CN112592884A - Human EGFR20ins lung cancer organoid and culture method, culture medium and application thereof - Google Patents
Human EGFR20ins lung cancer organoid and culture method, culture medium and application thereof Download PDFInfo
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Abstract
The invention relates to a human EGFR20ins lung cancer organoid and a culture method, a culture medium and application thereof, wherein the method comprises the following steps: carrying out enzymolysis on human EGFR20ins lung cancer tissues taken from individual patients to obtain lung cancer cells; carrying out clone culture on the lung cancer cells by adopting a clone-promoting culture medium and matrigel to obtain a clone culture product; taking out the clone cells which grow faster and have human EGFR20ins from the clone culture products; performing first amplification culture on the cloned cells by using an amplification culture medium and matrigel to obtain an amplification culture product; wherein the amplification medium contains NSC 228155; and carrying out first separation treatment on the amplification culture product, and carrying out second amplification culture on the obtained amplification cells to obtain the lung cancer organoid. The method disclosed by the invention has the advantages of high culture success rate and short period, and the cultured human EGFR20ins lung cancer organoid has strong stability.
Description
Technical Field
The disclosure relates to the technical field of biological medicines, in particular to a human EGFR20ins lung cancer organoid, a culture method, a culture medium and application thereof.
Background
EGFR exon20 insertion mutation (EGFR 20 ins), which is a type of EGFR gene mutation, accounts for about 4-12% of EGFR mutations. However, EGFR20ins structural diversity plus limitations in detection means may result in low detection rates. EGFR20ins, except p.a763_ Y764insFQEA, are generally insensitive to first and second generation EGFR TKIs. Experiments show that the next generation of EGFR TKIs such as EGF816, AP32788, osimertinib and poziotinib have great clinical potential for EGFR exon20ins mutation, and the drugs are in the clinical development stage. However, currently there are no approved EGFR TKIs for EGFR exon20 ins. Due to structural diversity (up to 200 or more mutant subtypes), different insertions of EGFR20ins may respond differently to EGFR TKIs. Therefore, establishing a cell model with EGFR20ins mutation in vitro is of great significance for research on personalized medicine and targeted drug development.
Research shows that tumor organoids can be used as a drug screening model for drug effectiveness detection, but no report on a method for establishing rare mutant organoids of lung cancer EGFR20ins exists at present.
Disclosure of Invention
The method has the advantages of high culture success rate and short period, and the cultured human EGFR20ins lung cancer organoid has strong stability.
In order to achieve the above object, a first aspect of the present disclosure provides a method for culturing human EGFR20ins lung cancer organoids, comprising:
s1, carrying out enzymolysis on human EGFR20ins lung cancer tissues taken from individual patients to obtain lung cancer cells;
s2, carrying out clone culture on the lung cancer cells by adopting a clone promoting culture medium and matrigel to obtain a clone culture product;
s3, taking out the clone cells in the clone culture product;
s4, performing first amplification culture on the cloned cells by adopting an amplification culture medium and matrigel to obtain an amplification culture product; the amplification medium contains NSC228155, and the content of NSC228155 is 10-55 mu M, preferably 20-50 mu M;
s5, carrying out first separation treatment on the amplification culture product to obtain an amplification cell;
and S6, performing second amplification culture on the amplified cells by using the amplification culture medium.
Optionally, the cloning promoting culture medium contains 30-50 mu M of Y-27632, 5-10 mu g/mL of amphotericin B, 50-100 mu g/mL of streptomycin, 1-3 vol% of B27, 3-5 mM of Nicotinamide, 15-30 mu M of EGF, 450-550 ng/mL of R-spondin, 90-110 ng/mL of Noggin, 45-55 ng/mL of Hydrocortisoninum, 3-8 mM of L-glutamine and 2-5 mu M of Insulin;
the amplification medium further comprises 8-12 mM of E2, 45-55 ng/mL of LPS, 5-10 mu g/mL of amphotericin B, 50-100 mu g/mL of streptomycin, 1-3 vol% of B27, 3-5 mM of Nicotinamide, 15-30 mu M of EGF, 15-20 mu M of bFGF, 45-55 ng/mL of hydrocortisone, 3-8 mM of L-glutamine and 2-5 mu M of Insulin.
