CN117757746A

CN117757746A - Culture medium, culture method and application for salivary gland polymorphous adenoma cells

Info

Publication number: CN117757746A
Application number: CN202311145308.0A
Authority: CN
Inventors: 顾挺; 张春叶; 陈宛灵; 田臻; 石超吉; 李江
Original assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2023-09-06
Filing date: 2023-09-06
Publication date: 2024-03-26

Abstract

The invention discloses a culture medium for salivary gland polymorphous adenoma cells, a culture method and application. The medium comprises a basal medium; the medium further comprises the following components in concentration based on the total volume of the medium: rock inhibitor 5-15 mu mol/L, cholera toxin 5-15 mu L/L. The invention also provides a method for culturing salivary gland polymorphous adenoma cells by using the culture medium. By adopting the culture medium or the culture method, cells with high biological characteristics with the parent tumor can be obtained through multiple passages, and the problem of difficult culture of benign salivary gland polymorphous adenoma cells is solved, so that personalized treatment of patients is facilitated.

Description

Culture medium, culture method and application for salivary gland polymorphous adenoma cells

Technical Field

The invention relates to the technical field of medicines, in particular to a culture medium for salivary gland polymorphous adenoma cells, a culture method and application.

Background

Salivary gland polymorphous adenoma (pleomorphic adenoma, PA) is the most common salivary gland epithelial benign tumor, well developed in the parotid gland, followed by the submandibular gland. Although PA belongs to benign tumor, it has characteristics of borderline tumor, and has recurrence and malignant potential. The recurrence rate of PA is about 2% -3%; PA patients with multiple recurrent history and longer disease course have 3% -15% of malignant rate and become Malignant PA (MPA). The malignant transformation of PA is a comprehensive pathological process of change of polygene, multifactorial and the like, but the interaction mechanism of the PA is not completely clear, and needs to be further studied. Therefore, the mechanism of occurrence, development and malignant transformation of salivary gland PA is further explored, so that a new therapeutic means is found and a new therapeutic target point is found, and a new clinical diagnosis and treatment idea is provided for treating PA and preventing the deterioration of PA.

Primary culture is a method of first culturing harvested tissue in vitro. The hereditary of the donor is reserved for the primary cultured cells, and the reserved primary genes can reach more than 90%, so that the method has good applicability to related researches of drug sensitivity tests and mechanisms, and is favored by scientific researchers. The cell line established on the basis can provide convenience for mechanism researches of malignant transformation, recurrence, metastasis and the like, and has important clinical significance.

Benign tumor has the growth characteristics of slow growth, self-limiting property and the like, and has the defects of easy pollution, low survival rate, high test condition requirement and the like in the culture process, so that the benign tumor is difficult to culture, and the wide application of the benign tumor is limited.

No report on the method of primary culture of PA cells is currently seen. The invention not only successfully constructs the primary cells of the PA, but also provides a culture medium for culturing the primary cells of the PA.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a medium for salivary gland polyoma cells, a culture method and use.

In order to achieve the above purpose, the present invention adopts the following technical scheme:

the first aspect of the invention provides a salivary gland polyoma cell culture medium comprising a basal medium; the medium further comprises the following components in concentration based on the total volume of the medium:

rock inhibitor 5-15 mu mol/L

Cholera toxin 5-15 mu L/L.

The culture medium of the invention contains Rock inhibitor and cholera toxin simultaneously, has high amplification efficiency, and can grow up a culture dish within 3-7 days, namely 5 multiplied by 10 ⁴ Cells of the order of magnitude can be successfully amplified to 5X 10 in about 3 to 7 days ⁶ Cells of the order of magnitude; the cells still keep stable proliferation speed after continuous passage, and can be continuously passed to more than 17 generations; in addition, positive cell localization, staining intensity and PA markers in both the parental tumor cells and primary cells remained consistent; the specific gene PLAG1 is broken, and the chromosome 8 is abnormal; the copy number variation of the exon is consistent with the variation of the mononucleotide, the common mutation characteristics and the mutation frequency, and the comprehensive indication that the salivary gland polymorphic adenoma cells still have the characteristics of parent tumors after passage is provided, so that the culture medium is used for culturing the salivary gland polymorphic adenoma cells, is very suitable for high-throughput screening of new candidate compounds and provides high-throughput in vitro sensitivity functional tests of drugs for patients.

In a first aspect of the invention, the Rock inhibitor is Y-27632.Rock inhibitors can prevent stem cell apoptosis and increase the efficiency of colony formation. The concentration of ROCK inhibition may be 5 to 11. Mu. Mol/L, 7 to 12. Mu. Mol/L, or 8 to 15. Mu. Mol/L based on the total volume of the medium. In a specific embodiment, the concentration is 10. Mu. Mol/L.

