CN113881635A - Culture medium and culture method for pleural mesothelioma organoid - Google Patents

Culture medium and culture method for pleural mesothelioma organoid Download PDF

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CN113881635A
CN113881635A CN202111334146.6A CN202111334146A CN113881635A CN 113881635 A CN113881635 A CN 113881635A CN 202111334146 A CN202111334146 A CN 202111334146A CN 113881635 A CN113881635 A CN 113881635A
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pleural mesothelioma
culture medium
organoid
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徐晓玲
毛伟敏
陈泽新
范云
徐艳珺
李晖
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Zhejiang Cancer Hospital
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Abstract

The invention discloses a culture medium and a culture method for pleural mesothelioma organoids. The culture medium comprises a basal medium, Advanced DMEM/F12 and specific additive factors; the specific additive factor comprises the following components: vitamin A-free B27, N2, N-acetylcysteine, EGF, Noggin, R-spondin 1, Wnt3a, CHIR99021, Thiazovivin, VEGFR, PDGFR, penicillin streptomycin mixed solution and Primocin. The culture medium contains the minimum components required by culturing the pleural mesothelioma organoid, can effectively culture mesothelial cell-derived tumor tissues, does not contain FBS, saves the cost, and reduces cytotoxicity and inhibitors brought by the FBS. The pleural mesothelioma organoid cultured by the invention maintains the morphological structure and the genetic characteristics of primary tissues.

