CN114736869B - Mucous membrane melanoma 3D organoid and culture method and application thereof - Google Patents

Mucous membrane melanoma 3D organoid and culture method and application thereof Download PDF

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CN114736869B
CN114736869B CN202210419167.6A CN202210419167A CN114736869B CN 114736869 B CN114736869 B CN 114736869B CN 202210419167 A CN202210419167 A CN 202210419167A CN 114736869 B CN114736869 B CN 114736869B
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melanoma
organoid
tissue
mucosal
mucosal melanoma
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CN114736869A (en
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石超吉
顾子悦
周榕
陈宛灵
徐声铭
韩永
张志愿
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
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Abstract

The invention belongs to the field of biological medicine, and discloses a mucous membrane melanoma 3D organoid and a culture method and application thereof. The invention also discloses a culture medium for culturing the 3D organoids of the mucosal melanoma, and the organoids of the mucosal melanoma cells can be effectively formed in a 3D environment by selecting specific culture medium components and compounding according to a certain proportion aiming at the phenotypic characteristics of the mucosal melanoma cells. The mucous membrane melanoma organoid obtained by the culture medium and the method can be used for screening medicines, researching action targets and the like, and has wide application prospects.

Description

Mucous membrane melanoma 3D organoid and culture method and application thereof
Technical Field
The invention relates to the technical field of organoid culture, and relates to a 3D organoid for mucous membrane melanoma, a culture method and application thereof.
Background
Mucosal Melanoma (MM) is a rare subtype of melanoma that is more aggressive and can occur in mucosal tissues such as the oral cavity, oropharynx, nasal cavity/sinuses, oesophagus, rectal anal canal, genital tract, urinary tract, etc. MM accounts for about 1% of the total melanoma, and compared with skin melanoma, melanoma occurring in mucous membranes such as head and neck has higher malignancy, high recurrence rate and distant metastasis rate, poor prognosis and high death rate, and most reports show that the survival rate of melanoma in 5 years is lower than 25%.
Melanoma onset has a certain race specificity: white people are more prone to malignant melanoma of skin, and mucosal melanoma is less common; and yellow mankind's mucosa and acromelamas are more common. In Asian population, the mucosa melanoma is the second largest subtype of melanoma, and the incidence rate of mucosa malignant melanoma accounts for 22.6% -30% of that of systemic malignant melanoma, which is higher than that of European and American population.
The good tumor preclinical model is an important basic stone for tumor research, and research, development and verification of various tumor new targets and new therapies including mucous membrane melanoma are all independent of the application of the preclinical tumor model. The in-vitro and in-vivo tumor model which can simulate the tumor heterogeneity of clinical patients simultaneously and keeps the genetic background and biological behavior of the tumor of the patients is an effective tool for tumor research.
The preclinical research model of skin melanoma is rich and various, and comprises most preclinical model types at present, and compared with the preclinical model of mucosa melanoma is especially lacking, so that the research progress of the disease is blocked to a certain extent. Recently, international journal CANCER CELL reported a systematic discussion of the preclinical model of melanoma by the professor Elizabeth pattern, et al, from the university of edinburgh, united states National Institutes of Health (NIH), glenn Merlino, which states the progress of model development in recent years leading to melanoma models, and underscores the five important challenges faced by current new therapies for melanoma, one of which is the treatment dilemma for rare types of melanoma (particularly mucosal melanoma), and the lack of preclinical models is a significant reason impeding the recognition and treatment strategy for this type of disease. Therefore, constructing different types of preclinical models of the mucous membrane melanoma can assist in drug screening and drug effect evaluation and simulate clinical characteristics such as tumor invasion and metastasis, so that evaluation, transformation and application of a targeted treatment strategy are the problems to be solved urgently.
For Malignant Melanoma (OMM) of mucous membrane occurring in the oral maxillofacial head and neck, the subject group of the invention has been striving to develop related preclinical models, which include a head and neck mucous membrane melanoma patient-derived transplantation tumor model (PDX) and a patient-derived mouse in situ transplantation tumor model (PDOX), a patient-derived tumor cell (PATIENT DERIVED tumor cell, PDC), and the like, and the study group has obtained relatively mature experience in the construction of OMM-PDx, OMM-PDC, and developed preclinical exploration experiments. On the basis of earlier research results of a subject group and reports of related documents, a mucous membrane melanoma patient-derived tumor cell model is constructed to screen drug sensitivity, and drugs aiming at specific mutation sensitivity are selected, so that a selection basis is provided for targeted treatment of c-kit mutant patients. By constructing malignant melanoma PDX of mucous membrane, ponatinib is found to inhibit the growth of c-kit V560D, K642E and D816V mutant transplantable tumor more effectively than Imatinib, thus providing potential choice for targeting treatment of malignant melanoma of mucous membrane of head and neck.
The definition of organoids (organoids) is the same as the name of organoids, and is a model of organs, and is a microscopic three-dimensional structure capable of self-assembly formed when various cells are cultured under certain conditions in vitro. The development of Patient-derived tumor organoid (PDO) technology greatly promotes the research of tumors, achieves a plurality of exciting achievements, also brings opportunities for constructing a mucosa black preclinical model, and provides new possibilities for the basic and clinical transformation research of the mucosa melanoma. However, the current technical methods for constructing PDO using normal epithelial tissue or malignant tumor tissue derived from epithelium have been relatively mature, and PDO has been widely reported if oral mucosa epithelium and head and neck squamous cell carcinoma tissue are constructed. However, there are few reports on PDO construction methods and studies on malignant tumors derived from mesenchymal tissues. The research report related to the mucosal melanoma organoids is not related to the mucosal melanoma origin mesenchymal cells at present. Although there are current reports on preclinical models of oral mucosal melanoma, such as cell lines, PDC, and PDX, there is still a lack of effective reference experience for the construction of organoid models of oral mucosal melanoma. The main reasons for this may be the relatively difficult availability of materials for mucosal melanoma, the biological behaviour and genetic background of mucosal melanoma being significantly different from other melanoma subtypes.