In the first aspect of the present invention, the cholera toxin may be present in a concentration of 5 to 10. Mu.L/L, 7 to 12. Mu.L/L, or 8 to 15. Mu.L/L, based on the total volume of the medium. Cholera toxin (cholera toxin) is an adenylate cyclase activator, which is biochemically involved in the transport of calcium. It has growth stimulating effect on cells from breast, colon, lung, prostate and skin. In a specific embodiment, 4.3. Mu.L/500 mL, i.e., 8.6. Mu.L/L.

In a first aspect of the invention, the basal medium is selected from one or more of DMEM medium, RPMI 1640 medium, MEM medium and F12 medium.

Preferably, the basal medium may have a ratio of 70 to 90v/v%, or 70 to 78v/v%, or 75 to 82v/v%, or 81 to 90v/v%, based on the total volume of the medium. In a specific embodiment, the ratio is 80v/v%.

Preferably, the basal medium is a mixture of DMEM medium and F12 medium.

More preferably, the volume ratio of the DMEM medium to the F12 medium may be (2 to 4): 1, or (2 to 2.8): 1, or (2.5 to 3.5): 1, or (3.4 to 4): 1. In a specific embodiment, the ratio is 3:1.

In a first aspect of the invention, the medium further comprises a growth promoting factor selected from one or more of insulin, hydrocortisone and epidermal growth factor EGF.

Preferably, the growth promoting factor is a mixture of insulin, hydrocortisone and epidermal growth factor EGF.

Preferably, the concentration of insulin may be 1 to 6mg/mL, 1 to 3mg/mL, 2 to 4.8mg/mL, or 3 to 6mg/mL, based on the total volume of the medium. In a specific embodiment, the concentration is 2mg/mL.

Preferably, the hydrocortisone may have a concentration of 20 to 70. Mu.g/mL, 20 to 55. Mu.g/mL, 40 to 60. Mu.g/mL, or 50 to 70. Mu.g/mL, based on the total volume of the medium. In a specific embodiment, 50 μg/mL.

Preferably, the concentration of the EGF is 0.1 to 5. Mu.g/mL, or 0.1 to 1.2. Mu.g/mL, or 1 to 3. Mu.g/mL, or 2 to 5. Mu.g/mL, based on the total volume of the culture medium. In a particular embodiment, 0.125 μg/mL.

In a first aspect of the invention, the medium further comprises serum selected from the group consisting of fetal bovine serum or calf serum.

Preferably, the serum is fetal bovine serum.

Preferably, the concentration of the serum may be 1 to 20v/v%, 1 to 11v/v%, 6 to 15v/v%, or 11 to 20v/v% based on the total volume of the medium. In a specific embodiment, the ratio is 10v/v%.

In the first aspect of the present invention, the medium further comprises an antibiotic selected from one or both of penicillin and streptomycin.

Preferably, the antibiotic is a mixture of penicillin and streptomycin.

More preferably, the concentration of the antibiotic may be 0.5 to 1.5v/v%, or 0.5 to 0.9v/v%, or 0.8 to 1.3v/v%, or 1.1 to 1.5v/v%, based on the total volume of the medium. In a specific embodiment, the ratio is 1.0v/v%.

In a first aspect of the invention, the medium comprises 1 volume of mixture a, 3 volumes of DMEM medium, and 1-fold volume of Ham F12 medium. Based on the total volume of the culture medium, the mixture A contains 2mg/mL insulin, 0.125 mug/mL epidermal growth factor, 50 mug/mL hydrocortisone, 10 mug/L Rock inhibitor, 4.3 mug/500 mL cholera toxin, 1v/v% double antibody (penicillin and streptomycin) and 10v/v% serum.

The second aspect of the invention protects the use of a medium as described above in the culture of salivary gland polymorphous adenoma cells.

In a third aspect the invention provides a method of culturing salivary gland polymorphous adenoma cells comprising the steps of:

salivary gland polymorphous adenoma cells were cultured using the media as described above.

In the third aspect of the present invention, the temperature of the culture may be 20 to 40℃or 20 to 26℃or 25 to 35℃or 30 to 40 ℃. In a specific embodiment, 37 ℃.

In the third aspect of the present invention, the time for the cultivation may be 4 to 10 hours, may be 4 to 8 hours, may be 5 to 8 hours, or may be 7 to 10 hours. In a specific embodiment, 10h.

In a third aspect of the present invention, the salivary gland polymorphous adenoma cells have an inoculation density of 5X 10 based on the total volume of the culture medium ⁶ And each mL.

In a third aspect of the invention, the method for obtaining salivary gland polymorphous adenoma cells comprises the steps of: tissue culture or digestion culture is adopted.

Preferably, the method for obtaining salivary gland polymorphous adenoma cells further comprises subculturing, wherein the subculturing method comprises the following steps: pancreatin is added for digestion and then cultivation in the medium is continued. The number of times of digestion is 2, and the time of each digestion is 3min. Specifically, after the first digestion with pancreatin for 3min, the digestion was terminated and the digested cell suspension was transferred to a centrifuge tube. Washing the cells in the petri dish once with PBS; and (3) continuing to add pancreatin for secondary digestion for 3min, transferring the cell suspension to a centrifuge tube after digestion is stopped, centrifuging the cell suspension, discarding the supernatant, and continuing to culture in the culture medium.