Description

Culture medium and culture method for pleural mesothelioma organoid
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to a culture medium and a culture method for pleural mesothelioma organoids.
Background
Mesothelioma (Mesothelioma) is a cancer that originates from thin-layered tissue (mesothelial cells) covering the surface of the viscera. The most commonly affected site is the pleura. The peritoneum is also present, but less commonly. In rare cases, it occurs in the pericardium or theca testis. Pleural mesothelioma (pleurometers thelioma) is a primary tumor derived from pleural mesothelial cells, accounts for 5% of pleural tumors, and is rarely seen clinically. Pleural mesothelioma may occur in any part of visceral and parietal pleura, 80% occurring in visceral pleura and 20% occurring in parietal pleura; can occur at any age, and is usually between 40 and 60 years old; the incidence of disease is now on the rise.
Over 80% of pleural mesothelioma results from exposure to asbestos-containing environments. The group with a high disease rate includes: miners of asbestos ores, persons who process asbestos into products, persons who work in contact with asbestos products, persons who live with the same, and persons who work in asbestos-containing buildings. Other risk factors include inheritance and infection by the SV40 virus. Mesothelioma may be preliminarily diagnosed by chest X-ray and X-ray computed tomography and confirmed by cytobiology or biopsy. The prognosis of the pleural mesothelioma is poor, the five-year survival rate is lower than 10%, and the establishment of a scientific and rigorous study model is not only beneficial to basic research, but also beneficial to diagnosis and treatment of the pleural mesothelioma and improvement of the survival rate.
Organoids are organ-specific collections of cells derived from stem cells or precursor cells. Organoids cultured in vitro are highly similar to the corresponding organs in terms of cellular composition and tissue architecture, and possess corresponding functional characteristics. Unlike conventional cell culture in two-dimensional environment, organoid culture is a three-dimensional environment in which multiple cell populations contained in a particular tissue or organ are cultured, and the culture system is more similar to the in vivo microenvironment. Therefore, the compound has a huge application prospect in the aspects of basic research of various organ physiopathologies, precise medical treatment, drug screening and development, gene therapy, regenerative medicine and the like.
Although many tumor tissues can be successfully cultured in vitro into organoids by using different methods and different culture conditions, few studies are currently conducted on the culture method of pleural mesothelioma organoids, and particularly, the specific test procedures, operation steps, culture conditions and culture medium formulas are not reported.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium and a culture method for pleural mesothelioma organoids.
The invention is realized by the following technical scheme.
A culture medium of pleural mesothelioma organoid, comprising basal medium Advanced DMEM/F12 and specific additive factor; the specific addition factor comprises the following components in final concentration: vitamin a-free B27, 1-5 ×; n2, 1-5 ×; n-acetyl cysteine, 0.2-5 μ M; EGF, 10-500 ng/ml; noggin, 20-500 ng/ml; r-spondin 1, 100-1000 ng/ml; wnt3a, 50-200 ng/ml; CHIR99021, 1-10. mu.M; thiazovivin, 0.5-5 μ M; VEGFR, 5-50 ng/ml; PDGFR, 5-50 ng/ml; penicillin streptomycin mixed solution, 1-5 times; primocin, 0.5-5 mg/ml.
Further, a culture medium of a pleural mesothelioma organoid, which comprises a basal medium Advanced DMEM/F12 and a specific additive factor; the specific addition factor comprises the following components in final concentration: vitamin a-free B27, 1-2 ×; n2, 1-2 ×; n-acetyl cysteine, 1-3 μ M; EGF, 10-100 ng/ml; noggin, 20-100 ng/ml; r-spondin 1, 100-800 ng/ml; wnt3a, 50-150 ng/ml; CHIR99021, 1-5. mu.M; thiazovivin, 1-5 μ M; VEGFR, 10-30 ng/ml; PDGFR, 10-40 ng/ml; penicillin streptomycin mixed solution, 1-2 x; primocin, 1-3 mg/ml.
A method for culturing a pleural mesothelioma organoid, comprising the steps of:
s1, preprocessing a pleural mesothelioma specimen or biopsy tissue, and centrifuging to remove a supernatant;
s2, mixing the culture medium of claim 1 with a biological scaffold material, and using the mixed solution to resuspend the cell mass precipitate obtained in the step S1;
s3, the gel mixed with the cells obtained in the step S2 is processed at 37 ℃ and 5% CO2Standing for 2-5min under the condition of (1), and then reversing and solidifying for 30-40 min;
s4, adding the culture medium of claim 1, and performing 5% CO treatment at 37 DEG C2Culturing under the condition;
s5, replacing the liquid culture medium every 2-3 days, and culturing for 4-7 days to obtain the pleural mesothelioma organoid.
Further, in step S1, the pretreatment step includes washing with PBS buffer and chopping to 1mm3Digesting the tissue blocks with pancreatin, lysing erythrocytes with erythrocyte lysate, and filtering to remove impurities.
Further, in step S1, the biological scaffold material is extracellular matrix glue.
The present application has the following advantageous effects.
The culture medium contains the minimum components required by culturing pleura mesothelioma organoid, and can effectively culture mesothelial cell-derived tumor tissues;
the culture medium does not contain the most common component bovine serum albumin (FBS) in cell culture, so that the cost is saved, and the cytotoxicity and inhibitors brought by the FBS are reduced;
the culture medium is suitable for culturing pleura mesothelioma organoid, and the cultured pleura mesothelioma organoid maintains the morphological structure and gene characteristics of primary tissues;
the invention contains composite antimicrobial components, can effectively inhibit the growth of microorganisms including bacteria and fungi, and can effectively reduce the risk of microbial contamination in the culture of pleural mesothelioma;
the culture medium and the culture method comprise components such as growth factors, small molecule inhibitors and the like optimized for the pleural mesothelioma, and the success rate and the survival rate of culturing pleural mesothelioma organoids are improved.
Drawings
FIG. 1 is a light field micrograph of pleural mesothelioma organoids from pleural effusion of the invention cultured for 0 and 4 days;
FIG. 2 is a light field micrograph of a pleuromutilin organoid of a punctured tissue source of the present invention cultured for 0 and 4 days.
Detailed Description
The invention is further described below with reference to the figures and examples.
Example 1
A culture medium of pleural mesothelioma organoid, comprising basal medium Advanced DMEM/F12 and specific additive factor; the specific addition factor comprises the following components in final concentration: b27 (no vitamin a), 1 ×; n2, 1 ×; n-acetyl cysteine, 2. mu.M; EGF, 50 ng/ml; noggin, 50 ng/ml; r-spondin 1, 100 ng/ml; wnt3a, 50 ng/ml; CHIR99021, 1 μ M; thiazovivin, 2. mu.M; VEGFR, 10 ng/ml; PDGFR, 10 ng/ml; penicillin streptomycin mixed solution, 1 x; primocin, 1 mg/ml.
A method for culturing pleural mesothelioma derived from pleural effusion, comprising the steps of:
s1, taking pleural effusion of a fresh pleural mesothelioma patient, centrifuging at 4 ℃ and 500 rpm for 5min, and removing a supernatant for later use after centrifugation;
s2, taking the culture medium and extracellular matrix glue according to a volume ratio of 1: 1.5, then resuspending the pellet of cells obtained in step S1 with the mixture, and dropping the gel mixed with the cells into a 60mm petri dish by using a pipette, wherein each drop is about 50 ul;
s3, placing the culture dish after the glue drops are inoculated into the culture dish at 37 ℃ and 5% CO2Standing in the incubator for 2min, carefully reversing after no obvious flow of light shaking glue drops, and fully solidifying for 30 min;
s4, 3ml of the culture medium is placed in a culture dish and then placed in a constant temperature incubator at 37 ℃ and 5% CO2Culturing under the concentration;
s5, replacing the liquid culture medium every 2-3 days, and culturing for 4-7 days to obtain the pleural mesothelioma organoid (shown in figure 1).
Example 2
A pleural mesothelioma culture medium comprises a basal medium Advanced DMEM/F12 and specific additive factors; the specific addition factor comprises the following components in final concentration: b27 (no vitamin a), 1 ×; n2, 1 ×; n-acetyl cysteine, 5. mu.M; EGF, 10 ng/ml; noggin, 50 ng/ml; r-spondin 1, 500 ng/ml; wnt3a, 100 ng/ml; CHIR99021, 1 μ M; thiazovivin, 5. mu.M; VEGFR, 10 ng/ml; PDGFR, 10 ng/ml; penicillin streptomycin mixed solution, 1 x; primocin, 1 mg/ml.
A method for culturing pleurodermatoma derived from punctured tissue, comprising the steps of:
s1, collecting a puncture biopsy tissue (the length is about 2 mm) from a fresh source of pleural mesothelioma, washing for 2 times by using a PBS (phosphate buffer solution) containing antibiotics, shearing the tissue into small blocks with the size of 0.5mm, digesting for 5min at room temperature by using pancreatin, filtering by using a 70-micron filter screen to obtain a cell mass with the cell number of 3-50 cells, and centrifuging at 500 rpm for 5min at 4 ℃ to remove a supernatant for later use;
s2, taking the culture medium and extracellular matrix glue according to a volume ratio of 1: 1.5, then resuspending the pellet of cells obtained in step S1 with the mixture, and dropping the gel mixed with the cells into a 24-well plate with a pipette, wherein each drop is about 30 μ l;
s3, placing the 24-hole plate after the glue drops are connected into a chamber at 37 ℃ and 5% CO2Standing in the incubator for 2min, carefully reversing after no obvious flow of light shaking glue drops, and fully solidifying for 30 min;
s4.24 well plate, adding 1ml of the above culture medium into each well, placing in a constant temperature incubator at 37 deg.C and 5% CO2Culturing under the concentration;
s5, replacing the liquid culture medium every 2-3 days, and culturing for 4-7 days to obtain the pleural mesothelioma organoid (shown in figure 2).
The embodiments of the present invention are preferred embodiments of the present invention, and the scope of the present invention is not limited by these embodiments, so: all equivalent changes made according to the structure, shape and principle of the invention are covered by the protection scope of the invention.