Therefore, the construction method for searching the oral mucosa melanoma organoids provides more effective preclinical research tools for the basic and transformation research of oral mucosa melanoma, and has important significance for comprehensively knowing the oral refractory malignant tumors.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide a mucosal melanoma 3D organoid, and a culture method and application thereof, which are used for solving the problems of a culture medium and a culture method for culturing a mucosal melanoma 3D organoid, and achieving the purposes of obtaining a 3D organoid with a consistent form and melanoma marker expression and primary mucosal melanoma expression through culture.
To achieve the above and other related objects, the present invention provides a mucosal melanoma 3D organoid, and a culturing method and application thereof.
It is an object of the present invention to provide a medium for culturing a mucosal melanoma 3D organoid, said medium comprising: basal medium, the following components based on basal medium: 2-20mM HEPES buffer, 1-5mM Glutamax, 0.2-2mg/ml Primocin, 0.5-5v% B27 supplement 、0.75-2mM N-Acetylcysteine、20-100ng/ml rhEGF、0.05-0.3uM A83-01、2-20mM Nicotinamide、300-800ng/ml Wnt3a、0.05-0.3μg/ml Noggin、0.05-0.3μg/ml R-spondin-1, and 0.5-5v% fetal bovine serum.
The basal medium is at least one selected from ADVANCED DMEM/F12 medium, DMEM, high-sugar DMEM medium, alpha MEM medium and RPMI1640 medium.
The concentration component ranges can be adjusted according to the actual culture.
It is another object of the present invention to provide the use of a medium as described above for culturing mucosal melanoma 3D organoids.
Another object of the present invention is to provide a method for culturing a mucosal melanoma 3D organoid, which comprises culturing the crushed mucosal melanoma tissue cells with the culture medium as described above, thereby obtaining the mucosal melanoma 3D organoid.
In the present invention, the mucosal melanoma tissue may be selected from tumor tissue of a patient suffering from mucosal melanoma. In some more preferred embodiments, the mucosal melanoma tissue is selected from mucosal melanoma PDX tissue that has been successfully constructed.
It is another object of the present invention to provide a mucosal melanoma 3D organoid cultured by the above culture method.
It is another object of the present invention to provide the use of the organoids described above as a drug evaluation model for antitumor drug evaluation.
Another object of the present invention is to provide an evaluation method of an antitumor drug, comprising the steps of:
(1) Culturing the obtained organoids using said method;
(2) Evaluation of antitumor drug sensitivity was performed using organoid tissues.
As described above, the 3D organoid for mucous membrane melanoma and the culture method and application thereof have the following beneficial effects:
Aiming at the growth characteristics of cells derived from the mesenchymal tissue such as the mucosal melanoma, the culture medium of the mucosal melanoma tissue organoid provided by the invention selects a plurality of cytokine components to be compounded according to a specific proportion, and the obtained culture medium contains a proper amount of cytokines and signal path regulatory factors, so that the mucosal melanoma tissue can form a 3D organoid which is close to the shape and the property of a real tissue in a short time.
The culture medium and the culture method can finish subculture of the mucous membrane melanoma, can keep the morphology and the property of the 3D organoids, and meet the requirement of copying the mucous membrane melanin tissue organoids on a certain scale.
The mucosa melanoma 3D organoid obtained by the culture medium or the method can simulate the physiological environment of mucosa melanoma cells, can effectively maintain the specificity of tissue cells, and has high consistency of tissue morphology and genotyping; the method can be used for screening therapeutic drugs for the mucous membrane melanoma, researching occurrence and development mechanisms of the mucous membrane melanoma, not only meeting the requirements of scientific research, but also providing beneficial choices for drug administration guidance of patients in clinical drug administration guidance, such as in vitro 3D culture of biopsy samples.
Drawings
FIG. 1 is a photograph of a mixture of cells and BME after inoculation of a 24-well plate, see black chocolate-like gel drops.
Figure 2 morphological observations under a mucosal melanoma PDO mirror. Day 7, B, C, under day 21.
FIG. 3 is a pellet 21 days after inoculation, with black cell spheres visible to the naked eye in part of the glue droplets, indicated by the red arrows.
Fig. 4: AEC red staining results positive or negative controls. Mucosal epithelium in oral mucosal tissue significantly expresses the epithelial marker AE1/AE3, while submucosal black tumor tissue and submucosal connective tissue expression are negative; melanoma tissue expressed HMB45, whereas mucosal tissue expression was negative.
Fig. 5: the tumor tissue source in example 1 was constructed mucosal melanoma PDX tumor tissue (melanoma body).
Fig. 6 is a morphological observation under a PDO mirror of the melanoma of mucosa in example 1. Day 7, B, C, under day 21.
Fig. 7: for the organoids that were balled 21 days after inoculation and culture in example 1, the spheroids were collected, and the melanoma organoid spheroids were wrapped with agarose gel for paraffin embedding, with the black organoid spheroids visible to the naked eye in the gel.
Fig. 8: mucosal melanoma organoids HE staining.