Preferably, the tissue culture method comprises the following steps: the tumor tissue and the suspension of the culture medium are cultured in a culture flask in an inverted mode, and then are cultured in an upright mode.

More preferably, the tumor tissue has a size of 1mm×1mm.

More preferably, the time of the inversion culture is 5 to 15 hours.

More preferably, the temperature of the inversion culture is 20-40 DEG C

More preferably, the time of the forward culture is 4 to 6 hours.

More preferably, the temperature of the forward culture is 20 to 40 ℃.

Preferably, the digestion culture method is as follows: the tumor tissue is digested with digestive enzymes.

More preferably, the digestion is followed by filtration. The filtration is carried out by sequentially passing through a filter screen with the pore diameter of 90-120 mu m, 60-80 mu m and 30-50 mu m. Preferably, the filter screens of 100 μm, 70 μm and 40 μm are passed sequentially. The application finds that the cell pellet obtained in the follow-up process contains more medium-sized cell clusters and is fast without filtering by adopting a filter screen with the size of 30-50 mu m, and single-cell suspension is not easy to form.

More preferably, the tumor tissue has a size of 1mm×1mm.

More preferably, the digestion temperature may be 20 to 40 ℃, or 20 to 28 ℃, or 25 to 35 ℃, or 31 to 40 ℃. In a specific embodiment, 37 ℃.

More preferably, the digestion time may be 0.5 to 4 hours, or 0.5 to 1.5 hours, or 1 to 3 hours, or 2 to 4 hours. In a specific embodiment, 1h.

More preferably, the digestive enzyme is selected from one or two of type II collagenase and deoxyribonuclease I, and the mass ratio of the type II collagenase to the deoxyribonuclease I is (10-30): 1. The present application found that digestion of tumor tissue with type II collagenase and Dispase II Dispase resulted in insufficient digestion with large amounts of tissue fragments, failure to obtain cell pellet and reduced levels of viable cells.

More preferably, the digestive enzymes are mixed with the culture medium to form a digestive fluid to digest the tumor tissue.

Further preferably, the medium is selected from DMEM high sugar medium.

Still more preferably, the concentration of the type II collagenase may be 0.01 to 5mg/mL, or 0.01 to 1.5mg/mL, or 0.8 to 2.3mg/mL, or 2.2 to 5mg/mL, based on the total volume of the digestive juice. In a specific embodiment, the concentration is 2mg/mL.

Still more preferably, the concentration of the DNase I is 0.01 to 0.5mg/mL, or 0.01 to 0.15mg/mL, or 0.12 to 0.3mg/mL, or 0.2 to 0.5mg/mL, based on the total volume of the digestive juice. In a specific embodiment, 0.1mg/mL.

In one embodiment, the digestive juice is composed of DMEM high sugar medium, type ii collagenase and deoxyribonuclease I; the concentrations of the type II collagenase and the deoxyribonuclease I are respectively 2mg/mL and 0.1mg/mL based on the total volume of the digestive juice.

In one embodiment, tumor tissue and digestive juice are mixed and digested, then digestive terminating solution is added to obtain digested suspension, and the digested suspension is filtered by using 100 mu m, 70 mu m and 40 mu m filter screens in sequence to obtain single cell suspension, supernatant is removed by centrifugation, sediment is resuspended, supernatant is removed by centrifugation, and then the single cell suspension is cultured in salivary gland polymorphous gonadal tumor cell culture medium.

A fourth aspect of the invention protects the use of salivary gland polymorphic adenoma cells obtained by the culture method as described above in the manufacture or screening of a medicament for the treatment of malignant, polymorphic adenoma of a salivary gland.

Compared with the prior art, the invention has the following beneficial effects:

1) By adopting the culture medium or the culture method, the amplification efficiency is high, the culture plate can be fully paved after 7 days of primary cells, the culture plate can be fully paved after 3 days of passage, and the primary salivary gland polymorphoadenoma cells in 1:2 (namely, the primary salivary gland polymorphous adenoma cells in 1 hole are continuously subcultured into 2 holes in a new 24-hole plate) are passaged, so long as the primary salivary gland polymorphous adenoma cells have 5 multiplied by 10 ⁴ The cell number of the grade can be amplified to 5 multiplied by 10 in about 3 to 7 weeks ⁶ Salivary gland polyoma cells of the order of magnitude.

2) By adopting the culture medium or the culture method, the cells can still keep stable proliferation speed after continuous passage, and can be continuously passed to more than 17 generations.

3) By adopting the culture medium or the culture method, the positive cell localization, the staining intensity and the PA markers of the parent tumor and the primary cells are highly consistent; meanwhile, translocation and cleavage of the specific PLAG1 gene are consistent; in addition, the copy number variation, single nucleotide variation, and common sequencing analysis mutation characteristics and mutation frequencies of the exons are consistent, and the mutant has the characteristics of salivary gland polymorphic adenoma cells.