Claims (5)

1. A culture medium of a pleural mesothelioma organoid, comprising: comprises a basal medium, Advanced DMEM/F12 and specific additive factors; the specific addition factor comprises the following components in final concentration: vitamin a-free B27, 1-5 ×; n2, 1-5 ×; n-acetyl cysteine, 0.2-5 μ M; EGF, 10-500 ng/ml; noggin, 20-500 ng/ml; r-spondin 1, 100-1000 ng/ml; wnt3a, 50-200 ng/ml; CHIR99021, 1-10. mu.M; thiazovivin, 0.5-5 μ M; VEGFR, 5-50 ng/ml; PDGFR, 5-50 ng/ml; penicillin streptomycin mixed solution, 1-5 times; primocin, 0.5-5 mg/ml.
2. A culture medium of a pleural mesothelioma organoid according to claim 1, wherein: comprises a basal medium, Advanced DMEM/F12 and specific additive factors; the specific addition factor comprises the following components in final concentration: vitamin a-free B27, 1-2 ×; n2, 1-2 ×; n-acetyl cysteine, 1-3 μ M; EGF, 10-100 ng/ml; noggin, 20-100 ng/ml; r-spondin 1, 100-800 ng/ml; wnt3a, 50-150 ng/ml; CHIR99021, 1-5. mu.M; thiazovivin, 1-5 μ M; VEGFR, 10-30 ng/ml; PDGFR, 10-40 ng/ml; penicillin streptomycin mixed solution, 1-2 x; primocin, 1-3 mg/ml.
3. A method for culturing a pleural mesothelioma organoid, the method comprising: the method comprises the following steps:
s1, preprocessing a pleural mesothelioma specimen or biopsy tissue, and centrifuging to remove a supernatant;
s2, mixing the culture medium of claim 1 with a biological scaffold material, and using the mixed solution to resuspend the cell mass precipitate obtained in the step S1;
s3, the gel mixed with the cells obtained in the step S2 is processed at 37 ℃ and 5% CO2Standing for 2-5min under the condition of (1), and then reversing and solidifying for 30-40 min;
s4, adding the culture medium of claim 1, and performing 5% CO treatment at 37 DEG C2Culturing under the condition;
s5, replacing the liquid culture medium every 2-3 days, and culturing for 4-7 days to obtain the pleural mesothelioma organoid.
4. A method of culturing a pleural mesothelioma organoid according to claim 1, wherein: in step S1, the pretreatment step includes washing with PBS buffer and chopping to 1mm3Digesting the tissue blocks with pancreatin,And (3) cracking the erythrocytes by adopting erythrocyte lysate and filtering to remove impurities.
5. A method of culturing a pleural mesothelioma organoid according to claim 1, wherein: in step S1, the biological scaffold material is extracellular matrix glue.
CN202111334146.6A 2021-11-11 2021-11-11 Culture medium and culture method for pleural mesothelioma organoid Withdrawn CN113881635A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115232792A (en) * 2022-09-19 2022-10-25 杭州艾名医学科技有限公司 Culture medium and culture method for pleural fluid source organoid

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115232792A (en) * 2022-09-19 2022-10-25 杭州艾名医学科技有限公司 Culture medium and culture method for pleural fluid source organoid

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Application publication date: 20220104