Optionally, in step S2, the performing clone culture on the lung cancer cells by using a cloning-promoting medium and matrigel comprises:
performing first mixing on the cloning promotion culture medium, the matrigel and the lung cancer cells to obtain a primary culture, wherein the matrigel content is 2-10 vol% and the concentration of the lung cancer cells is 3 x 10 based on the total volume of the cloning promotion culture medium3～5×103Per mL;
at 36-38 ℃ and CO2And performing clonal culture on the primary culture for 5-7 days under the condition that the concentration is 4-6 vol%.
Optionally, in step S3, the removing of the clonal cells in the clonal culture product comprises: picking out a first cell mass with the diameter of 50-200 mu m in the clone culture product under a mirror, wherein the first cell mass has an irregular budding form;
mixing the cell clusters with irregular budding shapes with a first cell digestive juice to obtain a first cell digestive product;
and carrying out second separation treatment on the first cell digestion product to obtain the clone cells.
Optionally, in step S4, the performing a first amplification culture on the cloned cells by using an amplification medium and matrigel comprises:
performing second mixing on the amplification culture medium, the matrigel and the cloned cells to obtain a second-generation culture, wherein the content of the matrigel is 2-10 vol% and the concentration of the cloned cells is 200-500/mL based on the total volume of the amplification culture medium;
at 36-38 ℃ and CO2And (3) carrying out first amplification culture on the second-generation culture for 3-5 days under the condition that the concentration is 4-6% by volume, and picking out a second cell mass with the diameter of 50-200 mu m under a microscope, wherein the second cell mass has an irregular budding form, so as to obtain an amplification culture product.
Optionally, in step S5, the subjecting the amplification culture product to a first separation process includes:
carrying out first separation treatment on the amplification culture product to obtain a cell precipitate;
mixing the cell sediment with a second cell digestive juice to obtain a second cell digestive product;
and carrying out third separation treatment on the second cell digestion product to obtain an expanded cell.
Optionally, in step S1, the subjecting the human EGFR20ins lung cancer tissue obtained from the individual patient to enzymolysis includes:
mixing the human EGFR20ins lung cancer tissue with enzymolysis liquid to obtain a to-be-enzymolyzed substance; the enzymolysis liquid is taken as a reference, and contains 2-10 vol% of FBS, 0.1-0.3 vol% of dispase, 0.01-0.03 vol% of DNAse, 1-2 mg/mL of I-type collagenase and 1-1.5 vol% of streptomycin;
carrying out enzymolysis on the to-be-enzymolyzed substance at 36-38 ℃ for 1-2h to obtain an enzymolysis product;
and carrying out fourth separation treatment on the enzymolysis product to obtain the lung cancer cell.
A second aspect of the present disclosure provides a culture medium for culturing human EGFR20ins lung cancer organoids, the culture medium comprising a pro-cloning medium and an amplification medium;
the amplification culture medium contains 10-55 mu M of NSC228155, preferably contains 20-50 mu M of NSC 228155;
preferably, the amplification medium further comprises 8-12 mM of E2, 45-55 ng/mL of LPS, 5-10 μ g/mL of amphotericin B, 50-100 μ g/mL of streptomycin, 1-3 vol% of B27, 3-5 mM of Nicotinamide, 15-30 μ M of EGF, 15-20 μ M of bFGF, 45-55 ng/mL of hydrocortisone, 3-8 mM of L-glutamine and 2-5 μ M of Insulin;
the clone promoting culture medium contains 30-50 mu M Y-27632, 5-10 mu g/mL amphotericin B, 50-100 mu g/mL streptomycin, 1-3 vol% B27, 3-5 mM Nicotinamide, 15-30 mu M EGF, 450-550 ng/mL R-spondin, 90-110 ng/mL Noggin, 45-55 ng/mL hydrocortisone, 3-8 mM L-gluutamine and 2-5 mu M Insulin.
In a third aspect of the present disclosure, a human EGFR20ins lung cancer organoid cultured by the method provided in the first aspect of the present disclosure is provided.
The fourth aspect of the disclosure provides a use of the human EGFR20ins lung cancer organoid provided by the third aspect of the disclosure in drug screening and precise drug administration detection.
According to the technical scheme, the method for culturing the human EGFR20ins lung cancer organoids provided by the disclosure adopts the cloning promoting culture medium and the NSC 228155-containing amplification culture medium to carry out three-dimensional culture on the human EGFR20ins lung cancer cells, can culture the human EGFR20ins lung cancer organoids with strong stability in a short period when different patients are diagnosed, adopts the culture to obtain the human EGFR20ins lung cancer organoids to carry out drug screening, and can formulate personalized diagnosis and treatment schemes for different patients.
