CN114736865A - Salivary gland malignant polymorphic adenoma 3D organoid and culture method and application thereof - Google Patents
Salivary gland malignant polymorphic adenoma 3D organoid and culture method and application thereof Download PDFInfo
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- CN114736865A CN114736865A CN202210226798.6A CN202210226798A CN114736865A CN 114736865 A CN114736865 A CN 114736865A CN 202210226798 A CN202210226798 A CN 202210226798A CN 114736865 A CN114736865 A CN 114736865A
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Abstract
The invention belongs to the technical field of organoid culture, and discloses a 3D organoid of malignant polymorphic adenoma of salivary glands and a culture method and application thereof. The invention also discloses a culture medium for culturing the malignant pleomorphic adenoma 3D organoid, aiming at the growth characteristics of malignant pleomorphic adenoma cells, a plurality of cell factor components are selected and compounded according to a certain proportion, and the organoid can be effectively formed in a 3D environment. The culture medium and the method are particularly suitable for culturing salivary gland malignant polymorphic adenoma organoids, and have important significance for treating targets of salivary gland malignant polymorphic adenomas, screening drugs, researching tumorigenesis, malignant transformation mechanisms and the like.
Description
Technical Field
The invention relates to the technical field of organoid culture, and relates to a 3D organoid of malignant polymorphic adenoma of salivary glands and a culture method and application thereof.
Background
Pleomorphic Adenomas (PA) are the most common benign tumors of salivary glands, accounting for 73.8% of all salivary gland benign tumors [ Tian Z; li L; wang L; hu Y; li J; a23-year responsive output test of 6982cases in an enterprise Chinese patent publication, International Journal of industry and maxilla responsive output 2010,39(3):235-242 ]. Although it is benign, the adenomas are prone to recurrence, multiple recurrence or long-standing, are highly susceptible to malignant transformation, and become malignant adenomas (MPA), also called malignant mixed tumor (refractory tumor) or carcinoma among the adenomas are known as pleomorphic adenomas (carcinosoma in pleomorphic adenoma). Malignant polymorphic adenomas are not uncommon in our country, and statistics of this subject group show that the incidence of malignant polymorphic adenomas (accounting for 8.0% (first) of all malignant tumors of salivary glands) is much higher than WHO (3.6%) [ EI-Naggar AK; chan JKC; grandis JR; takashi T; slootweg PJ; the World Health Organization classification of head and wick tumors, IARC: Lyon,2017, 159-. After malignant adenoma is malignant, once malignant cells break through the outside of the envelope by more than 1.5mm, invasive malignant adenoma is formed, the prognosis of the patient becomes worse suddenly, the biological behavior turns into high malignancy, regional lymph node metastasis and liver, lung, bone and other distant metastasis can occur, and the 5-year survival rate is only 25%. Currently, malignant polymorphic adenomas still lack effective treatment means, mainly including surgical resection, combined with postoperative radiotherapy and chemotherapy, but have poor curative effect. Therefore, the search of prognosis evaluation and effective therapeutic targets of malignant polymorphic adenomas to improve the survival rate of patients is an important subject of research in the field.
The tumor preclinical model can reproduce the original genomics information of tumor patients, combines the genomics information with pharmacogenomics, is one of the important means for screening preclinical drugs, excavating new treatment targets and optimizing treatment strategies, and highlights the bridge function of deep integration of the preclinical model as basic research result and conversion application research. The preclinical model mainly comprises a cell line, a human-derived tissue xenograft model (PDX), Organoids (Organoids) and the like.
Organoids are 3D cell clusters that are formed by organ-specific adult stem cells, embryonic stem cells, or induced pluripotent stem cells that differentiate and self-assemble, and that replicate certain characteristics and functions of tissues and organs. Organoid technology can be organically integrated with other biotechnology, including gene editing, single cell genomics, real-time imaging and microfluidics, thereby providing a new visual angle for understanding pathogenesis and development process of diseases and transforming new diagnosis and treatment technology. Researchers in various fields simulate various tissue types to construct various organs, such as developmental biology, precise medicine, disease modeling, regenerative medicine, toxicology and drug development. The newly released guiding principles (trial implementation) of gene therapy product non-clinical research and evaluation techniques in medicine evaluation and assessment by the national food and drug administration (CDE) clearly indicates that organoids can be used as a substitute model of non-clinical research animal models and as an evaluation model of effectiveness and safety of therapeutic drugs.
Although various human tissue 3D organoids can be obtained by in vitro culture, no study report on the culture method of salivary gland malignant polymorphic adenoma 3D organoids is available. Because the incidence of malignant polymorphic adenomas in Chinese population is significantly higher than that reported by WHO, and the treatment means for malignant polymorphic adenomas is lacking and the prognosis of patients is poor at present, the development of organoid models of malignant polymorphic adenomas and related diseases is urgent, and the establishment of such models lays a solid foundation for the successful development of malignant polymorphic adenoma treatment targets and drug screening work.
Disclosure of Invention
In view of the above disadvantages of the prior art, the present invention aims to provide a salivary gland malignant polymorphic adenoma 3D organoid, and a culture method and applications thereof, which are used to solve the problems of the prior art that there is no culture medium and culture method for salivary gland malignant polymorphic adenoma 3D organoid, and achieve the purpose of obtaining 3D organoid with tissue cell morphology, protein expression, etc. highly consistent with the real tumor of malignant polymorphic adenoma of a patient.
In order to achieve the above objects and other related objects, the present invention provides a 3D organoid of malignant polymorphic adenoma of salivary glands, a culture method and applications thereof.