Fig. 9: mucosal melanoma PDO expressed the melanoma markers HMB45, melan-A and S100, and the epithelial marker AE1/3 was negative. The expression of PDO (C) molecule is consistent with the expression of tumor-derived PDX tissue (B) and primary patients, namely HMB45, melan A and S100 are positive, and AE1/3 is negative.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.
The invention provides an in vitro culture method of a mucous membrane melanoma organoid, which is characterized in that organoids are induced and constructed by extracting PDX-derived mucous membrane melanoma cells, and the organoids which are highly consistent with the tissue morphology and genotyping of living mucous membrane melanoma are obtained by HE staining and immune group assay of the morphology and cytokine expression of the mucous membrane melanoma organoids.
The invention provides a culture medium for culturing a 3D organoid of a mucous membrane melanoma, which comprises the following components: basal medium, the following components based on basal medium: HEPES buffer, glutaMAX, primocin, B supplement, N-ACETYLCYSTEINE, RHEGF, A83-01, nicotinamide, wnt a, noggin and R-spondin-1. Specifically, the culture medium for culturing the mucosa melanoma 3D organoids comprises a basal medium and the following components based on the basal medium: 2-20mM HEPES buffer, 1-5mM Glutamax, 0.2-2mg/ml Primocin, 0.5-5v% B27 supplement 、0.75-2mM N-Acetylcysteine、20-100ng/ml rhEGF、0.05-0.3μM A83-01、2-20mM Nicotinamide、300-800ng/ml Wnt3a、0.05-0.3μg/ml Noggin、0.05-0.3μg/ml R-spondin-1, and 0.5-5v% fetal bovine serum.
Wherein the basal medium is at least one selected from ADVANCED DMEM/F12 medium, DMEM, high-sugar DMEM medium, alpha MEM medium and RPMI1640 medium.
Wherein, v% refers to the volume percentage of some substance in ADVANCED DMEM/F12. For example, 0.5-2v% B27 means that the volume of B27 is 0.5-2mL in 100mL Advanced DMEM/F12.
Wherein the concentration of HEPES buffer solution in ADVANCED DMEM/F12 is 2-20mM, which can be 2-5mM, 5-10mM, 10-15mM, 15-20mM; specifically, the concentration may be 5, 10, 15 or 20mM. Preferably 10-15mM; still more preferably, it is 10mM.
Wherein, the concentration of Glutamax in ADVANCED DMEM/F12 is 1-5mM, which can be 1-1.5mM, 1.5-2mM, 2-2.5mM, 2.5-3mM, 3-4mM, 4-5mM, and can be 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mM. Preferably 0.5-3mM; further preferably, 1-2.5mM; still more preferably, it is 2mM.
Wherein Primocin in ADVANCED DMEM/F12 has a concentration of 0.2-2mg/ml, which may be 0.2-0.5mg/ml, 0.5-1mg/ml, 1-1.5mg/ml, 1.5-2mg/ml, specifically 0.2, 0.5, 1, 1.5, 2mg/ml. Preferably 0.5-1.5mg/ml; still more preferably, it is 1mg/ml.
Wherein the B27 supplement accounts for ADVANCED DMEM/F12 in volume percent of 0.5-5v%, can be 0.5-1.0v%, 1.0-2v%, 2.5-3v%, 3.5-4v%, 4-4.5v%, 4.5-5v%, and can be 0.5, 0.8, 1.0, 1.5, 2.0, 3.0, 4.0, 5v%. Preferably 1-3%; further preferably, 1.5 to 2.5%; still more preferably, it is 2%.
Wherein the concentration of N-ACETYLCYSTEINE in ADVANCED DMEM/F12 is 0.75-2mM, which can be 0.75-1mM, 1-1.2mM, 1.2-1.5mM, 1.5-2mM, and specifically can be 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5mM. Preferably 0.8-1.2mM; still more preferably, it is 1mM.
Wherein, the concentration of rhEGF in ADVANCED DMEM/F12 is 20-100ng/ml, which can be 20-40ng/ml, 40-60ng/ml, 60-80ng/ml, 80-100ng/ml, and can be 20, 30, 40, 50, 60, 70, 80, 90, 100ng/ml. Preferably, 20-80ng/ml; further preferably, it is 35-70ng/ml; still more preferably 50ng/ml.
Wherein the concentration of A83-01 in ADVANCED DMEM/F12 is 0.05-0.3. Mu.M, which can be 0.05-0.08. Mu.M, 0.08-0.1. Mu.M, 0.1-0.15. Mu.M, 0.15-0.2. Mu.M, 0.2-0.25. Mu.M, 0.25-0.3. Mu.M, and can be specifically 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.22, 0.25, 0.3. Mu.M. Preferably 0.08-0.15. Mu.M; still more preferably 0.1. Mu.M.
Wherein, the concentration of Nicotinamide in ADVANCED DMEM/F12 is 2-20mM, which can be 2-5mM, 5-8mM, 8-10mM, 10-12mM, 12-15mM, 15-20mM, and can be 2, 4,6, 8, 10, 12, 14, 16, 18, 10mM. Preferably 5-15mM; preferably 8-12mM; still more preferably, it is 10mM.
Wherein the concentration of Wnt3a in ADVANCED DMEM/F12 is 300-800ng/ml, which can be 300-350ng/ml、350-400ng/ml、400-450ng/ml、450-500ng/ml、500-550ng/ml、550-600ng/ml、600-650ng/ml、650-700ng/ml、700-750ng/ml、750-800ng/ml,, and specifically 300, 400, 500, 600, 700 and 800ng/ml. Preferably 380-650ng/ml; further preferably, it is 400-600ng/ml; still more preferably 500ng/ml.