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.
In a first aspect, the present disclosure provides a method of culturing human EGFR20ins lung cancer organoids, the method comprising:
s1, carrying out enzymolysis on human EGFR20ins lung cancer tissues taken from individual patients to obtain lung cancer cells;
s2, carrying out clone culture on the lung cancer cells by adopting a clone promoting culture medium and matrigel to obtain a clone culture product;
s3, taking out the clone cells in the clone culture product;
s4, performing first amplification culture on the cloned cells by adopting an amplification culture medium and matrigel to obtain an amplification culture product; wherein the amplification culture medium contains NSC228155, and the content of NSC228155 is 10-55 mu M, preferably 20-50 mu M;
s5, carrying out first separation treatment on the amplification culture product to obtain an amplification cell;
and S6, performing second amplification culture on the amplified cells by using the amplification culture medium.
The inventors of the present disclosure unexpectedly found that human EGFR20ins lung cancer organoids cultured in three dimensions are morphologically distinct from wild type, have a small clonal morphology and an irregular budding morphology, and that NSC228155 can promote proliferation of human EGFR20ins lung cancer organoids cells, but does not promote wild type lung cancer organoids cells. The method adopts the cloning-promoting culture medium and the amplification culture medium containing NSC228155 to culture the human EGFR20ins lung cancer cells, has short culture period and high success rate, and the cultured human EGFR20ins lung cancer organoid has high stability, can be used for solving the problems of EGFR20ins sample deficiency, the lack of heterogeneous research data of a lung cancer patient and the like, and provides a more effective and diversified research model for scientific research, drug screening, clinical treatment and other aspects of lung cancer.
In a preferred embodiment, the NSC228155 content in the amplification medium is 25-50 μ M, and more preferably 45-50 μ M.
According to the present disclosure, the kind of the cloning promoting medium is not particularly limited, and for example, the cloning promoting medium may contain 30 to 50 μ M of Y-27632 (ROCK inhibitor), 5 to 10 μ g/mL of amphotericin B, 50 to 100 μ g/mL of streptomycin, 1 to 3 vol% of B27 (cell culture additive), 3 to 5mM of Nicotinamide (Nicotinamide), 15 to 30 μ M of EGF (epidermal growth factor), 450 to 550ng/mL of R-spondin, 90 to 110ng/mL of Noggin (recombinant human Noggin), 45 to 55ng/mL of Hydrocortissum (hydrocortisone), 3 to 8mM of L-glutamine (L-glutamine), and 2 to 5 μ M of Insulin.
According to the present disclosure, the amplification medium may further comprise other components, which may be selected from a wide range, and in one embodiment, the medium may further comprise 8-12 mM of E2 (estradiol), 45-55 ng/mL of LPS (lipopolysaccharide), 5-10 μ g/mL of amphotericin B, 50-100 μ g/mL of streptomycin, 1-3 vol% of B27 (cell culture additive), 3-5 mM of Nicotinamide (Nicotinamide), 15-30 μ M of EGF (epidermal growth factor), 15-20 μ M of bFGF (basic fibroblast growth factor), 45-55 ng/mL of Hydrocortison (hydrocortisone), 3-8 mM of L-glutamine (L-glutamine), and 2-5 μ M of Insulin. Wherein, the above components can be purchased and obtained, and the composition, dosage form, specification and the like of the components are known in the field and are not described herein again. In the embodiment of the disclosure, the components are reasonably matched to obtain an amplified culture medium, and the amplified culture medium and a cloning-promoting culture medium are used for three-dimensional culture of human EGFR20ins lung cancer cells, so that human EGFR20ins lung cancer organoids with good stability can be obtained through culture.