4) The salivary gland polymorphous adenoma primary cells obtained by the culture medium or the culture method have the advantages of large number, few other miscellaneous cells, high uniformity, high survival rate and good cell proliferation activity and morphology, and are suitable for high-throughput screening of new candidate compounds and in-vitro sensitivity functional test of high-throughput drugs for patients.

Drawings

FIG. 1 shows the morphology of the salivary gland polyoma culture cells of the P2, P4 and P17 generation in the present application.

Fig. 2 shows a histopathological verification of salivary gland polymorphous adenoma in the present application. Wherein A is parent tumor HE staining, HE×20; b is the HE staining of the P5 generation cell climbing sheet, and HE is multiplied by 20.

FIG. 3 is a graph showing the results of IHC and P10 generation cell IF identification of parental tumor tissue in the present application.

FIG. 4 shows a validation graph of FISH results for parental tumor tissue and P6 generation cells in the present application.

FIG. 5 shows a chromosome karyotype analysis of the P2 cell line of the present application.

FIG. 6 shows a chart of the results of sequencing exons of the P2 generation cells in the present application. Wherein, chromosome banding, sequencing coverage, indel distribution, single nucleotide variation distribution and copy number variation distribution are sequentially carried out from outside to inside.

FIG. 7 shows a profile of mutations shared by the P2-generation cells and the parental tumors in this application.

FIG. 8 shows a graph of mutation frequencies of P2 generation cells and parent tumors in the present application.

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.

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.

Epidermal growth factor (Epidermal Growth Factor, EGF)

Cholera toxin (CAS number 9012-63-9).

The ROCK inhibitor is Y-27632, the CAS number is 146986-50-7, and the structural formula is as follows:

example 1

1.1 materials

Tumor samples: a49-year-old female with a pathological diagnosis of primary oral salivary gland polymorphic adenoma is obtained from 1 patient affiliated to Shanghai university medical school and subjected to oral maxillofacial-head and neck oncology department operation, a primary range is positioned on the right parotid gland, and the patient signs an informed consent.

Treatment of the parental tumors: and preparing HE sections from the resected tumor tissues, and subsequently performing histogroup observation, immunohistochemical staining and fluorescence in situ hybridization detection analysis.

The major components of the polyoma cell culture medium are: 2mg/mL insulin, 0.125. Mu.g/mL epidermal growth factor (EGF, MCE), 50. Mu.g/mL Hydrocortisone (Hydrocrisone, MCE), 10. Mu. Mol/L Rock inhibitor (MCE), 1v/v% diabody (penicillin and streptomycin, gibco), 10v/v% serum (Gibco), cholera toxin 4.3. Mu.L/500 mL, the above ingredients were added to 3 volumes of DMEM medium (Shanghai source culture) and 1 volumes of Ham F12 medium (Gibco). The formula of the digestive juice is as follows: 2mg/mL type II Collagenase (Collagenase II, gibco) and 0.1mg/mL deoxyribonuclease I (DNase I, gibco) in DMEM high-sugar medium.

The formula of the digestion stopping solution is as follows: DMEM high sugar medium containing 10wt% fetal bovine serum (fetal bovine serum, FBS) and 1wt% diabody.

Washing liquid: PBS solution containing 5% of neomycin double antibody (500 IU/mL penicillin and 500mg/mL streptomycin).

1.2 methods

1.2.1 dissociation of tumor tissue samples

1) Fresh tumor samples were rapidly placed in centrifuge tubes containing commercial tissue preservation fluid (MACS, germany) in less than 30 minutes ex vivo.

2) Centrifuge tubes were loaded into ice boxes at 4 ℃ and transported to the laboratory within 1 hour. Digestion is carried out in the sterilized biosafety cabinet.

Treatment of tumor sample tissue: tumor sample tissue was transferred to a petri dish and immersed in the wash solution for 10 minutes. After soaking, the tumor sample tissue is transferred to a new culture dish containing washing liquid, and the tumor sample tissue is clamped by a sterile instrument (ophthalmic forceps, ophthalmic scissors, etc.), and then the envelope tissue is carefully sheared off. Tissue was cut into approximately 1mm tissue pieces using sterile surgical scissors.

1.2.2, P0 Generation obtaining and culturing

Tissue block culture method: then the sheared tissue blocks obtained in the step 1.2.1 are moved to a new culture bottle containing salivary gland polymorphous adenoma cell culture medium to obtain suspension of the tissue blocks; cutting 1mL of a pipetting gun head into an inclined plane, then sucking suspension of a tissue block into a T25 culture bottle, uniformly distributing the suspension at the bottom, adding 1-1.5 mL of salivary gland polymorphous adenoma cell culture medium, screwing a bottle mouth, inversely placing the bottle mouth into a 37 ℃ cell culture box for culturing for 10 hours, then rightly placing the T25 culture bottle, supplementing 2mL of salivary gland polymorphous adenoma cell culture medium, continuously culturing for 4-6 days after rightly placing, and adding 4-6 mL of salivary gland polymorphous adenoma cell culture medium to obtain the generation P0. And the generation P0 is continued to be cultivated in an incubator, and the generation P0 reaches more than 80% of the culture flask, so that the culture flask can be passaged.