One of the objects of the present invention is to provide a medium for culturing salivary gland malignant pleomorphic adenoma 3D organoids, the medium comprising: advanced DMEM/F12, and the following components based on Advanced DMEM/F12: 0.5-2 v% of antimicrobial-antimicrobial, 0.5-2 v% of GlutaMAX, 0.5-2 v% of HEPES buffer solution, 50-300 ng/ml of fiber forming growth factor, 50-300 ng/ml of EGF, 5-25 mu g/ml of bFGF, 300-800 nmol/L of TGF-beta I type receptor inhibitor, 300-800 ng/ml of Wnt3a recombinant protein, 0.05-0.3 mu g/ml of Noggin recombinant protein, 0.05-0.3 mu g/ml of R-spondin-1 recombinant protein, 0.75-2.5 mmol/L N-acetylcysteine, 5-30 mmol/L of nicotinamide, 0.5-5 v% of B27 supplement, 0.2-5 mu mol/L of dexamethasone and 0.2-5 mg/ml of Primocin.
The culture medium preferably further comprises one or more of N-2 supplement, FGF2 and Prostaglandin E2.
Another object of the present invention is to provide the use of the culture medium as described above for culturing salivary gland malignant polymorphic adenoma 3D organoids.
Another objective of the present invention is to provide a method for culturing malignant polymorphic adenoma 3D organoid of salivary gland, which comprises culturing the disrupted malignant polymorphic adenoma cells in the above culture medium to obtain the malignant polymorphic adenoma 3D organoid of salivary gland.
In the present invention, the salivary gland malignant polymorphic adenoma tumor tissue is selected from human.
As mentioned above, the salivary gland malignant polymorphic adenoma 3D organoid, the culture method and the application thereof have the following beneficial effects:
the culture medium of the salivary gland malignant multiform adenoma organoid aims at the growth characteristics of salivary gland malignant multiform adenoma source cells, multiple cell factor components are selected and compounded according to a specific proportion, and the obtained culture medium contains appropriate content of cell factors and signal channel regulation factors, so that malignant multiform adenoma cells can form 3D organoids which are close to the shape and the property of real tumor tissues in a short time.
The culture medium and the culture method can complete the subculture of the salivary gland malignant polymorphic adenoma tissues, can keep the form and the property of a 3D organoid, and meet the requirement of massively replicating the salivary gland malignant polymorphic adenoma organoid.
The 3D organoid obtained by the culture medium or the method can simulate the survival microenvironment of the real salivary gland malignant pleomorphic adenoma tumor cells, can effectively maintain the cell tissue specificity, and has highly consistent cell tissue morphology and protein expression; the kit can be used for screening therapeutic drugs for malignant polymorphic adenomas, researching the occurrence and development mechanism of malignant polymorphic adenomas, screening biomarkers related to malignant polymorphic adenomas and the like, not only meets the requirements of scientific research, but also provides beneficial choices in the aspect of clinical medication guidance, such as providing medication guidance for patients after in vitro 3D culture of tumor biopsy sample tissues of the patients and the like.
Drawings
FIG. 1 is a photograph of example 1 after inoculation of step (1.5) into a culture plate.
FIG. 2 is the microscopic representation (10X magnification) of primary cultured organoids of malignant polymorphic adenomas of example 1 at day 10.
FIG. 3 is the microscopic representation (10X magnification for both) of malignant polymorphic adenomas in example 1 at day 6 in different fields of view in organoid culture of passage 2.
FIG. 4 is the microscopic representation (all 4X magnifications) of malignant polymorphic adenomas in example 1 under different visual fields at day 6 in 3 rd generation organoid culture.
FIG. 5A is the results of HE staining (HE, 400X) at day 4 of organoid culture of passage 2 of malignant polymorphic adenoma of example 1.
FIG. 5B is the pathological HE staining results (HE, 400X) of the tumor tissue samples of the patient of example 1.
FIG. 6A shows the immunohistochemical staining results (immunohistochemistry, 400X) of CK7, CK19, SMA protein expression and cell proliferation index Ki67 on day 4 of organoid culture of passage 2 of malignant polymorphic adenoma of example 1.
Fig. 6B is the immunohistochemical staining results (immunohistochemistry, 400 ×) for CK7, CK19, SMA protein expression and cell proliferation index Ki67 of the patient's tumor tissue samples of example 1.
FIG. 7 is the microscopic expression (10X magnification) of malignant pleomorphic adenoma primary organoid cultured in culture medium on day 7 in comparative example 4.
FIG. 8 is the microscopic expression (10X magnification) of malignant pleomorphic adenoma primary organoid cultured with Medium A on day 7 in comparative example 4.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and those skilled in the art can easily understand the advantages and effects of the present invention from the disclosure of the present specification. The invention is capable of other and different embodiments and its several details are capable of modifications and various changes in detail, which may be made without departing from the spirit of the invention.
The invention provides an in vitro construction method and application of salivary gland malignant pleomorphic adenoma organoid, which comprises the steps of extracting human parotid malignant pleomorphic adenoma cells, inducing and constructing organoids, observing the form under a mirror, and obtaining organoids highly consistent with the cell form, protein expression and the like of tumor samples of patients through HE staining, immunohistochemical observation and verification of the tissue cell form and protein expression condition of the malignant pleomorphic adenoma organoids.
The invention provides a culture medium for culturing 3D organoids of malignant polymorphic adenomas of salivary glands, which comprises:
advanced DMEM/F12, and the following components based on Advanced DMEM/F12: 0.5-2 v% of antimicrobial-antimicrobial, 0.5-2 v% of GlutaMAX, 0.5-2 v% of HEPES buffer solution, 50-300 ng/ml of fibroblast growth factor (FGF-10), 50-300 ng/ml of EGF, 5-25 mu g/ml of bFGF, 300-800 nmol/L of TGF-beta I type receptor inhibitor, 300-800 ng/ml of Wnt3a recombinant protein, 0.05-0.3 mu g/ml of Noggin recombinant protein, 0.05-0.3 mu g/ml of R-spondin-1 recombinant protein, 0.75-2.5 mmol/L N-Acetylcysteine (N-Acetylcyeine, NACC), 5-30 mmol/L of Nicotinamide (Nicotinamide), 0.5-5 v% of B27 supplement, 0.2-5 mu mol/L of dexamethasone, and 0.2-5 mg/ml of dexamethasone.