In one embodiment, the step S2 of performing clonal culture on the lung cancer cells by using a cloning-promoting medium and matrigel may comprise: carrying out first mixing on a cloning promotion culture medium, matrigel and lung cancer cells to obtain a primary culture, wherein the content of the matrigel is 2-10% by volume based on the total volume of the cloning promotion culture medium, and the concentration of the lung cancer cells is 3 multiplied by 103~5×103Per mL; at 36-38 ℃ and CO2CO concentration of 4-6 vol%, preferably at 37 deg.C2At a concentration of 5 vol.% on the primary cultureAnd performing cloning culture for 5-7 days. The kind of Matrigel is not particularly limited in the present application, and may be well known to those skilled in the art, and may be, for example, Matrigel. In a preferred embodiment, the primary culture is placed in culture wells of a culture plate (e.g., a 24-well plate) such that 0.3-0.4 mL of primary culture is present in each well, followed by clonal culture. In the process of cloning culture, the culture plate can be firstly placed in an incubator with the temperature of 37 ℃ and 5 vol% of carbon dioxide for culturing for 0.5-1 hour, then 0.1-0.2 mL of the cloning promotion culture medium is added into each culture hole, and then 0.1-0.2 mL of the cloning promotion culture medium is supplemented into each hole every 3-5 days until the diameter of the cloning culture in the culture hole reaches 50-200 mu m.
In one embodiment, the step S3 of removing the clonal cells from the clonal culture product can include: taking out cell clusters with diameters of 50-200 mu m and irregular sprouting forms in the clone culture product, and preferably, picking out a first cell cluster with diameters of 50-200 mu m and irregular sprouting forms by using a sterile gun head (for example, a 10-200 mu L sterile gun head) under a microscope; mixing the taken first cell mass with a first cell digestive juice to carry out cell digestion treatment to obtain a first cell digestion product; and carrying out second separation treatment on the first cell digestion product to obtain a clone cell. The type of the first cell digestive juice is not particularly limited, the first cell digestive juice may be Trypsin-EDTA Solution, Acctuase, etc., and the amount of the cell digestive juice may be selected according to actual needs, preferably, 5 × 105The dosage of the cell mass is 0.5-1 mL, and the digestion treatment time is 1-5 min. The second separation treatment may be a method conventionally employed by those skilled in the art, and may be, for example, centrifugation.
In one embodiment, in step S4, performing a first amplification culture on the cloned cells using an amplification medium and matrigel may include: carrying out second mixing on the amplification culture medium, the matrigel and the cloned cells to obtain a second-generation culture, wherein the content of the matrigel is 2-10% by volume based on the total volume of the amplification culture medium, and the concentration of the cloned cells is200-500 pieces/mL; at 36-38 ℃ and CO2CO concentration of 4-7 vol%, preferably at 37 deg.C2The second generation culture was subjected to the first amplification culture at a concentration of 5 vol% for 3 to 5 days. In a preferred embodiment, the second generation culture is placed in culture wells of a culture plate (e.g., a 96-well plate) such that the second generation culture is 0.1 to 0.5mL per well, and then subjected to an amplification culture. In the amplification culture process, the culture plate can be placed in an incubator with carbon dioxide of 36-38 ℃, preferably 37 ℃, 4-7 vol% and preferably 5 vol% for 3-5 days, 0.1-0.2 mL of amplification culture medium is supplemented into each hole every 2-5 days in the culture process, and a second cell mass with the diameter of 50-200 mu m and irregular budding form is picked out under the microscope to obtain an amplification culture product.
In one embodiment of the present application, the step S5 of subjecting the amplified culture product to a first separation process may include: carrying out first separation treatment on the amplification culture product to obtain cell sediment; mixing the cell sediment with a second cell digestive juice to obtain a second cell digestive product; and carrying out third separation treatment on the second cell digestion product to obtain the expanded cells. The specific type of the second cell digest may be the same as that of the first cell digest, and the type of the third separation treatment may be, for example, a centrifugal separation treatment.
In a specific embodiment, in step S6, performing a second amplification culture on the amplified cells by using the amplification culture medium until the cell mass diameter of the second amplification culture is 50-200 μm, so as to obtain human EGFR20ins lung cancer organoids.
In one embodiment of the present application, the step S1 of performing enzymolysis on the human EGFR20ins lung cancer tissue obtained from an individual patient may include: mixing human EGFR20ins lung cancer tissues with the enzymolysis liquid to obtain a to-be-enzymolyzed substance; wherein the enzymolysis liquid is taken as a reference, and contains 2-10 volume percent of FBS, 0.1-0.3 volume percent of dispase, 0.01-0.03 volume percent of DNAse, 1-2 mg/mL of I-type collagenase and 1-1.5 volume percent of streptomycin; carrying out enzymolysis on the to-be-enzymed substances at 36-38 ℃, preferably at 37 ℃ for 1-2h to obtain enzymolysis products; and carrying out fourth separation treatment on the enzymolysis product to obtain the lung cancer cell.