Wherein the concentration of Noggin in ADVANCED DMEM/F12 is 0.05-0.3 μg/ml, which can be 0.05-0.1 μg/ml, 0.1-0.15 μg/ml, 0.15-0.2 μg/ml, 0.2-0.25 μg/ml, 0.25-0.3 μg/ml, and can be specifically 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.22, 0.25, 0.28, 0.3 μg/ml. Preferably 0.05-0.15. Mu.g/ml; further preferably, 0.08-0.12. Mu.g/ml; still more preferably, it is 0.1. Mu.g/ml.
Wherein the concentration of R-spondin-1 in ADVANCED DMEM/F12 is 0.05-0.3 μg/ml, which can be 0.05-0.1 μg/ml, 0.1-0.15 μg/ml, 0.15-0.2 μg/ml, 0.2-0.25 μg/ml, 0.25-0.3 μg/ml, and can be specifically 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.22, 0.25, 0.28, 0.3 μg/ml. Preferably 0.05-0.15. Mu.g/ml; further preferably, 0.08-0.12. Mu.g/ml; still more preferably, it is 0.1. Mu.g/ml.
In some preferred embodiments, the medium for culturing a mucosal melanoma 3D organoid comprises: 500ml Advanced DMEM/F12 (Gibco), and the following based on ADVANCED DMEM/F12: 10mM HEPES buffer, 2mM Glutamax, 1mg/ml Primocin, 2v% B27 supplement, 1mM N-ACETYLCYSTEINE, 50ng/ml rhEGF, 0.1. Mu. M A83-01, 10mM Nicotinamide, 500ng/ml Wnt3a, 0.1. Mu.g/ml Noggin, and 0.1. Mu.g/ml R-spondin-1.
The organoid model derived from the epithelial tissue of the fetal bovine serum is an optional additive, and the addition of the fetal bovine serum is beneficial to the growth of the organoid of the mucous membrane melanoma in the culture of the organoid of the mucous membrane melanoma. Wherein, the fetal bovine serum accounts for ADVANCED DMEM/F12 of 0.5-5v percent. Can be 0.5-1.0v%, 1.0-1.5v%, 1.5-2v%, 2.5-3v%, 3.5-4v%, 4-4.5v%, 4.5-5v%, and specifically can be 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0, 2.5, 3, 3.5, 4, 4.5, 5%. Preferably 0.8-3v%; further preferably, 1.5 to 2.5v%; still more preferably, it is 2v%.
In some more preferred embodiments, the medium comprises: 500ml Advanced DMEM/F12 (Gibco), and the following based on ADVANCED DMEM/F12: 10mM HEPES buffer, 2mM Glutamax, 1mg/ml Primocin, 2v% B27 supplement 、1mM N-Acetylcysteine、50ng/ml rhEGF、0.1μM A83-01、10mM Nicotinamide、500ng/ml Wnt3a、0.1μg/ml Noggin、0.1μg/ml R-spondin-1, and 2v% fetal bovine serum.
The culture medium for culturing the mucosa melanoma 3D organoids, which is preferred by the invention, further comprises at least one of Y-27632, FGF-10, dexamethasone, N2, FGF-2, prostaglandin E2, CHIR 99021, forskolin, nicotinamide and dexamethasone. Preferably, the medium further comprises Y-27632, FGF-10, dexamethasone, N2, FGF-2, prostaglandin E2, CHIR 99021 and Forskolin. In some preferred embodiments, the medium comprises: basal medium, the following components based on basal medium: HEPES buffer, glutaMAX, primocin, B supplement, N-ACETYLCYSTEINE, RHEGF, A83-01, nicotinamide, wnt3a, noggin, R-spondin-1, fetal bovine serum, Y-27632, FGF-10, dexamethasone, N2, FGF-2, prostaglandin E2, CHIR 99021, forskolin. In other preferred embodiments, the medium comprises: basal medium, the following components based on basal medium: HEPES buffer, glutaMAX, primocin, B supplement, N-ACETYLCYSTEINE, RHEGF, A83-01, nicotinamide, wnt3a, noggin, R-spondin-1, fetal bovine serum, Y-27632, FGF-10, dexamethasone, N2, FGF-2, prostaglandin E2, CHIR 99021, forskolin, nicotinamide and dexamethasone.
Wherein the concentration of Y-27632 in ADVANCED DMEM/F12 is 2-20. Mu.M, which can be 2-5. Mu.M, 5-8. Mu.M, 8-10. Mu.M, 10-12. Mu.M, 12-15. Mu.M, 15-20. Mu.M, and can be 2, 4, 6, 8, 10, 12, 14, 16, 18, and 10. Mu.M. Preferably 5-15. Mu.M; preferably 8-12. Mu.M; still more preferably, it is 10. Mu.M.
Wherein, the concentration of FGF-10 in ADVANCED DMEM/F12 is 50-300ng/ml, which can be 50-100ng/ml, 100-150ng/ml, 150-200ng/ml, 200-250ng/ml, 250-300ng/ml, and can be 50, 80, 100, 120, 150, 180, 200, 220, 250, 280, 300ng/ml. Preferably, 60-180ng/ml; further preferably, it is 80-120ng/ml; still more preferably, it is 100ng/ml.
Wherein Dexamethasone has a concentration of 0.75-2. Mu.M, which may be 0.75-1. Mu.M, 1-1.2. Mu.M, 1.2-1.5. Mu.M, 1.5-2. Mu.M, and specifically 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5. Mu.M in ADVANCED DMEM/F12. Preferably 0.8 to 1.2. Mu.M; still more preferably, 1. Mu.M.