Wherein v% refers to the volume percentage of a substance in Advanced DMEM/F12. For example, 0.5-2 v% of the antimicrobial-antimicrobial means that the volume of the antimicrobial-antimicrobial is 0.5-2 mL in 100mL Advanced DMEM/F12.
Wherein, the volume percentage of the Antibiotic-Antibiotic in the Advanced DMEM/F12 is 0.5-2%, can be 0.5-1.0%, 1.0-1.5%, 1.5-2%, and specifically can be 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0%. Preferably, 0.6 to 1.6%; further preferably, the content is 0.8-1.2%; still more preferably, it is 1%.
Wherein, the volume percentage of GlutaMAX in Advanced DMEM/F12 is 0.5-2%, which may be 0.5-1.0%, 1.0-1.5%, 1.5-2%, and specifically may be 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0%. Preferably, 0.6 to 1.6%; more preferably, 0.8 to 1.2%; still more preferably, it is 1%.
The HEPES buffer solution accounts for 0.5-2% of Advanced DMEM/F12 by volume, and can be 0.5-1.0%, 1.0-1.5% and 1.5-2%, and specifically can be 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0%. Preferably, 0.6 to 1.6%; further preferably, the content is 0.8-1.2%; still more preferably, it is 1%.
Wherein, the concentration of the fibroblast growth factor (FGF-10) in Advanced DMEM/F12 is 50-300 ng/ml, can be 50-100 ng/ml, 100-150 ng/ml, 150-200 ng/ml, 200-250 ng/ml and 250-300 ng/ml, and can be 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 300 ng/ml. Preferably, the concentration is 60-240 ng/ml; further preferably, the concentration is 80-160 ng/ml; further preferably, the concentration is 80-120 ng/ml; even more preferably, it is 100 ng/ml.
Wherein, the concentration of EGF in Advanced DMEM/F12 is 50-300 ng/ml, can be 50-100 ng/ml, 100-150 ng/ml, 150-200 ng/ml, 200-250 ng/ml and 250-300 ng/ml, and specifically can be 50, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 and 300 ng/ml. Preferably, the concentration is 60-240 ng/ml; further preferably, the concentration is 80-160 ng/ml; further preferably 80-120 ng/ml; even more preferably, it is 100 ng/ml.
Wherein the concentration of the bFGF in the Advanced DMEM/F12 is 5-25 mu g/ml, can be 5-10 mu g/ml, 10-15 mu g/ml, 15-20 mu g/ml and 20-25 mu g/ml, and can be 5, 8, 10, 12, 15, 18, 20, 22 and 25 mu g/ml. Preferably, the concentration is 6-18 mu g/ml; more preferably, the concentration is 8-12 mu g/ml; still more preferably, it is 10. mu.g/ml.
Wherein the concentration of the TGF-beta I type receptor inhibitor (preferably A83-01) in Advanced DMEM/F12 is 300-800 nmol/L, and can be 300-350 nmol/L, 350-400 nmol/L, 450-500 nmol/L, 500-550 nmol/L, 550-600 nmol/L, 600-650 nmol/L, 650-700 nmol/L and 750-800 nmol/L. Preferably, the concentration is 400-600 nmol/L; more preferably, the concentration is 450-550 nmol/L; still more preferably, it is 500 nmol/L.
Wherein the concentration of the Wnt3a recombinant protein in Advanced DMEM/F12 is 300-800 ng/ml, 300-350 ng/ml, 350-400 ng/ml, 450-500 ng/ml, 500-550 ng/ml, 550-600 ng/ml, 600-650 ng/ml, 650-700 ng/ml and 750-800 ng/ml. Preferably, the concentration is 400-600 ng/ml; more preferably, 450-550 ng/ml; even more preferably, it is 500 ng/ml.
Wherein the concentration of the Noggin recombinant protein in Advanced DMEM/F12 is 0.05-0.3 μ g/ml, 0.05-0.1 μ g/ml, 0.1-0.15 μ g/ml, 0.15-0.2 μ g/ml, 0.2-0.25 μ g/ml and 0.25-0.3 μ g/ml, and specifically 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.22, 0.25, 0.28 and 0.3 μ g/ml. Preferably, the concentration is 0.07-0.2 mu g/ml; more preferably, the concentration is 0.08 to 0.15 mu g/m; still more preferably, it is 0.1. mu.g/ml.
Wherein, the concentration of the R-spondin-1 recombinant protein in Advanced DMEM/F12 is 0.05-0.3 mu g/ml, can be 0.05-0.1 mu g/ml, 0.1-0.15 mu g/ml, 0.15-0.2 mu g/ml, 0.2-0.25 mu g/ml and 0.25-0.3 mu g/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 and 0.3 mu g/ml. Preferably, the concentration is 0.07-0.2 mu g/ml; more preferably, the concentration is 0.08 to 0.15 mu g/m; still more preferably, it is 0.1. mu.g/ml.
Wherein, the concentration of N-Acetylcysteine (NAC) in Advanced DMEM/F12 is 0.75-2.5 mmol/L, and can be 0.75-1 mmol/L, 1-1.25 mmol/L, 1.25-1.5 mmol/L, 1.5-1.75 mmol/L, 1.75-2 mmol/L, 2-2.25 mmol/L, 2.25-2.5 mmol/L, and specifically can be 0.75, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.5 mmol/L. Preferably, the concentration is 0.9-1.8 mmol/L; further preferably, the concentration is 1-1.5 mmol/L; still more preferably, it is 1.25 mmol/L.
