room temperature for 20 minutes, washing off redundant matrigel, and drying in an incubator at 37 ℃.
(4) And (3) stamping the PDMS stamp on the inner bottom surface of the cell non-attachment culture dish, applying 0.2 Newton force for 10 minutes, and removing the stamp after the pattern is formed on the bottom surface of the culture dish.
(5) Pluronic F-127 (Pluronic F-127), a polypropylene glycol/ethylene oxide addition polymer (polyether), was added at a mass fraction of 1% and incubated at room temperature for 2 hours, and the space on the bottom surface of the remaining dish was closed to obtain an "extracellular matrix pattern microarray" having an adhesive effect on cells. The prepared microarray culture dish is washed 3 times by PBS before use, so that the culture dish substrate cannot be completely dried, and then cells are inoculated.
(6) The culture dish was inoculated with human lung cancer cells (H1299, inoculum size 1.5X 10)5Culture at 37 deg.c for 6 hr, washing off unattached cells, and culturing for 3 days to form macroscopic lung tumor organs. The lung tumor organoids are all on the same plane, and have uniform size and controllable quantity.
Human lung cancer cells are defined to grow attached to a "microarray of extracellular matrix patterns". The culture medium used in the step is a DMEM high-sugar culture medium containing 10% fetal calf serum and 1% double antibody.
The decellularized lung extracellular matrix pattern microarray chip is prepared by a micropattern printing technology, lung tumor cells are further cultured, and the lung tumor organoid can be formed after 3 days of culture.
The advantageous effects of the present invention are demonstrated by specific test examples below.
Test example 1 screening of drugs by the Lung tumor organoids of the present invention
Experimental methods
The 100 μm diameter lung tumor organoids cultured in example 1 were selected and screened with paclitaxel, doxorubicin hydrochloride and cisplatin at different concentrations (5 μ g/ml, 10 μ g/ml, 50 μ g/ml, 100 μ g/ml) for 24h and 48h, respectively. Analysis of dead and live staining was performed using calcein with propidium iodide fluorescence double staining and analyzed using confocal microscopy imaging.
Results of the experiment
From the results of fig. 4, it can be seen that: the lung tumor organoids cultured by the method can effectively screen the influence of the drug species, the drug dosage and the drug action time on the lung tumor. The results show that the activity of lung tumor organoids is related to the type of drug, the dose of drug and the culture time. Doxorubicin had the best inhibitory activity against lung tumor organoids, which stopped growing as doxorubicin concentration and culture time increased. And the cisplatin has better inhibitory activity on lung tumor organoids, and the lung tumor organoids stop growing along with the increase of concentration and culture time. Paclitaxel has relatively weak inhibitory activity against lung tumor organoids. The research finds that the size of the organoids is controlled and arranged, so that the drug screening result can be better analyzed and observed. The lung tumor organoid pattern microarray chip has potential value in personalized treatment and antitumor drug screening.
Test example 2 screening of Pattern microarray in the Lung tumor organoid culture method of the present invention
Earlier studies show that in the pattern microarray, if rectangular lattice points are used, the effect of preparing lung tumor organoids is poor, and even lung tumor organoids cannot be generated; the lung tumor organs prepared by using the circular lattice are obviously superior to the rectangular lattice. Thus, an extracellular matrix pattern microarray was prepared by the method described in example 1, varying the size of circular array dots (the diameter of the circular array dots are 50 μm, 100 μm, 150 μm, 200 μm, respectively) in the pattern microarray, the interval between each circular array dot was 50 μm, and the microarray size was 4cm2. Each patterned microarray is shown in FIGS. 5A and 5B.
The above procedure was followed as described in example 1Respectively culturing lung tumor organs with extracellular matrix pattern microarray, washing with PBS 1 time before use to ensure that culture substrate can not be completely dried, and inoculating human lung cancer cell (H1299, inoculum size is 1.5 × 10)5A/dish). The patterned microarray was placed on the cell culture dish bottom to confine the growth and adhesion space of the cells, so that the cells were confined to be attached to the matrix spots in the extracellular matrix gel patterned microarray to grow, and the lung tumor organoids were formed after 3 days of culture (fig. 5D).