Wherein, the N2 accounts for ADVANCED DMEM/F12 with the volume percentage of 0.5-3v%, can be 0.5-1.0v%, 1.0-1.5v%, 1.5-2v%, 2.5-3v%, and can be 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0, 2.5 and 3%. Preferably 0.6-2%; further preferably, 0.8 to 1.5%; still more preferably, 1%.
Wherein the concentration of FGF-2 in ADVANCED DMEM/F12 is 3-12ng/ml, and can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12ng/ml. Preferably 3-9ng/ml; further preferably, it is 4-7ng/ml; still more preferably, it is 5ng/ml.
Wherein Prostaglandin E.about.2 in ADVANCED DMEM/F12 has a concentration of 0.5 to 3. Mu.M, which may be 0.5 to 0.75. Mu.M, 0.75 to 1. Mu.M, 1 to 1.2. Mu.M, 1.2 to 1.5. Mu.M, 1.5 to 2. Mu.M, 2 to 2.5. Mu.M, 2.5 to 3. Mu.M, and particularly may be 0.5, 0.8, 1, 1.2, 1.5, 2, 2.5, 3. Mu.M. Preferably 0.8 to 1.2. Mu.M; still more preferably, 1. Mu.M.
Wherein the concentration of CHIR 99021 in ADVANCED DMEM/F12 is 0.1-0.3. Mu.M, specifically 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.22, 0.24, 0.26, 0.28, 0.3. Mu.M. Preferably 0.15 to 0.25. Mu.M; still more preferably 0.2. Mu.M.
Wherein the concentration of Forskolin in ADVANCED DMEM/F12 is 0.5-3. Mu.M, which can be 0.5-0.75. Mu.M, 0.75-1. Mu.M, 1-1.2. Mu.M, 1.2-1.5. Mu.M, 1.5-2. Mu.M, 2-2.5. Mu.M, 2.5-3. Mu.M, and can be 0.5, 0.8, 1, 1.2, 1.5, 2.5, 3. Mu.M. Preferably 0.8 to 1.2. Mu.M; still more preferably, 1. Mu.M.
Wherein Antibiotic-Antimycotic accounts for ADVANCED DMEM/F12 in volume percent of 0.5-3v%, can be 0.5-1.0v%, 1.0-1.5v%, 1.5-2v%, 2.5-3v%, and can be specifically 0.5, 0.8, 1.0, 1.2, 1.5, 1.8, 2.0, 2.5, 3v%. Preferably 0.6-2v%; further preferably, 0.8 to 1.5v%; still more preferably, 1v%.
Wherein the concentration of the nicotinamide in ADVANCED DMEM/F12 is 5-30mmol/L; can be 5-10, 10-15, 15-20, 20-25, 25-30mmol/L.
Wherein the concentration of dexamethasone in ADVANCED DMEM/F12 is 0.2-2 mu mol/L.
In some more preferred embodiments, the medium comprises: 500ml Advanced DMEM/F12 (Gibco), and the following based on ADVANCED DMEM/F12: 10mM HEPES buffer, 2mM Glutamax, 1mg/ml Primocin, 2v% B27 supplement 、1mM N-Acetylcysteine、50ng/ml rhEGF、0.1μM A83-01、10mM Nicotinamide、10μM Y-27632、100ng/ml FGF-10、1μM Dexamethasone、1v％N2、5ng/ml FGF-2、1μM Prostaglandin E2、0.2μM CHIR 99021、1μM Forskolin、500ng/ml Wnt3a、0.1μg/ml Noggin、0.1μg/ml R-spondin-1、1v％Antibiotic-Antimycotic、, and 2v% fetal bovine serum.
The culture medium and the method can obtain the organoids with tissue morphology and genotyping which are highly consistent with that of real tissues. In the culture process, the proportion of living cells is ensured to the greatest extent, and the culture time can be shortened. The cell factors and the regulating factors are directly and closely affected, are coordinated and matched, and are combined with unique digestive enzymes and digestion methods, and an inoculation culture method, so that the mucous membrane melanoma tissue cells can better show the inherent activity characteristics in the culture process, and the comprehensive characteristics highly approximate to that of living mucous membrane melanoma tissues are realized. The mucosal melanoma organoids obtained by culturing with the culture medium of the invention are aggregated into clusters, which are similar to the morphology and structure of active mucosal melanoma tissues.
The invention also provides application of the culture medium in culturing the mucosa melanoma 3D organoids. Aiming at the culture and growth characteristics of cells derived from the mucosal melanoma tissues, the culture medium of the mucosal melanoma organoids selects a plurality of cytokine components to compound according to a specific proportion, and the obtained culture medium contains a proper amount of cytokines and signal path regulating factors, so that the mucosal melanoma cells can form 3D organoids which are close to the shape and the properties of real tissues in a short time. The culture medium has low cost, strong operability and good repeatability, and the organoids subjected to multiple subculture can still ensure that the morphology of the organoids and the expression level of cytokines are close to that of real tissues.
Preferably, the mucosal melanoma tissue of the invention is derived from mucosal melanoma PDX tissue or human mucosal melanoma tumor tissue.
The invention also provides a mucous membrane melanoma 3D organoid cultured by the culture method. The 3D organoids of the mucous membrane melanoma can simulate the physiological environment of mucous membrane melanoma cells, can effectively maintain the specificity of tissue cells, and have high consistency of tissue morphology and genotyping;
The invention also provides application of the organoid in serving as a drug evaluation model, wherein the drug evaluation model is an antitumor drug evaluation.