Tissue block digestion: the tissue mass obtained in step 1.2.1 is transferred to a 50mL centrifuge tube containing a digestion liquid, preferably covering the tissue mass, and digested for 1h at 37℃in a shaker. Then adding a digestion stopping solution to stop digestion, and filtering the digested suspension by using a 100 μm filter screen (biosharp) and a 70 μm filter screen (biosharp) in sequence to obtain single-cell suspension; washing the single cell suspension with PBS, and centrifuging to obtain cell precipitate; the cell pellet was resuspended in new salivary gland polymorphous adenoma cell culture medium and inoculated into 10cm dishes to give the P0 generation. After 3 days of culture, the cells were changed. After 2-3 days, the liquid is changed once. The replacement of the liquid refers to replacement of the liquid with a new salivary gland polyoma cell culture medium. The generation P0 reaches more than 80% of the culture dish, and the culture dish can be passaged. In this example 1, a tissue block digestion method was used.

1.2.3 subculture (P1-P17)

P1 generation (purification):

1) Pancreatin digestion to isolate fibroblasts. I.e. adding pancreatin into the P0 generation obtained in the step 1.2.2, spreading the bottom of the whole culture dish or the culture bottle, digesting for 1min, tapping the culture bottle or the culture dish, adding a digestion stopping solution to stop digestion, sucking out fibroblast suspension, and adding PBS for 1 time to remove the fibroblasts. The procedure of digestion with pancreatin for 1min was repeated to further eliminate the fibroblasts, to obtain purified adherent cells.

2) And digested with pancreatin to passaging. Performing 1 st digestion for 3min by using pancreatin on the purified adherent cells obtained in the step 1), adding a digestion stopping solution to stop digestion, transferring to a centrifuge tube, and washing with PBS for one time; and (3) continuing to add pancreatin for 2 nd digestion for 3min, adding a digestion stopping solution to stop digestion, transferring to a centrifuge tube, centrifuging, discarding the supernatant to obtain a cell sediment, and placing the cell sediment in a culture dish containing a culture medium for culturing for 4-6 days to obtain the P1 generation cells.

Observing the cell fusion degree under a microscope with a magnification of 10 times, and when the cell migration area of the P1 generation cell exceeds the visual field range, adopting pancreatin digestion for 2 times to carry out subculture to obtain the P2 generation cell.

After the generation of P2 is grown to be converged to 80%, the generation is carried out, and after the cell growth is stable, the generation is carried out according to the proportion of 1:2 (namely, the generation of primary salivary gland polymorphous adenoma cells in 1 hole is carried out to 2 holes in a new 24-hole plate), so that the generation P3, the generation P4, the generation P5, the generation P6, the generation P7, the generation P8, the generation P9, the generation P10, the generation P11, the generation P12, the generation P13, the generation P14, the generation P15, the generation P16 and the generation P17 are obtained. If the generation P2 has 5×10 ⁴ Cells of the order of magnitude can successfully amplify the generation P3 in 3 days to obtain 5 multiplied by 10 ⁶ Cells of the order of magnitude.

1.3 analysis of cell morphology, histology, fluorescence in situ hybridization, chromosome karyotype, and exon sequencing

1.3.1 morphological observations

Morphology of P2, P4 and P17-substituted cells was observed using a split microscope and recorded by photographing, which is a graph of the cells cultured until day 7, and the results are shown in fig. 1.

As can be seen from FIG. 1, the morphology of the cells of the P2, P4 and P17 passages are all: the slender fusiform contains a small amount of irregular polygonal cells, the cells still keep stable cell morphology after continuous passage, the same culture dish can be grown up 3 days after passage according to the ratio of 1:2, the next generation is obviously more and denser than the previous generation, and the proliferation speed of salivary gland polymorphous adenoma in the culture medium is stable.

1.3.2 histological analysis

The P5 generation cells obtained in the step 1.2.2 are taken as a study object.

By observing HE sections of the P5 generation cells and their corresponding parental tumor tissues, it was verified whether the P5 generation cells remained histopathologically characteristic of the parental tumor.

The HE slicing method comprises the following steps: after 48 hours of P5 generation cell slide, the slide was fixed with 10% neutral buffered formaldehyde solution for 30min-1H, washed with PBS, stained with Hematoxylin-Eosin (Hematoxylin & Eosin, H-E), observed in conventional histology, and compared with the Parental tumor (Parentil), the results are shown in FIG. 2.

As can be seen from fig. 2, the P5 generation cells have the same pathological characteristics as the parent tumor cells: are composed of myoepithelial cells (blue arrow in the figure) and glandular epithelial cells (yellow arrow in the figure), and the myoepithelial cells are rich and locally see mucus-like components.

1.3.3 immunofluorescence detection assay

The P10 generation cells obtained in the step 1.2.2 are taken as a study object.