Wherein the concentration of Nicotinamide (Nicotinamide) in Advanced DMEM/F12 is 5-30 mmol/L, 5-10 mmol/L, 10-15 mmol/L, 15-20 mmol/L, 20-25 mmol/L, 25-30 mmol/L, specifically 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 mmol/L. Preferably, the concentration is 6-20 mmol/L; further preferably, the concentration is 8-16 mmol/L; still more preferably, it is 10 mmol/L.
Wherein the concentration of the B27 supplement in Advanced DMEM/F12 is 0.5-5%, and can be 0.5-1%, 1-1.5%, 1.5-2%, 2.5-3%, 3.5-4%, 4.5-5%, specifically 0.5, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5%. Preferably, the content is 1-3.5%; more preferably, the content is 1.5-2.5%; still more preferably, it is 2%.
Wherein, the concentration of dexamethasone in Advanced DMEM/F12 is 0.2-5 μmol/L, can be 0.2-0.5 μmol/L, 0.5-1 μmol/L, 1-1.5 μmol/L, 1.5-2 μmol/L, 2.5-3 μmol/L, 3.5-4 μmol/L, 4.5-5 μmol/L, and specifically can be 0.2, 0.5, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5 μmol/L. Preferably, the concentration is 0.5-3.5 mu mol/L; further preferably, the concentration is 0.8-2.5 mu mol/L; still more preferably, it is 1. mu. mol/L.
Wherein the concentration of Primocin in Advanced DMEM/F12 is 0.2-5 mg/ml, and may be 0.2-0.5 mg/ml, 0.5-1 mg/ml, 1-1.5 mg/ml, 1.5-2 mg/ml, 2.5-3 mg/ml, 3.5-4 mg/ml, 4.5-5 mg/ml, and specifically may be 0.2, 0.5, 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5 mg/ml. Preferably, the concentration is 0.5-3.5 mg/ml; further preferably, the concentration is 0.8-2.5 mg/ml; even more preferably, it is 1 mg/ml.
In some preferred embodiments, the culture medium further comprises one or more of N-2 supplement, FGF2, Prostaglandin E2. May comprise the N-2 supplement alone, or FGF2, or Prostaglandin E2, or N-2 supplement and FGF2, or N-2 supplement and Prostaglandin E2, or FGF2 and Prostaglandin E2, or N-2 supplement, FGF2 and Prostaglandin E2. In some preferred embodiment modes, the culture medium comprises N-2 supplement, FGF2 and Prostaglandin E2.
When the culture medium contains an N-2 supplement, the N-2 supplement accounts for 0.5-3% of the Advanced DMEM/F12 by volume, and can be 0.5-1.0%, 1.0-1.5%, 1.5-2%, 2-2.5% and 2.5-3%, and specifically can be 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.5, 2.8 and 3.0%. Preferably, 0.6-1.6%; more preferably, 0.8 to 1.2%; still more preferably, it is 1%.
When the culture medium contains FGF2, the volume percentage of FGF2 in Advanced DMEM/F12 is 2-10 ng/ml, and can be 2, 3, 4, 5, 6, 7, 8, 9 and 10 ng/ml. Preferably, the concentration is 3-8 ng/ml; further preferably, the concentration is 4-6 ng/ml; even more preferably, it is 5 ng/ml.
When the culture medium contains Prostagladin E2, the volume percentage of Prostagladin E2 in Advanced DMEM/F12 is 0.5-3 μmol/L, and can be 0.5-1.0 μmol/L, 1.0-1.5 μmol/L, 1.5-2 μmol/L, 2-2.5 μmol/L, and 2.5-3 μmol/L, specifically 0.5, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.5, 2.8, and 3.0 μmol/L. Preferably, the concentration is 0.6-1.6 mu mol/L; more preferably, the concentration is 0.8 to 1.2 mu mol/L; still more preferably, it is 1. mu. mol/L.
In some more preferred embodiments, the medium comprises: 500ml Advanced DMEM/F12(Gibco), 1 v% Antibiotic-Antibiotic (Gibco), 1 v% GlutaMAX (Gibco), 1 v% HEPES buffer (Gibco), fibroblast growth factor (FGF-10)100ng/Ml (MCE), EGF 100ng/Ml (MCE), bFGF 10. mu.g/Ml (MCE), A83-01500 nmol/L (MCE), Wnt3a recombinant protein 500ng/Ml (MCE), Noggin recombinant protein 0.1. mu.g/Ml (MCE), R-spondin-1 recombinant protein 0.1. mu.g/Ml (MCE), N-Acetylcysteine (N-Acetylcysteine, NAC)1.25mmol/L (MCE), Nicotinamide (Nicotinamide) 10/L, dexamethasone 1.42% MCE, dexamethasone 1.32 mg/L (MCE), dexamethasone 1.32. mu.1. mu.g/Ml (MCE), dexamethasone 2. mu.32. mu.g/Ml (MCE), FGF2 is 5ng/Ml (MCE), Prostaglandin E21. mu. mol/L (MCE); wherein, in the culture medium, except Advanced DMEM/F12, the dosage of other components such as percentage content or concentration is explained by taking Advanced DMEM/F12 as a reference; the B27 supplement is a 50X B27 supplement; the N-2 supplement is 100X N-2 supplement.
The invention also provides the application of the culture medium in culturing malignant pleomorphic adenoma 3D organoid. The culture medium is used for aiming at the culture growth characteristics of malignant polymorphic adenoma cells, various cell factor components are selected and compounded according to a specific proportion, the obtained culture medium contains appropriate contents of cell factors and signal pathway regulatory factors, and the salivary gland malignant polymorphic adenoma cells can form 3D organs which are close to the shapes and the characteristics of real tumor tissues in a short time. The culture medium has low cost, strong operability and good repeatability, and the organoid cultured by multiple subcultures still can keep the cell morphology of the organoid, and the protein expression is close to the real tumor tissue.