The invention also provides an evaluation method of the antitumor drug, which comprises the following steps:
(1) Culturing the obtained organoids using said method;
(2) Evaluation of antitumor drug sensitivity was performed using organoid tissues.
Because the 3D organoid tissue morphology and genotyping of the mucous membrane melanoma constructed by the invention are highly consistent with those of in-vivo tumors, the method can be used for screening the therapeutic drugs of mucous membrane melanoma, truly reflects the therapeutic effects of the drugs, greatly simplifies the drug screening process and meets the scientific research needs.
Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts.
Materials and methods
1. Step of culturing mucosal melanoma organoid
The collected tissue sample is prepared into single cell suspension according to an enzyme digestion method, and the sample can be derived from clinical samples and PDX tumor bodies, and the specific steps are as follows.
1. Obtaining single cell suspension by enzyme digestion method
Preparing digestive juice in advance: a tissue digest (1640 medium containing 1mg/ml type IV collagenase, 200U/L hyaluronidase, 200U/L Dnase I,0.05% Tyrpsin-EDTA) was prepared.
The specific flow is as follows:
i. if the mucosa melanoma sample is derived from a biopsy sample of a bacterial part such as the alimentary canal, the nasal cavity and the oral cavity, the tumor tissue is placed in 75% alcohol for 3 seconds, if the sample has necrotic burst, the sample is soaked in 0.05% sodium hypochlorite for 10 seconds (if the mucosa melanoma is transplanted tumor PDX-derived tumor tissue sample, the tumor tissue sample is not treated by alcohol and sodium hypochlorite). Surface necrotic tissue was removed and then washed once with PBS containing 5% penicillin-streptomycin, 1 x Primocin antibiotics; the inside of the tumor was observed for necrosis, and if so, the tumor was removed and washed once with PBS as described above.
Placing the cleaned tumor tissue in a small 6cm dish, and shearing the tissue into tissue blocks with the size of 1-3mm 3 by using fibrous surgical scissors; 1-2 pieces of tissue were placed in 10% formalin or 4% paraformaldehyde for subsequent histological analysis;
centrifuging the sheared tissue at 4 ℃ for 200g and 3min, and removing the supernatant.
Tissue digestion: the digestion liquid is prepared by the following steps: tumor tissue 10:1 volume ratio tissue digest was added to tumor tissue, followed by C-tube (meitian gentle) and mixed well. The Meitian gentle tissue separation instrument was adjusted to Tumor.03.01 mode (suitable for softer texture tumors) for coarse tissue separation. If the device is not used for beauty and gentle, the tissue can be sheared by scissors as much as possible, and the experimental requirements can be generally met.
And v, placing the c-tube after the program is run on a shaking table at 37 ℃ to digest for 30-60min, closely observing the digestion condition of tumor tissues, slightly vibrating the c-tube every 15-20min, and avoiding the influence of tissue agglomeration on digestion.
When the digestive juice becomes turbid, the general suspension of the mucous membrane melanoma sample is black brown, the centrifuge tube is shaken, and when no obvious residual tumor tissue is found, the digestion is stopped, an equal volume of 1640 culture medium containing 10% FBS,1% penicillin-streptomycin and 1X Primocin antibiotics is added, and the supernatant is removed by centrifugation at 200g for 3min at 4 ℃.
Resuspension with about 10ml1640 medium, filtering the incompletely dissociated tissue with 100 μm sieve. The filtration was carried out twice with a 70 μm sieve. The cell suspension obtained by filtration was centrifuged at 4℃for 200g and 3min, and the supernatant was removed.
Re-suspension of cells with 10ml 1640 medium, counting. The cell suspension was centrifuged and the cells were resuspended to 5 x 10 5/mL of cell suspension for use with pre-chilled mucosal melanoma medium, depending on cell density and experimental requirements.
2. Organoid inoculation
During the above steps of digesting cells, organoid inoculation experiment reagent consumables are prepared.
The culture plate (such as 24-hole or 48-hole plate) can be placed into an incubator for preheating for at least 30min before a series of operations are started;
The culture medium of the mucous membrane melanoma organoid is placed in an incubator in advance for preheating;
The BME glue is put on ice to be melted;
The gun head for experiments needs to be refrigerated and precooled in advance;
The specific steps of inoculating the mucosal melanoma organoid are as follows:
i. And mixing a proper amount of the cell suspension stock solution prepared in the steps with BME glue according to the volume ratio of 1:1, and resuspending cells.
Note that: and (3) operating on ice, and soaking the pipette tip by using a conventional culture medium containing serum before sucking cells, namely, sucking the culture medium and then beating out the culture medium to prevent adhesion loss of cells.
Taking out the preheated 24-well plate, dripping the mixture of cells and BME at the middle position of the bottom of the 24-well plate culture plate, wherein each drop is about 30-40 mu l (20-50 ul) (shown in figure 1), and the pigment is gradually reduced in the later period with the increase of passage times due to the fact that melanoma generally contains a large amount of pigment, and the gel drops are generally black chocolate-like due to a large amount of pigment, cell fragments and the like during the first inoculation. And (3) injection: the operation is rapid, and the glue is prevented from solidifying.
Inverting the plates in a 37 ℃ incubator for 30min, solidifying the BME droplets, inverting the plates and carefully adding mucosal melanoma organoid medium along the side wall in each well, typically 500 μl in 24 plates, based on the gel-free spheres.
Placing the culture plate in a 37 ℃ incubator for culture, replacing the culture medium every 3-5 days, carefully sucking out the culture medium from each hole, and then adding fresh preheated mucous membrane melanoma organoid culture medium (the glue which can be prevented from solidifying by preheating is in a liquid state), and ideally, carrying out first passage (different tissue growth speeds) after 14-21 days.