The expression of the PA markers AE1/AE3, CK7, CK14, P63, S-100, calponin and Smooth Muscle Actin (SMA) in the P10 generation cells was examined by immunofluorescent staining (IF).

CK7 is a marker of normal salivary duct cells, is weakly positive expressed in acinar cells, and is not expressed in myoepithelial cells.

CK14 is partially expressed in the acinar and periductal myoepithelial cells and basal lamina cells of the excretory ducts.

SMA is a marker of normal salivary gland myoepithelial cells and S-100 is a low molecular weight calcium dependent binding protein.

1) Fixing: taking out the P10 generation cell slide after 48 hours, washing twice by PBS, then fixing for 30 minutes at room temperature by using 4% neutral buffer formaldehyde, and penetrating for 15 minutes by using a membrane rupture liquid.

2) Closing: after blocking the cells lh with blocking buffer (5% nonfat milk in PBS), antibodies (AE 1/AE3, CK7, calponin, S-100, p63, SMA, CK 14) were added dropwise and incubated overnight at 4 ℃.

3) Dyeing and sealing: after PBS washing, anti-mouse IgG (H+L), F (ab') 2Fragment (Alexa)488 Conjugate) #4408 secondary antibody 30min, pbs wash 3 times. DAPI cappers containing anti-quenchers were added dropwise and spread over cells, and the results were observed under a fluorescence microscope, as shown in FIG. 3. Wherein, P10 is the 10 th generation cell, the nucleus of the cell emits dark blue, and the positive cell emits yellow fluorescence.

From fig. 3, the immunofluorescence results of positive cell localization and staining intensity were highly consistent in the parent tumor and P10 generation cells, indicating that P10 generation cells have the same PA markers as the parent tumor cells, and that P10 generation cells maintain highly consistent biological characteristics with the parent tumor cells.

1.3.4 immunohistochemical staining analysis

The parent tumor (Parentil) obtained in step 1.2.2 was the subject of the study.

Immunohistochemical (IHC) method is as follows:

continuously slicing paraffin-embedded parent tumor tissues for a plurality of pieces at a thickness of 3 mu m, and baking the pieces at a temperature of 70 ℃ for 1h; dewaxing and hydrating, and then carrying out antigen retrieval in EDTA buffer solution with pH of 8.0; by 3%H ₂ O ₂ The solution is blocked off from the sections to eliminate the endogenous catalase activity in the tissue. After rinsing 3 times with PBS, primary antibodies (AE 1/AE3, CK7, CK14, S-100, SMA, calponin, P63) were added dropwise and incubated at room temperature for 1 hour. After rinsing 3 times with PBS, secondary antibody (immunohistochemical secondary antibody) is added dropwise, incubation is carried out for 30min at room temperature, after rinsing 3 times with PBS, DAB is used for developing and hematoxylin counterstaining, and the result is shown in figure 3 after sealing and observation with neutral resin. Wherein Parentil is the parent tumor, and the positive part of the cells is brown yellow after being stained.

As can be seen from FIG. 3, the positive cell localization and staining intensity were highly consistent with the immunohistochemical staining results and P10 generation cellular immunofluorescence results of the parent tumor of the patient.

1.3.5 fluorescent in situ hybridization assay

The P6 generation cells obtained in the step 1.2.2 are taken as a study object.

The fluorescence in situ hybridization (Fluorescence in Situ Hybridization, FISH) method is as follows:

1) After culturing the cell climbing sheet for 48 hours, fixing the cell climbing sheet by 10% neutral buffer formaldehyde solution for 30min-1h, and after PBS cleaning, placing the climbing sheet in 90 ℃ distilled water for pretreatment for 30min.

2) The tissue is digested in a water bath in pepsin digestion solution at 54 ℃ for 15-20 min, and the glass slide is dehydrated and air-dried by gradient ethanol. Under the condition of light shielding, 5-10 mu L of probe (PLAG 1) is dripped, and a slide glass rubber cement sealing piece is covered. Denaturation by hybridization instrument: the hybridization temperature was at 76℃for 6min, 42℃for 16h 30min.

3) The next day, the slide glass was taken out, the rubber cement was removed, and the slide glass was immersed in a 2 XSSC/0.3% NP-40 (pH 7.0-7.5) solution preheated to 72℃for 2 minutes, and then the slide glass was placed in a 2 XSSC/0.1% NP-40 solution at room temperature for 2 minutes. Gradient ethanol dehydration, natural airing in dark, dropwise adding 10 mu L of DAPI counterstain liquid into a probe hybridization corresponding region, covering a cover glass, and observing under a fluorescence microscope. Tumor cells were searched sequentially with 10-fold, 40-fold and 100-fold oil mirrors, and red and green signals were counted in 20 cells under 100-fold oil mirrors, and the results are shown in fig. 4. Wherein ParentalParental is the parent tumor and P6 is the 6 th generation cell.

As can be seen from FIG. 4, the PLAG1 gene related to PA was disrupted in both the parent tumor and the P6 cell, and the results showed that the PLAG1 gene was disrupted in both the parent tumor and the P6 cell, and remained consistent.