The invention also provides a method for culturing the salivary gland malignant pleomorphic adenoma 3D organoid, which comprises the step of culturing the broken salivary gland malignant pleomorphic adenoma cells by adopting the culture medium to obtain the salivary gland malignant pleomorphic adenoma 3D organoid.
Specifically, the method comprises the following steps:
(a) tissue disruption
Crushing a salivary gland malignant polymorphic adenoma tumor tissue to obtain a tissue fragment;
(b) tissue digestion
Digesting the tissue fragments by using digestive enzymes to obtain a digested suspension;
the digestive enzyme comprises collagenase type II and DNase I; preferably, the digestive enzyme comprises 0.8-4 mg/ml collagenase type II and 0.05-0.3 mg/ml DNase I;
(c) filtration
Filtering the digested suspension, discarding the supernatant, resuspending the precipitate to obtain cell suspension, inoculating, and culturing.
In the digestive enzyme in the step (b), the concentration of collagenase II is 0.8-4 mg/ml; may be 0.8 to 1mg/ml, 1 to 1.5mg/ml, 1.5 to 2mg/ml, 2 to 2.5mg/ml, 2.5 to 3mg/ml, 3 to 3.5mg/ml, 3.5 to 4mg/ml, and specifically may be 0.8, 1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4 mg/ml. Preferably, the concentration is 0.8-3 mg/ml; further preferably, the concentration is 1.5-2.5 mg/ml; even more preferably, it is 2 mg/ml.
In the digestive enzyme in the step (b), the concentration of DNase I is 0.05-0.3 mg/ml; may be 0.05 to 0.1mg/ml, 0.1 to 0.15mg/ml, 0.15 to 0.2mg/ml, 0.2 to 0.25mg/ml, 0.25 to 0.3mg/ml, and specifically may be 0.05, 0.08, 0.1, 0.12, 0.15, 0.18, 0.2, 0.22, 0.25, 0.28, 0.3 mg/ml. Preferably, the concentration is 0.07-0.2 mg/ml; further preferably, the concentration is 0.08-0.15 mg/ml; even more preferably, it is 0.1 mg/ml.
In the step (b), the digestion is carried out in a shaking table, and the rotating speed of the shaking table is 150-500 rrpm; can be 150-200 rrpm, 200-250 rrpm, 300-500 rrpm, 350-400 rrpm, 450-500 rrpm; preferably, the rotating speed is 180-400 rpm; preferably, the rotating speed is 200-260 rpm; more preferably, 300 rpm.
In the step (c), the filtering step comprises filtering the digested suspension through a filter membrane of 90-120 μm and 60-80 μm in sequence; preferably, filtration is carried out sequentially through a 100 μm, 70 μm filtration membrane.
In step (c), after the cell suspension is obtained, the cell suspension is mixed with matrigel and inoculated into the culture medium as described above for culture. Wherein the mixing operation is carried out at 0-10 ℃; can be at 0 deg.C, 2 deg.C, 4 deg.C, 6 deg.C, 8 deg.C, 10 deg.C.
In the step (c), the culture temperature is 28-40 ℃; can be at 28 deg.C, 30 deg.C, 32 deg.C, 34 deg.C, 36 deg.C, 38 deg.C, 40 deg.C. Preferably, the temperature is 32-38 ℃; further preferably 37 deg.c.
In the step (c), the culture temperature is 5-10 days; may be 5, 6, 7, 8, 9, 10 d. Preferably, the time is 6-8 d; further preferably 6 d.
In the step (c), the inoculated culture plate is firstly cultivated in an inverted mode until matrigel forms a dome-shaped structure, and then the culture plate is placed in an incubator in an upright mode for cultivation.
According to the method, the salivary gland malignant pleomorphic adenoma 3D organoid, namely primary salivary gland malignant pleomorphic adenoma 3D organoid can be obtained through culture, and the primary salivary gland malignant pleomorphic adenoma 3D organoid is subjected to subculture continuously to obtain a subculture salivary gland malignant pleomorphic adenoma 3D organoid; the number of passages is not limited, and for example, one, two, three or more passages may be performed as long as the culture of the salivary gland malignant pleomorphic adenoma 3D organoid having the desired properties is achieved, and preferably, one, two or three passages may be performed.
The step of passaging comprises:
melting matrigel, centrifuging, removing supernatant, adding pancreatin for digestion to obtain cell suspension containing cells or cell masses with proper sizes, and subculturing, wherein the culture steps and the method are the same as those of the primary salivary gland malignant polymorphic adenoma 3D organoid.
In some preferred embodiments, the step of passaging comprises: abandoning the original culture medium, melting matrigel by using precooled PBS-1, slightly blowing the mixture of the matrigel and the organoid, transferring the mixture into a centrifuge tube, centrifuging the mixture to abandon the supernatant, adding PBS-1 for the first time for resuspension, centrifuging the mixture to abandon the supernatant to remove the matrigel completely, adding 1-2 ml of pancreatin (Suzhou Xinsaimei biotechnology limited) for resuspending and precipitating the digestion spheres for 3min, and terminating the digestion. The supernatant was discarded by centrifugation, and the pellet was resuspended a second time with PBS-1. Counting by a counting plate under a microscope, and suspending cell sediment by adding a proper amount of culture medium according to a counting result; then, the step of inoculation and culture is performed in the same way as the step of primary malignant pleomorphic adenomatoid organs. In some more preferred embodiments, after the PBS-1 is resuspended for the first time, if part of the beads are in suspension and part of the macrospheres sink, the supernatant containing the beads can be transferred to another centrifuge tube, the PBS-1 suspension containing the macrospheres can be centrifuged to remove the supernatant, and then the supernatant can be digested with pancreatin and then transferred to the centrifuge tube containing the beads for centrifugation; thus, part of the original small balls and single cells can be reserved to a certain extent, the large balls can be digested into small balls or single cells, and the small balls can be beneficial to growing out the organoids more quickly and easily to a certain extent.