Organoid morphology observations were periodically made during the culture period, recorded by photographing, and small cell spheres were generally visible around 7 days (fig. 2A), with a significant increase in cell spheres seen for 21 days (fig. 2B-C), and partially visible to the naked eye (fig. 3).
3. Mucosal melanoma organoid passaging
The nature of organoid passage is that the original glue is destroyed and removed, and on the basis, the cell spheres formed by the previous generation culture are changed into single-cell multi-micro cell clusters by a mechanical blowing method or an enzyme digestion method, and the organoid is formed again by passage growth. The spheres are typically passaged or used for identification as long as 50-100 μm in size.
Mechanical blowing method
I. The culture medium in the well plate was aspirated, pre-chilled PBS was added thereto, the colloid was blown using a 1ml gun head, the colloid was broken by mechanical disruption, PBS was aspirated together with the broken colloid and the cell pellet into a 15ml centrifuge tube, and a small amount of pre-chilled PBS was added to the centrifuge tube.
Centrifuging at low temperature of 4deg.C, and centrifuging at 500-700g for 3min; after centrifugation, the supernatant was aspirated (without dumping), resuspended in 5ml of ice PBS, and blown off to blow off the cell pellet until no apparent cell pellet was visible to the naked eye.
Centrifuging at low temperature of 4deg.C, and centrifuging at 500-700 (g) for 3min; the supernatant was discarded, and a proper amount of pre-chilled medium was added to mix with matrigel, and the cell suspension stock and BME gel resuspended cells at 1:1.
If the spheres cannot be broken mechanically, the spheres are passaged by enzyme digestion, and the method is as follows:
i. Sucking out the culture medium in the pore plate, adding precooled PBS, blowing the blue gun head against the colloid, crushing the colloid by a mechanical crushing method, sucking the PBS together with the crushed colloid and cell balls into a 15ml centrifuge tube, and adding a small amount of precooled PBS into the centrifuge tube in advance. (the glue was discarded along with the PBS).
Resuspension with ice PBS, gentle blowing, standing until the big pellet is precipitated to the bottom, and sucking most supernatant into a new 15ml centrifuge tube A (because some pellets in the supernatant contain single cells generated by the mechanical blowing in the last step); 2ml of pancreatin is added into the centrifuge tube, and the mixture is digested for 3 to 4 minutes, and the mixture is gently blown for a few minutes when the mixture is digested for 2 minutes. The digestion was then stopped with serum medium and the liquid in centrifuge tube A was transferred to an old centrifuge tube for centrifugation, 300-500g,3min, and the supernatant discarded.
Resuspension with medium, the density is the same as that of inoculation, and the cells are resuspended according to the ratio of 1:1 according to the cell suspension stock solution and BME gel, and then inoculated.
4. Mucous membrane melanoma organoid cryopreservation
The procedure for obtaining the cell suspension was as described in point1 above. Centrifuging, removing supernatant, resuspending organoid cell suspension with 10%DMSO+90%FBS 1ml, transferring into a freezing tube, placing into a gradient cooling box, cooling in a refrigerator at-80deg.C, and transferring to liquid nitrogen tank for storage the next day.
5. Embedding of mucosal melanoma organoids
When the length of the organoid tube sphere is 50-100 mu m, the sphere is collected and can be identified by adopting a frozen section or paraffin embedding method. The paraffin embedding method mainly comprises the steps of wrapping agar and then embedding the agar. The method comprises the following steps:
i. drawing and fixing: the spheroids to be passaged and identified are collected according to the anterior segment method, washed and centrifuged by PBS, and PBS is removed. The 10% formalin solution was fixed at room temperature for 2h.
The cell spheres were transferred to a disposable plastic embedding mold using a 1ml pipette, with the tip minus 5mm. The fixing liquid in the grinding tool is sucked as much as possible, so that the ball body is prevented from being sucked away.
Pre-embedding and demolding: the agar solution is preheated to a viscous state at 70 ℃ in advance by a water bath kettle, hot agar is slowly dripped into a disposable plastic embedding mould containing spheres along four corners of the mould, and the mixture is slowly added onto tissues until the agar wraps all the tissues.
And iv, after the agar solidifies at room temperature, sequentially placing the pre-embedded tissue together with the mold into 70% alcohol, 85% alcohol, 95% alcohol and 100% alcohol for dehydration for 5-10min each time. This facilitates demolding of the agar block.
V. dehydration: sequentially adding 70% alcohol 30min,85% alcohol 30min,95% alcohol 30min, and 100% alcohol 2 hr into the agar block.
Transparent and wax-dipping: the process is the same as the conventional embedding method.
Staining of the embedded sections.
6. Identification of consistency of PDO (PDO) of mucous membrane melanoma and primary focus
Macroscopic tumor tissues obtained by PDO growth are embedded according to the method, the sections are subjected to HE staining, and the tissue morphology comparison is carried out with the clinical patient tumor tissue HE staining. Meanwhile, the mucosa melanoma and the corresponding patient primary focus mucosa melanoma related markers (S100, HMB45, melan-A) and the epithelial markers AE1/AE3 were subjected to immunohistochemical staining (red staining) comparison.