In a specific embodiment, the method may further include step S7: and screening the human EGFR20ins lung cancer organoids with the same gene mutation type as the human EGFR20ins lung cancer tissues. In a preferred embodiment, the human EGFR20ins lung cancer tissue obtained in step S1 and the lung cancer organoid obtained in step S6 are subjected to second-generation sequencing respectively, and the gene mutation types of the two are compared, and if the mutation types are consistent, the lung cancer organoid obtained in step S6 is the human EGFR20ins lung cancer organoid of the individual patient in step S1.
In a specific embodiment, step S1 may further include: carrying out surgical excision or puncture on individual patients to obtain human EGFR20ins lung cancer tissues, and carrying out enzymolysis treatment on the human EGFR20ins lung cancer tissues to obtain lung cancer cells; the human EGFR20ins lung cancer tissue is preserved in the lung cancer tissue protective solution, and the lung cancer tissue protective solution contains 1-5 vol% of Amphotericin B, 0.5-2 vol% of streptomycin and 5-20 vol% of RPMI1640 of FBS on the basis of the lung cancer tissue protective solution.
According to the present disclosure, the enzymatic treatment may include: mixing human EGFR20ins lung cancer tissues with the enzymolysis liquid to obtain a to-be-enzymolyzed substance; wherein the enzymolysis liquid is taken as a reference, and contains 2-10 volume percent of FBS, 0.1-0.3 volume percent of dispase, 0.01-0.03 volume percent of DNAse, 1-2 mg/mL of I-type collagenase and 1-1.5 volume percent of streptomycin; carrying out enzymolysis on the to-be-enzymed substances at 36-38 ℃, preferably at 37 ℃ for 1-2h to obtain enzymolysis products; and carrying out fourth separation treatment on the enzymolysis product to obtain the lung cancer cell.
A second aspect of the present disclosure provides a culture medium for culturing human EGFR20ins lung cancer organoids, the culture medium comprising a pro-cloning medium and an amplification medium; the amplification medium contains 10-55 mu M of NSC228155, preferably 20-50 mu M of NSC 228155;
in a preferred embodiment of the present disclosure, the amplification medium further comprises 8-12 mM of E2, 45-55 ng/mL of LPS, 5-10 μ g/mL of amphotericin B, 50-100 μ g/mL of streptomycin, 1-3 vol% of B27, 3-5 mM of Nicotinamide, 15-30 μ M of EGF, 15-20 μ M of bFGF, 45-55 ng/mL of Hydrocortison, 3-8 mM of L-glutamine, and 2-5 μ M of Insulin.
In a preferred embodiment of the present disclosure, the cloning promotion culture medium contains 30-50 μ M Y-27632, 5-10 μ g/mL amphotericin B, 50-100 μ g/mL streptomycin, 1-3 vol% B27, 3-5 mM Nicotinamide, 15-30 μ M EGF, 450-550 ng/mL R-spondin, 90-110 ng/mL Noggin, 45-55 ng/mL hydrocortisone, 3-8 mM L-glutamine and 2-5 μ M Insulin.
In a third aspect of the present disclosure, a human EGFR20ins lung cancer organoid cultured by the method provided in the first aspect of the present disclosure is provided.
The fourth aspect of the disclosure provides a use of the human EGFR20ins lung cancer organoid provided by the third aspect of the disclosure in drug screening and precise drug administration detection.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The raw materials, reagents, instruments and equipment mentioned in the examples of the present disclosure can be purchased without specific description, and when the specific test temperature is not specified in the examples of the present disclosure, the test temperature is room temperature (20 to 25 ℃).
The cloning-promoting medium used in the examples of the present disclosure contains: 20 μ M Y-27632, 10 μ g/mL amphotericin B, 100 μ g/mL streptomycin, 2% B27, 5mM Nicotinamide, 20 μ M EGF, 500ng/mL R-spondin, 100ng/mL Noggin, 50ng/mL hydrocortisone, 5mM L-glutamine, and 4 μ M Insulin in DMEM/F12 medium.