In the present invention, PBS-1 is PBS to which 5 v% of the dual resistance to streptomycin has been added.
The method of the invention can obtain organoids with tissue cell morphology and protein expression highly consistent with that of real salivary gland malignant polymorphic adenomas. In the culture process, the proportion of living cells is ensured to the maximum extent, and the culture time can be shortened.
Various cytokines and regulatory factors are directly and closely influenced with each other, are coordinated and matched, and are combined with unique digestive enzymes, a digestion method and an inoculation culture method, so that the salivary gland malignant polymorphic adenoma cells can better show the inherent activity characteristics in the culture process, and the comprehensive characteristics which are highly similar to those of real salivary gland malignant polymorphic adenoma tumors are realized. The salivary gland malignant pleomorphic adenoma organoids obtained by the culture medium of the invention converge into clusters, which are similar to the cell morphology and the tissue structure of real salivary gland malignant pleomorphic adenoma tumor.
In the present invention, the salivary gland malignant polymorphic adenoma tumor tissue is selected from human.
In the present invention, v% means a volume percentage of a certain substance. For example, 0.5-2 v% of the antimicrobial-antimicrobial based on Advanced DMEM/F12 means that the volume of the antimicrobial-antimicrobial is 0.5-2 mL in 100mL of Advanced DMEM/F12.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments, and is not intended to limit the scope of the present invention; in the description and claims of the present application, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. 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, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.
Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.
In this example, PBS refers to phosphate buffered saline (brand Gibco).
PBS-1 refers to PBS with 5 v% of the dual resistance to streptomycin.
EXAMPLE 1 salivary gland malignant polymorphic adenoma organoid culture
The source of the specimen is the department of orofacial head and neck oncology of the ninth national hospital affiliated to Shanghai university of transportation, one example is a 62-year-old male patient with malignant pleomorphic adenoma, and the primary focus is located in the parotid gland. Before the frozen biopsy operation, obtaining the consent of the patient and the guardian, signing an informed consent, and carrying out postoperative pathological diagnosis as follows: malignant polymorphic adenomas of the parotid gland.
(1) Specific culture method
(1.1) tissue preservation
The biopsy samples were quickly loaded into a centrifuge tube containing a tissue preservation solution containing DMEM (shanghai culture biotechnology limited) as a main component, to which amphotericin B (500 ng/ml in the preservation solution) (suzhou xinsai biotechnology limited) and penicillin-streptomycin double antibody (Gibco) 5 v% of the preservation solution were added. The centrifuge tubes were loaded into ice boxes and sent to the laboratory.
(1.2) tissue disruption
In a biosafety cabinet procedure, malignant polymorphic adenoma tissue was transferred to petri dishes and the tissue was soaked for about 10 minutes in PBS-1 solution containing 5 v% penicillin-streptomycin dual antibody. The cells were transferred to a new petri dish containing the above tissue wash solution to remove the coated tissue. The tissue is sheared into tissue fragments of about 1mm ^3 in size.
(1.3) tissue digestion
The tissue fragments were transferred to 50ml centrifuge tubes containing the digestive enzymes (2mg/ml collagenase type II (Gibco) and 0.1mg/ml DNase I (Applichem)), and digested for 1.5h at 37 ℃ in a shaker at 300rpm, resulting in more cell pellet and no cell adhesion. Then adding serum DMEM culture medium to stop digestion, and blowing and beating for 10-20 times up and down to further promote tissue disruption.
(1.4) filtration
Filtering the digested suspension by using 100-micrometer and 70-micrometer filter screens in sequence to obtain cell suspension; centrifuging at 200g for 3min, discarding the supernatant, resuspending the cell pellet with PBS-1, counting with a counting plate under a microscope, and resuspending the cell pellet by adding an appropriate amount of culture medium according to the counting result.
Wherein the culture medium comprises the following components in parts by weight: 500ml Advanced DMEM/F12(Gibco), 1 v% Antibiotic-Antibiotic (Gibco), 1 v% GlutaMAX (Gibco), 1 v% HEPES buffer (Gibco), fibroblast growth factor (FGF-10)100ng/Ml (MCE), EGF 100ng/Ml (MCE), bFGF 10. mu.g/Ml (MCE), A83-01500 nmol/L (MCE), Wnt3a recombinant protein 500ng/Ml (MCE), Noggin recombinant protein 0.1. mu.g/Ml (MCE), R-spondin-1 recombinant protein 0.1. mu.g/Ml (MCE), N-Acetylcysteine (N-Acetylcysteine, NAC)1.25mmol/L (MCE), Nicotinamide (Nicotinamide) 10/L2 v% B2 v/L, dexamethasone 1.42 (MCE) 2mg/ml, dexamethasone 1. mu.32 mg/L (MCE), dexamethasone 1. mu.1. mu.g/Ml (MCE), dexamethasone 2. v/L (MCE) supplement (MCE), FGF2 is 5ng/Ml (MCE), Prostaglandin E21. mu. mol/L (MCE); wherein, in the culture medium, except Advanced DMEM/F12, the dosage of other components such as percentage content or concentration is explained by taking the Advanced DMEM/F12 as a reference; the B27 supplement is a 50X B27 supplement; the N-2 supplement was 100X N-2 supplement.
(1.5) inoculation
An appropriate amount of cell suspension was selected and mixed with Matrigel (Low growth factor Matrigel, Corning), wherein about 10^4 cells per 40. mu.l of Matrigel mixture, seeded in 24-well cell culture plates (NEST), and 40. mu.l of Matrigel mixture was added per well. After inoculation as shown in FIG. 1.