The immunohistochemical staining procedure was as follows:
Cutting wax block into 4 μm thick slices, performing immunohistochemical staining, heating in a sodium citrate repair liquid water bath, performing antigen repair, removing pigment with 3% hydrogen peroxide for 60min, adding the primary antibody after PBS washing, washing at room temperature (22-25 ℃) for 1.5 hours, washing the primary antibody with room temperature PBS for 10min×3 times, adding the secondary antibody for room temperature reaction for 30min, washing the secondary antibody with room temperature PBS for 10min3 times, performing AEC red development for 9min, counterstaining with hematoxylin for 9min, and sealing with water-soluble sealing tablet.
Mucosal melanoma tissues occurring in the oral cavity are selected for performing immunohistochemical staining of epithelium and melanoma tissues, and are used as positive and negative comparison references, including oral mucosal epithelium (Epithelium) and mucosal melanoma tissues (melanoma). Such as: HMB45 is expressed in melanoma tissue, while in mucosal epithelium this HMB45 expression is negative. The epithelial marker AE1/AE3 was positive in mucosal epithelium, while AE1/AE3 was negative in melanoma tissue (FIG. 4).
Example 1 mucosal melanoma organoid culture
The specimen source was mucosal melanoma PDX tumor tissue successfully constructed in previous studies (FIG. 5). Construction of PDX according to the present invention, a biopsy or surgical tissue derived from a patient is inoculated subcutaneously into an immunodeficient mouse. Specifically, a disposable sterile culture dish is poured with an appropriate amount of one portion of 0.04% sodium hypochlorite and three portions of PBS or serum-free medium with 1% penicillin-streptomycin double antibody. Taking out the tumor tissue of the in-vitro mucous membrane melanoma, cleaning the tumor tissue in a sodium hypochlorite solution for 10 seconds, rapidly putting the tumor tissue into PBS or a serum-free culture medium to wash away sodium hypochlorite on the surface of the tumor, trimming the necrosis and fibrous tissue on the surface of the tumor tissue by using a sterile instrument, cleaning the tumor tissue twice by using PBS or the serum-free culture medium for 2 minutes each time, and cutting the tissue into small blocks with the size of 2-3 mm. The anesthetized mouse flank was sterilized with alcohol, an incision approximately 5mm in size was made by surgical scissors, the flank was slowly blunt-separated from the flank to the armpit and inguinal using microscopic forceps, tumor tissue was inoculated to the armpit and inguinal using microscopic forceps, and approximately 2 to 3 tumor tissues were inoculated to each site. After transplanting the tumor subcutaneously, it was used in the subsequent experiments. The PDX tumor source patient is a female oral mucosa melanoma patient, the primary focus is positioned on the maxillary gingiva, and the postoperative pathological diagnosis is as follows: melanoma of mucous membrane.
Specific implementation steps referring to the experimental steps, this embodiment is briefly described as follows:
The collected tissue samples were prepared as single cell suspensions according to the enzymatic digestion method used above. After counting, cells were resuspended to 5 x 10 5/mL of cell suspension for use with pre-chilled mucosal melanoma medium.
The cell suspension was mixed with BME gel at 1:1, seeded in 24 well plates, 40 μl per well, and inverted in an incubator for 30 minutes. 500. Mu.L of mucosal melanoma medium was added. The specific ingredients added to ADVANCED DMEM/F12 medium are shown in the following Table (based on ADVANCED DMEM/F12 medium):
Organoid morphology observations were made periodically during the culture period, and photographic recordings were made, with cell clumps generally visible after 7 days of culture (FIG. 6A), and cell spheroids significantly increased after 21 days of culture (FIGS. 6B-C).
Further collecting organoid spheres and fixing paraffin by using an agar embedding method; the collected spheres were visible to the naked eye (fig. 7).
Identification of organoid spheres
After embedding the spheres, HE staining was performed on paraffin sections (fig. 8), as can be seen from fig. 8;
The organoids prepared in this example were consistent with primary focal tumor expression molecules as shown in fig. 9 by comparing organoid tissues with immunohistochemical staining (red staining) of the corresponding primary focal mucosal melanoma-associated markers (S100, HMB45, melan-a) and the epithelial markers AE1/AE 3.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims (7)

1. A culture medium for culturing a mucosal melanoma 3D organoid, the culture medium comprising: ADVANCED DMEM/F12 medium, the following components based on ADVANCED DMEM/F12 medium: 10mM HEPES buffer, 2mM Glutamax, 1mg/ml Primocin, 2v% B27 supplement, 1mM N-ACETYLCYSTEINE, 50ng/ml rhEGF, 0.1 μ M A-01, 10mM Nicotinamide, 500ng/ml Wnt3a, 0.1 μg/ml Noggin 0.1μg/ml R-spondin-1、10μM Y-27632、100ng/ml FGF-10、1μM Dexamethasone、1％N2、5ng/ml FGF-2、1μM Prostaglandin E2、0.2μM CHIR 99021、1μM Forskolin、1％Antibiotic-Antimycotic、2％FBS.
2. Use of the medium according to claim 1 for culturing mucosal melanoma 3D organoids.
3. A method for culturing a mucosal melanoma 3D organoid, comprising culturing crushed mucosal melanoma cells with the culture medium according to claim 1 to obtain the mucosal melanoma 3D organoid.
4. A culture method according to claim 3, wherein the mucosal melanoma is selected from a primary sample derived from a patient suffering from mucosal melanoma or a tumour tissue derived from mucosal melanoma PDX tissue.
5. A mucosal melanoma 3D organoid cultivated by the cultivation method of claim 3 or 4.
6. Use of the organoid of claim 5 as a drug evaluation model for antitumor drug evaluation.
7. The method for evaluating the antitumor drug is characterized by comprising the following steps of:
(1) Using the organoid of claim 5;
(2) Evaluation of antitumor drug sensitivity was performed using organoid tissues.
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