1.3.6 chromosome karyotyping

The P2 generation cells obtained in the step 1.2.2 are taken as a study object.

The karyotype analysis is as follows:

1) Observing the state of the P2 generation cells in the step 1.2.2 under an inverted microscope, culturing until the P2 generation cells grow vigorously, and harvesting when the confluence degree is about 70-80%.

2) 1-3 hours prior to harvesting cells, 20. Mu.g/mL colchicine 70. Mu.L (colchicine is toxic to cells, duration and concentration of action are determined according to cell type) was added per 10cm dish (10 mL medium). If the cell death is severe or the division phase is too small after the treatment, the concentration and time should be adjusted).

3) Gently pipette the medium into a new 15mL pointed bottom centrifuge tube, gently wash the petri dish with 1-2mL PBS, aspirate the PBS into the centrifuge tube (dividing cells are usually not firmly attached and are easy to fall off, and the medium and PBS are collected together and the harvested dividing phases are more in a centrifuge).

4) Adding 0.25% trypsin solution, digesting at 37deg.C for 1-2 min (time is determined according to cell type, too short cells will not digest, and chromosome morphology will not be good too much), gently blowing off cells stuck on the bottom of culture dish, adding serum-containing medium to stop (medium which can be recovered in the centrifuge tube before), and mixing thoroughly.

5) Centrifugation was carried out at 1500rpm for 10 minutes at room temperature, cell pellet was visible at the bottom of tube, and supernatant was aspirated.

6) Preheating 0.075mol/L KCL hypotonic solution in a 37 ℃ water bath box for at least 30 minutes, adding 6-8mL KCL hypotonic solution, gently and fully homogenizing, and hypotonic for 15 minutes in the 37 ℃ water bath box (when chromosome dispersion is poor, the time is prolonged properly). Excessively hypotonic cells are tacky and have poor chromosomal morphology).

7) Pre-fixing: directly to the hypotonic cell suspension, 1mL of freshly prepared fixative (methanol: glacial acetic acid=3:1) was slowly added and gently and repeatedly homogenized by blowing.

8) Centrifugation at 1500rpm for 10 min, after centrifugation the supernatant was discarded and the whole cell pellet was retained.

9) Fixing for the first time: 5mL of the fixative was added to the cell pellet, and the cells were gently and repeatedly blown until the cells were completely dispersed. The lid was closed and allowed to stand at room temperature for 30 minutes. Centrifugation was carried out at 1500rpm for 10 minutes at room temperature, the supernatant was aspirated, and the cell pellet was not to be discarded.

10 Secondary fixation): then adding 5mL of fixing solution, gently and repeatedly blowing the cells again, completely dispersing the cells, and covering the cells, if the cells cannot be detected in time and stored in a refrigerator at the temperature of minus 20 ℃.

11 Sealing the cover with a sealing film, placing into a inspection box, fixing, preventing shaking, and transporting at normal temperature or ice bag to Shanghai Zhen and Biotechnology limited company for chromosome karyotype analysis, wherein the result is shown in figure 5.

From fig. 5, the chromosome karyotyping results performed on the P2 generation cells were: 46, X, t (X; 8) (q 22; p 22.1), chromosome number 46, number normal; the long arm of sex chromosome X and the short arm of chromosome 8 undergo balanced translocation, and the chromosome breakage and the centromere point are q22 and p22.1. These abnormalities are consistent with the characteristics of human salivary gland polyoma-specific gene PLAG1 present and located on chromosome 8. The genetic abnormality of general PA is the presence of translocation on chromosome 8, further indicating that the karyotype analysis results are consistent with the parental tumors.

1.3.7 sequencing analysis

The P2 generation cells obtained in the step 1.2.2 are taken as a study object.

Genomic DNA was extracted from parental tumors and P2-generation cells using (QIAGEN Germany DNA extraction kit). DNA quantification and integrity were determined by Nanodrop spectrophotometry and 1% agarose electrophoresis, respectively.

Genomic DNA samples of parental tumors and P2 generation cells were captured using a Agilent SureSelect human whole exon v8 library (Agilent Technologies, USA) according to the manufacturer's protocol.

The copy number variation and single nucleotide variation, mutation characteristics and mutation frequency in PA were analyzed by whole exon sequencing in the corresponding parental tumors and P2 generation cells, and the results are shown in fig. 6, 7 and 8. In fig. 6 and 8, cells are P2 generation cells, and paramental is a parent tumor; in FIG. 7, signature A and signature B are common mutational characteristics of parental tumors and P2 generation cells.

As can be seen from FIG. 6, the cases of copy number variation and single nucleotide variation in exons of the parent tumor and P2 generation cells are highly consistent and highly consistent.

As can be seen from FIG. 7, the characteristics of signature A and signature B are close in the exon donation ratio of the parental tumor and P2 generation cells, and the common mutation characteristics are highest with C > T and T > A.