The matrigel is in a liquid state at 0-10 ℃ and is solidified at high temperature, so the relevant operations of the matrigel are required to be carried out on ice, and the mixing of the matrigel and the cell suspension is preferably completed by a pre-cooled gun head in a shortest time to prevent the matrigel from solidifying.
(1.6) Primary culture of malignant polymorphic adenomatoid organoids
The inoculated 24-well plate was placed upside down in a 37 ℃ incubator (Thermo Fisher) for about 45min, and after the gel solidified, the 24-well plate was placed upright with 450ul of the medium as described in step (4) above per well. The mixture was returned to the 37 ℃ incubator for culture. And then replacing the culture medium every 2-3 days.
The inversion operation is more beneficial to the formation of the dome-shaped structure of the matrigel and the adherence is firm. During which organoids are observed under an inverted microscope, typically within two weeks, and passaged if the organoids do not grow any more. The passage ratio was 1: 6 (i.e. primary malignant polymorphic adenomatoid organs in one well were passaged to 6 wells in a new 24-well plate).
(1.7) passage of malignant polymorphic adenomatoid organoids
Abandoning the original culture medium, melting matrigel by using precooled PBS-1, slightly blowing the mixture of the matrigel and the organoid, transferring the mixture into a centrifuge tube, centrifuging the centrifuge tube to abandon the supernatant, adding PBS-1 for resuspension, centrifuging the centrifuge tube to abandon the supernatant so as to remove the matrigel completely, adding 1-2 ml of pancreatin (Suzhou Xinsaimei biotechnology, Co., Ltd.) for resuspending the digestion spheres for 3min, and terminating the digestion. The supernatant was discarded by centrifugation, and the pellet was resuspended in PBS-1. Counting by a counting plate under a mirror, and suspending cell sediment by adding a proper amount of culture medium according to a counting result; then inoculating and culturing the primary malignant pleomorphic adenomatoid organ.
(2) Morphological observation of malignant pleomorphic adenomatoid organoids
The morphology of the malignant polymorphic adenomatous organoids of different growth generations was observed under an inverted microscope and photographed. The morphology of primary (P1) malignant polymorphic adenomatoid organoids cultured up to day 6 is shown in fig. 2: the organoids are spherical in shape with continuous edges. Passage 2 (P2) was obtained after passage and the microscopic appearance of P2 by day 6 is shown in fig. 3. Passage again, generation 3 (P3) malignant polymorphic adenomatous organoids were obtained and the microscopic appearance by day 6 of culture of generation P3 is shown in fig. 4. Fig. 3 and 4 show: after subculture, the tissue cell morphology of the P2 and P3 generation malignant polymorphic adenomatoid organoids is similar to that of the primary malignant polymorphic adenomatoid organoids and remains unchanged.
(3) Identification of malignant polymorphic adenoma organoid cell morphology and protein expression
The P2 generation malignant polymorphic adenoma organoids were cultured to day 4, and after organoid harvesting, HE staining was performed (fig. 5A), and cell morphology comparison was performed with HE staining of patient tumor tissues (fig. 5B) (patient tumor sample section data was retrieved from pathology archive library of oral pathology department in ninth national hospital affiliated to shanghai transportation university). The alignment result shows that the cell morphology of the malignant polymorphic adenoma organoid of the P2 generation is similar to that of the real tumor sample.
Immunohistochemical staining of P2 generation malignant polymorphic adenoma organoids cultured up to day 4 (CK7, CK14, SMA) for protein expression and cell proliferation index Ki67 expression was performed (fig. 6A), and the staining results were compared with immunohistochemical staining results of the above-mentioned markers for patient tumor tissues (fig. 6B) (patient tumor sample immunohistochemical data was retrieved from pathology archive of oral pathology department tissue bank of ninth national hospital affiliated to shanghai transportation university). The comparison result shows that the expression level of the P2 generation organoid protein and the expression level of the proliferation index are similar to those of a real tumor sample.
Comparative example 1 Using different digestive enzymes
The procedure was as in example 1 except that the digestive enzyme component in step (1.3) was replaced with 0.63mg/ml collagenase type II, 0.5mg/ml hyaluronidase (Gibco), 6.25mmol/L CaCl2(Gibco)。
By comparison, in example 1, the use of the type ii collagenase and DNase I containing digestive enzymes resulted in a large number of cell pellets, a large number of viable cells, and no cell adhesion. The cell pellet obtained after digestion in this example was reduced and cell adhesion occurred. It can be seen that example 1, based on collagenase type ii, with dnase i as the digestive enzyme component, can effectively lyse DNA released by cell death without affecting living cells.
Comparative example 2 different digestion regimes were used
The operation steps are the same as example 1, only the shaking table at 37 ℃ in the step (1.3) is digested for 1.5h at 300rpm, and the digestion is replaced by the incubator at 37 ℃ for 1.5h, and the centrifuge tube is manually shaken every 15-30 min.
The comparison result shows that in example 1, the mode of continuous digestion for 1.5 hours by using a shaking table can ensure that the enzyme has a larger contact area with the tissue fragments, the digestion is more sufficient, more cell precipitates are obtained, cell adhesion does not occur, the content of living cells in the cell precipitates can be ensured, and a better digestion effect is achieved. In the comparative example, after digestion is carried out in the incubator for 1.5h, digestion is not sufficient, a plurality of tissue fragments still exist, and ideal cell precipitation cannot be obtained; if further digestion is carried out by a method such as prolonged digestion, more cell pellet is obtained, but the content of viable cells is reduced.
Comparative example 3 different filtration mode
The procedure is as in example 1 except that in step (1.4) the digested suspension is filtered through a 100 μm, 70 μm filter (Biosharp) instead of through a 100 μm, 70 μm, 40 μm filter.
As a result of comparison, it was found that the cell pellet obtained by the filtration method used in example 1 contains a large amount of medium-sized cell aggregates, and based on these cell aggregates, organoids containing individual cell aggregates are grown more easily and more rapidly. In the comparative example, the obtained cell sediment contains more single cells by adopting a filtering mode, and the cell sediment is not easy to grow into organoids during subsequent inoculation culture.