The amplification medium used in the examples of the present disclosure contains: 50 μ M NSC228155, 10mM E2, 50ng/mL LPS, 10 μ g/mL amphotericin B, 100 μ g/mL streptomycin, 2% B27, 5mM Nicotinamide, 20 μ M EGF, 20 μ M bFGF, 50ng/mL hydrocortisone, 5mM L-glutamine, and 4 μ M Insulin in DMEM/F12 medium.
The sources of reagents used in the examples of the disclosure are as follows:
DMEM/F12 medium (cat. No. 12634010), FBS (cat. No. 10100147), LPS (cat. No. 0111: B4) B27 (cat. No. 17504044), bFGF (cat. No. PHG0360), collagenase type I (cat. No. C21900), L-glutamine (cat. No. 25030081) are available from Gibco, NSC228155 (cat. No. 113104-25-9) are available from APEXBIO, estradiol (E2) (cat. No. BP 1976-EKW) are available from Bailebo, EGF (cat. No. 4022-500) is available from BioVision, Y-27632 (cat. No. 146986-50-7) is available from MCE, penicillin G-streptomycin (cat. No. 15070063) is available from Invitrogen, Nicotinamide (cat. No. 98-92-0) is available from UuStandard, and Matrigel (cat. No. 356234) is available from BD.
Example 1
S1 pretreatment of lung cancer tissue sample
Human EGFR20ins lung cancer tissues obtained by surgical excision or puncture of a lung cancer patient clinically diagnosed with EGFR20ins are placed in pre-cooled RPMI1640 lung cancer tissue protective solution containing 2 volume percent of Amphotericin B, 1 volume percent of streptomycin and 5 volume percent of FBS, and are transported to a laboratory for treatment within 48h under the condition of low-temperature transportation.
Rapid transfer of human EGFR20ins Lung cancer tissue to 100mm in a sterile operating Table in the laboratory2The plate was washed twice with RPMI1640 medium containing 2 vol% Ampotericin B and 1 vol% streptomycin and cut to 1mm with sterile scissors3Adding a DMEM culture medium containing 2 vol% of FBS, 0.2 vol% of dispase, 0.02 vol% of DNAse, 1.5 mg/mL type I collagenase and 1 vol% of streptomycin into small blocks, uniformly mixing, and carrying out enzymolysis on the small blocks for 1-2h on a low-speed shaking table at the temperature of 37 ℃ to obtain an enzymolysis monocellular liquid;
filtering the single cell suspension obtained by enzymolysis into a sterile centrifuge tube through a 70-micron cell sieve, centrifuging for 5min according to 300g to obtain cell precipitates, washing by using a PBS buffer solution, and repeating the steps to obtain human EGFR20ins lung cancer cells;
s2, adding pre-cooled clone growth promoting medium containing 5 vol% Matrigel to the precipitate (based on the total volume of the clone growth promoting medium), and adjusting the cell concentration to 3X 103Slowly adding the mixture into a 24-well plate along the plate wall according to the amount of 0.3 mL/holeAfter Matrigel is solidified, 0.2mL of human lung cancer tumor organoid clone-promoting growth culture medium is slowly supplemented along the plate wall, and 5% CO is added at 37 DEG C2Culturing in an incubator, and observing cell proliferation every 3 days under a microscope during the culture process. After culturing for 7 days, observing the growth state of the cells under an inverted microscope, and if the cells have obvious proliferation and good growth state, obtaining a clone culture product;
s3, rapidly picking cell clusters with cell diameters of 50-200 mu m and irregular budding shapes into a freshly prepared amplification medium by using a 10-200 mu L sterile gun head under a microscope, and then concentrating the picked cell clusters in a 15mL sterile centrifuge tube by centrifuging for 5min according to 300 g. Adding 1-2mL of cell digestive juice into the precipitate, digesting for 2min, blowing, uniformly mixing, and centrifuging for 5min at 300g again to obtain cell precipitate, namely clone cells;
s4, adding a pre-cooled amplification culture medium (taking the total volume of the amplification culture medium as a reference) containing 5 vol% of Matrigel into the obtained cloned cells, gently mixing the cells uniformly by using a pipette to obtain a single cell suspension with the cell concentration of 200-500/mL, slowly adding the single cell suspension into a 96-well plate along the plate wall according to the amount of 0.1 mL/hole, slowly supplementing 0.1mL of amplification culture medium along the plate wall after the Matrigel is solidified, and slowly supplementing 5% CO at 37 ℃ along the plate wall2Culturing in an incubator, supplementing 0.1mL of amplification culture medium into each hole every 3 days in the culture process, and collecting culture solution with a budding state and a cell mass with the diameter of 200-;
s5, centrifuging for 5min according to 300g to obtain cell mass sediment, adding 1-2mL of cell digestive juice into the cell mass sediment for digesting for 2min, blowing, uniformly mixing, centrifuging for 5min again at 300g, and obtaining cell sediment, namely amplified cells;
s6, precipitating the obtained cells according to a ratio of 1: 3, passage, continuously culturing by using an amplification culture medium until the diameter of a cell mass reaches 50-200 mu m, and collecting the obtained cell mass, namely the human EGFR20ins lung cancer organoid;
s7, selecting tumor organoids with cell viability of more than 85% after 5 passages and diameters of 0.2 mm-0.5 mm, comparing gene mutation conditions of the human lung cancer tissues and human EGFR20ins lung cancer organoids from the tissues by adopting a second-generation sequencing method, and judging that the human EGFR20ins lung cancer organoids are human EGFR20ins lung cancer organoids of individual patients in step S1 if the mutation conditions are consistent.