As can be seen from FIG. 8, the mutation frequencies in exons of both the parental tumor and P2 generation cells are C > T/G > A and T > C/A > G.

Comparative example 1

In this comparative example 1, tumor tissue fragments were digested with collagenase II and dispase.

The specific digestive juice comprises the following components: 2.5mg/mL of collagenase II, 4mg/mL of mldipase II dispase, 1% diabody and alpha-MEM medium.

As a result, it was found that the tumor tissue was insufficiently digested, and there were still many fragments of tissue, and that the desired cell pellet could not be obtained; although further digestion is possible over extended periods of time, the viable cell content is reduced.

Comparative example 2

This comparative example 2 differs from example 1 in that the culture medium for the polymorphic adenoma cells does not contain cholera toxin, and is otherwise identical to example 1.

As a result, it was found that the cell state was easily developed by cell swelling necrosis after culturing for 10 passages without addition of a cholera toxin medium. Whereas the medium of example 1 can be passaged for at least 17 passages.

Comparative example 3

This comparative example 3 differs from example 1 in that the culture medium for the polymorphic adenoma cells does not contain a ROCK inhibitor, and is otherwise identical to example 1.

As a result, it was found that the growth of cells was slow in the medium without the addition of the ROCK inhibitor and required more than 10 days for the growth, whereas the addition of the ROCK inhibitor required only 7 days for the growth.

Comparative example 4

This comparative example 3 is different from example 1 in that 100 μm and 70 μm sieves (biosharp) were used for filtration, respectively, in sequence, and 40 μm sieves were not used for filtration, and the remainder was the same as example 1.

As a result, it was found that the obtained cell pellet contained a large amount of medium-sized cell aggregates, and single-cell suspensions were not easily formed.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. In addition, many modifications and variations of the methods and compositions of the invention set forth herein will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention.

Claims

1. A salivary gland polyoma cell culture medium, comprising a basal medium; the medium further comprises the following components in concentration based on the total volume of the medium:

rock inhibitor 5-15 mu mol/L

Cholera toxin 5-15 mu L/L.

2. The culture medium of claim 1, wherein the basal medium is selected from one or more of DMEM medium, RPMI 1640 medium, MEM medium, and F12 medium;

and/or the culture medium further comprises a growth promoting factor selected from one or more of insulin, hydrocortisone and epidermal growth factor EGF;

and/or the culture medium further comprises serum selected from the group consisting of fetal bovine serum or calf serum;

and/or the culture medium further comprises an antibiotic selected from one or both of penicillin and streptomycin;

and/or, the Rock inhibitor is Y-27632.

3. The culture medium of claim 2, wherein the basal medium is a mixture of DMEM medium and F12 medium;

and/or, the antibiotic is a mixture of penicillin and streptomycin;

and/or, the serum is selected from the group consisting of fetal bovine serum;

and/or the growth promoting factor is a mixture of insulin, hydrocortisone and epidermal growth factor EGF.

4. The culture medium according to claim 3, wherein the volume ratio of the DMEM medium to F12 medium is (2-4): 1;

and/or, the concentration of the fetal bovine serum is 1-20 v/v% based on the total volume of the culture medium;

and/or, the concentration of the antibiotic is 0.5-1.5 v/v% based on the total volume of the culture medium;

and/or, the concentration of the insulin is 1-6 mg/mL based on the total volume of the culture medium;

and/or, the concentration of hydrocortisone is 20-70 mug/mL based on the total volume of the culture medium;

and/or the concentration of the EGF is 0.1-5 mug/mL based on the total volume of the culture medium.

5. Use of the medium according to any one of claims 1-4 for culturing salivary gland polymorphous adenoma cells.

6. A method of culturing salivary gland polymorphous adenoma cells comprising the steps of:

culturing salivary gland polymorphous adenoma cells using a medium as claimed in any one of claims 1 to 4.

7. The method according to claim 6, wherein the temperature of the culture is 20 to 40 ℃;

and/or, the time of the culture is 3-7 d;

and/or, the salivary gland polymorphous adenoma cells are obtained by the following steps: adopting a tissue block culture method or a digestive enzyme culture method;

preferably, the digestive enzyme culture method is as follows: the tumor tissue is digested with digestive enzymes.

8. The culture method according to claim 7, wherein the digestive enzyme is one or both of collagenase type II and deoxyribonuclease I; preferably, the mass ratio of the type II collagenase to the deoxyribonuclease I is (10-30): 1;

and/or further comprises filtration after digestion, wherein the filtration is carried out by sequentially passing through a filter screen with the pore diameter of 90-120 mu m, 60-80 mu m and 30-50 mu m.

9. The method of claim 7, further comprising subculturing after obtaining salivary gland polyoma cells, wherein the method of subculturing is: pancreatin is added for digestion and then cultivation in the medium is continued.

10. Use of salivary gland polymorphous adenoma cells obtained by a culture process as claimed in any of claims 6 to 9 for the preparation or screening of a medicament for the treatment of malignant salivary gland polymorphous adenomas.