Comparative example 4 different Medium
The source of the specimens is the department of orofacial head and neck oncology of the ninth national hospital affiliated to Shanghai university of traffic, one example of middle-aged male patients with malignant pleomorphic adenoma, and the primary focus is located in the parotid gland. Before the frozen biopsy operation, obtaining the consent of the patient and the guardian, signing an informed consent, and carrying out postoperative pathological diagnosis as follows: malignant polymorphic adenomas of the parotid gland.
The procedure was as in example 1, and culture and alignment were performed using medium A from step (1.4) of example 1, which was the medium of step (1.4) of example 1 but did not contain the N-2 supplement, FGF2, Prostaglandin E2.
The results of culturing primary malignant polymorphic adenomatous organoids with medium up to day 7 are shown in FIG. 7. The results of culturing primary malignant polymorphic adenomatous organoids with medium a up to day 7 are shown in fig. 8. As can be seen from FIGS. 7 and 8, the growth of malignant polymorphic adenomatous organoids in culture medium is faster than in medium A, and the number of organoid spheres is larger and the volume is larger in the same period of culture. The culture of primary malignant polymorphic adenomatoid organoids in medium A requires a longer period of time, usually about 2w, for a given number of organoids to form.
While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.
Claims (10)
1. A medium for culturing salivary gland malignant polymorphic adenomas 3D organoids, said medium comprising:
advanced DMEM/F12, and the following components based on volume Advanced DMEM/F12: 0.5-2 v% of antimicrobial-antimicrobial, 0.5-2 v% of GlutaMAX, 0.5-2 v% of HEPES buffer solution, 50-300 ng/ml of fibroblast growth factor, 50-300 ng/ml of EGF, 5-25 mu g/ml of bFGF, 300-800 nmol/L of TGF-beta I type receptor inhibitor, 300-800 ng/ml of Wnt3a recombinant protein, 0.05-0.3 mu g/ml of Noggin recombinant protein, 0.05-0.3 mu g/ml of R-spondin-1 recombinant protein, 0.75-2.5 mmol/L N-acetylcysteine, 5-30 mmol/L of nicotinamide, 0.5-5 v% of B27 supplement, 0.2-5 mu mol/L of dexamethasone and 0.2-5 mg/ml of mocin.
2. The culture medium of claim 1, further comprising one or more of N-2 supplement, FGF2, Prostaglandin E2.
3. The culture medium of claim 2, comprising at least one of the following 1) to 3):
1) the content of the N-2 supplement accounts for 0.5-3% of the volume percentage of the Advanced DMEM/F12;
2) the concentration of the FGF2 in Advanced DMEM/F12 is 2-10 ng/ml;
3) the concentration of the Prostaglandin E2 in Advanced DMEM/F12 is 0.5-3 mu mol/L.
4. Use of the medium according to any one of claims 1 to 3 for culturing malignant polymorphic adenoma of salivary glands in 3D organoids.
5. A method for culturing malignant polymorphic adenoma 3D organoids from salivary glands, comprising culturing the disrupted malignant polymorphic adenoma tumor cells in the culture medium of any one of claims 1 to 3 to obtain the malignant polymorphic adenoma 3D organoids.
6. The culture method according to claim 5, wherein the method comprises:
(a) tissue disruption
Crushing malignant polymorphic adenoma tumor tissue to obtain tissue fragments;
(b) tissue digestion
Digesting the tissue fragments by using digestive enzyme to obtain a digested suspension;
the digestive enzyme comprises collagenase type II and DNase I; preferably, the digestive enzyme comprises 0.8-4 mg/ml collagenase type II and 0.05-0.3 mg/ml DNase I;
(c) filtration
Filtering the digested suspension, discarding the supernatant, resuspending the precipitate to obtain cell suspension, inoculating, and culturing.
7. The culture method according to claim 6, comprising at least one of the following 1) to 6):
1) in the step (b), the digestion is carried out in a shaking table, and the rotation speed of the shaking table is 150-500 rpm;
2) in step (c), the step of filtering comprises: filtering the digested suspension by a filter membrane of 90-120 mu m and 60-80 mu m in sequence; preferably, filtration is performed sequentially through a 100 μm, 70 μm filtration membrane;
3) after obtaining the cell suspension in the step (c), mixing the cell suspension with matrigel, and inoculating the mixture to the culture medium of any one of claims 1 to 3 for culture; preferably, the mixing operation is carried out at 0-10 ℃;
4) in the step (c), the culture temperature is 28-40 ℃;
5) in the step (c), the culture time is 5-10 days;
6) in the step (c), the inoculated culture plate is firstly inversely cultured until the matrigel forms a dome-shaped structure, and then is vertically placed in an incubator for culture.
8. The method of claim 5, wherein said malignant polymorphic adenoma 3D organoid obtained by culturing is a primary malignant polymorphic adenoma 3D organoid, and said primary malignant polymorphic adenoma 3D organoid is passaged to obtain a passaged malignant polymorphic adenoma 3D organoid;
the step of passaging comprises: and melting matrigel, centrifuging, removing the supernatant, removing the matrigel, adding pancreatin for digestion, and carrying out subculture, wherein the culture steps and the method are the same as those of the primary malignant pleomorphic adenoma 3D organoid.
9. Malignant polymorphic adenoma 3D organoids prepared by the method of any one of claims 5 to 8.
10. The malignant polymorphic adenoma 3D organoid of claim 9 for at least one of the following uses:
1) preparing or screening a medicament for treating salivary gland malignant polymorphic adenoma;
2) studying the occurrence and development mechanism of malignant polymorphic adenoma of salivary glands;
3) screening salivary gland malignant polymorphic adenoma related biomarkers.
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