The optimal size of the circular lattice in the microarray was selected for dead and live function based on lung tumor organoid morphology. The research finds that: when the diameter of the circular lattice point is too small (50 mu m), the circular lattice point cannot be cultured into lung tumor organoids; with the increase of the diameter of the circular lattice points, dead cells in lung tumor organs increase, and the functional activity is poor, so that the lung tumor organs are not suitable for being evaluated as a model. Comprehensively considering, finally selecting a circular lattice point with the diameter of 100 mu m as the optimal lattice point for culturing the lung tumor organoid pattern microarray, and obtaining the lung tumor organoid with optimal shape and function.
In conclusion, the invention provides a method for culturing lung tumor organoids, which adopts a lung decellularized ECM pattern microarray chip with specific lattice point shape, size and density to culture lung tumor cells to prepare the lung tumor organoids, shortens the in-vitro amplification culture time of the lung tumor organoids, simplifies the culture mode of the lung tumor organoids, effectively improves the defects of complex operation, high cost, small scale, inconsistent particle size, difficult analysis and observation and the like, effectively controls the particle size and arrangement of the lung tumor organoids, and improves the repeatability and stability of drug screening. The lung decellularized ECM provides a tumor microenvironment for lung tumor organoid culture, is beneficial to maintaining tumor heterogeneity and simulates lung cancer tissues in vivo. The lung tumor organoids are used for drug screening, so that the efficiency and the safety are higher, and meanwhile, the PDO can be used for personalized treatment and guiding clinical medication.
Claims (10)
1. A lung tumor organoid culture vessel, characterized in that: the device is a cell culture container with an array of lung extracellular matrix imprinting blocks on the inner bottom surface;
the regions of the basal surface not covered by the lung extracellular matrix are blocked by a blocking agent;
the lung extracellular matrix imprinted block array is composed of circular array points with the diameter of 50-200 mu m, and the distance between every two circular array points is 25-300 mu m.
2. The lung tumor organoid culture vessel of claim 1, wherein: the lung extracellular matrix imprinted block array is composed of circular array points with the diameter of 100 mu m, and the distance between every two circular array points is 50 mu m.
3. The lung tumor organoid culture vessel of claim 1 or 2, wherein: the area of the lung extracellular matrix imprinted block array is 1-10 cm2；
Preferably, the area of the lung extracellular matrix imprinted block array is 4cm2。
4. The lung tumor organoid culture vessel of claim 1 or 2, wherein: the container is a culture dish, a culture box, a culture plate or a culture bottle;
and/or, the sealant is Pluronic F-127;
preferably, the mass fraction of the Pluronic F-127 is 1-5%.
5. The lung tumor organoid culture vessel of claim 1 or 2, wherein: the lung extracellular matrix is a porcine lung extracellular matrix;
preferably, the preparation method of the porcine lung extracellular matrix comprises the following steps:
(1) preparing a porcine whole lung decellularized scaffold by adopting a Triton-SLES-Triton perfusion method;
(2) the obtained decellularized lung scaffold is obtained by freeze-drying, crushing and enzymolysis by adopting pepsin.
6. The lung tumor organoid culture vessel of claim 1 or 2, wherein: the concentration of the lung extracellular matrix is 0.05-0.5 mg/mL;
preferably, the concentration of the lung extracellular matrix is 0.1-0.5 mg/mL.
7. A method for culturing lung tumor organoids is characterized in that: the method comprises the following steps:
inoculating the lung cancer cells into the culture container of any one of claims 1 to 6, culturing for 4 to 6 hours, washing off the cells which are not attached, and culturing for 3 to 5 days to obtain the lung tumor organoid.
8. The culture method according to claim 7, wherein: the inoculation density of the lung cancer cells is 104～106A seed/culture vessel;
and/or, the lung cancer cell is derived from a human;
preferably, the lung cancer cells are seeded at a density of 105And (4) culturing the cells in a container.
9. A lung tumor organoid, characterized by: the lung tumor organoid prepared by the method of claim 7 or 8.
10. Use of the lung tumor organoid of claim 9 in the construction of a lung cancer model or drug screening.
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