Example 2
Human EGFR20ins lung cancer organoids were cultured in the same manner as in example 1, except that the NSC228155 content in the amplification medium was 20. mu.M.
Example 3
Human EGFR20ins lung cancer organoids were cultured in the same manner as in example 1, except that the NSC228155 content in the amplification medium was 10. mu.M.
Comparative example 1
Human EGFR20ins lung cancer organoids were cultured according to the method of example 1, except that, in step S4, NSC228155 was not included in the amplification medium.
Comparative example 2
Human EGFR20ins lung cancer organoids were cultured according to the method of example 1, except that human EGFR20ins lung cancer cells were subjected to clonal culture using an amplification medium and matrigel in step S2, and the clonal cells were subjected to a first amplification culture using a pro-clonal growth medium and matrigel in step S4.
Comparative example 3
Human EGFR20ins lung cancer organoids were cultured according to the method of example 1, except that human EGFR20ins lung cancer cells were subjected to clonal culture using an amplification medium and matrigel in step S2.
Comparative example 4
Human EGFR20ins lung cancer organoids were cultured according to the method of example 1, except that the clonal cells were subjected to a first expansion culture using a clonogenic growth medium and matrigel for the first expansion culture in step S4.
Test examples
1. Clone formation assay
Human EGFR20ins lung cancer organoids constructed according to the culture methods of examples 1-3, comparative examples 1, 2 and 3 are obtained, the human EGFR20ins lung cancer organoids successfully identified by the method of step S7 of examples 1-3 are respectively subjected to amplification culture according to the method of examples 1-3, 5 fields of the middle area in the culture plate are randomly selected under an inverted microscope after 3 days of culture, the number of organoids with the diameter of 0.05 mm-0.2 mm is calculated, clone precipitates are collected, cell counting is carried out after enzymolysis, and the statistical result is shown in Table 1.
TABLE 1 human EGFR20ins Lung cancer organoid clonogenic profiles
| Human EGFR20ins lung cancer organoid clone formation quantity/quantity | Organoid cell number/number | |
| Example 1 | 26 | 1.3×105 |
| Example 2 | 5 | 3.1×103 |
| Example 3 | 3 | 1.9×103 |
| Comparative example 1 | 0 | 0 |
| Comparative example 2 | 0 | 0 |
| Comparative example 3 | 0 | 0 |
| Comparative example 4 | 1 | 846 |
As can be seen from examples 1-3, comparative examples 1-4 and Table 1, under the condition that different culture media are used, the clone number and organoid cell number of human EGFR20ins lung cancer organoids cultured and formed in examples 1-3 are higher than those of comparative examples 1-4, which shows that the method can effectively promote the growth of human EGFR20ins lung cancer organoids.
2. Gene mutation detection
The lung cancer patients judged to contain EGFR exon20 insertion mutation were divided into eight equal size groups in 8 cases. After culturing according to the method of the embodiment and the comparative example, the 15 th generation cell is respectively detected the mutation condition of the original mutant gene by adopting the next generation sequencing technology, and the abundance result of each mutation is counted and recorded, wherein the result is expressed as M +/-SD, and the statistical result is shown in the table 2.
TABLE 2 statistical results of Gene mutation abundance
| Method | EGFR20ins (mutant abundance/%) |
| Lung cancer tissue | 22.11 |
| Example 1 | 19.32 |
| Example 2 | 13.25 |
| Example 3 | 11.10 |
| Comparative example 1 | 0 |
| Comparative example 2 | 0 |
| Comparative example 3 | 0 |
| Comparative example 4 | 10.36 |
As can be seen from Table 2, the 15 th generation lung cancer organoids cultured according to the methods of examples 1-3 have gene mutation abundances still close to the original tissues compared with the original lung cancer tissues, and the detection results of the three mutant genes all show that the mutation abundances are maintained, namely, example 1 > example 2 > example 3 > comparative example 4 > comparative example 1= comparative example 3, which shows that the method disclosed by the invention can effectively maintain the gene mutation conditions of the original tissues.
3. Modeling success ratio comparison
Taking lung cancer patient operation group clinically judged to contain EGFR No. 20 exon insertion mutation8 examples, construction of human EGFR20ins Lung cancer organoids according to the methods of examples 1 to 3 and comparative examples 1 to 4 to enable the number of organoid cells to be cultured to 1X 106For the success standard of model construction, the success rate of model construction is recorded and counted, and the result is shown in table 3.
TABLE 3 success rate comparison of different methods modeling
| Method | Number of examples | Success number | Success rate |
| Example 1 | 8 | 7 | 87.5% |
| Example 2 | 8 | 4 | 50% |
| Example 3 | 8 | 2 | 25% |
| Comparative example 1 | 8 | 0 | 0 |
| Comparative example 2 | 8 | 0 | 0 |
| Comparative example 3 | 8 | 0 | 0 |
| Comparative example 4 | 8 | 1 | 12.5% |
As can be seen from table 3, the success rate of the EGFR20 exon insertion-containing mutant lung cancer organoids cultured according to the method of example 1 is up to 87.5%, and compared with the comparative example, the modeling success rate is example 1 > example 2 > example 3 > comparative example 4 > comparative example 1= comparative example 2= comparative example 3.
4. Passage cycle study
8 cases of lung cancer patient operation groups judged to contain EGFR20 exon insertion mutation in clinical practice are taken, human EGFR20ins lung cancer organoids are constructed according to the methods of examples 1-3 and comparative example 4, the cell density reaches 80% of the implanted holes and is used as a passable standard, the time from step S4 to passable of cells is recorded and counted, and the results are shown in Table 4.
TABLE 4 human Lung cancer EGFR20ins mutant organoid passage cycle
| Method | Success number | Average time (day) |
| Example 1 | 7 | 7.29 |
| Example 2 | 4 | 10.75 |
| Example 3 | 2 | 21.5 |
| Comparative example 4 | 1 | 31 |
As can be seen from Table 4, the average time of the human EGFR20ins lung cancer organoids capable of continuous expansion in vitro cultured according to the method of example 1 is 7.29 days, and the number of days required for successful culture, example 1 < example 2 < example 3 < comparative example 4, indicates that the disclosed method can successfully establish the human EGFR20ins lung cancer organoids in vitro in a short time.
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 (3)
1. A culture medium for culturing human EGFR20ins lung cancer organoids, wherein the culture medium comprises a pro-cloning medium and an amplification medium;
the amplification culture medium contains 10-55 mu M of NSC 228155;
the clone promoting culture medium contains 30-50 mu M Y-27632, 5-10 mu g/mL amphotericin B, 50-100 mu g/mL streptomycin, 1-3 vol% B27, 3-5 mM Nicotinamide, 15-30 mu M EGF, 450-550 ng/mL R-spondin, 90-110 ng/mL Noggin, 45-55 ng/mL hydrocortisone, 3-8 mM L-gluutamine and 2-5 mu M Insulin.
2. The culture medium according to claim 1, wherein the amplification medium comprises 20-50 μ M NSC 228155.
3. The medium according to claim 1 or 2, wherein the amplification medium further comprises 8-12 mM E2, 45-55 ng/mL LPS, 5-10 μ g/mL amphotericin B, 50-100 μ g/mL streptomycin, 1-3 vol% B27, 3-5 mM Nicotinamide, 15-30 μ M EGF, 15-20 μ M bFGF, 45-55 ng/mL hydrocortisone, 3-8 mM L-glutamine, and 2-5 μ M Insulin.
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