WO2022227110A1 - 口腔癌原代细胞的培养基及培养方法 - Google Patents

口腔癌原代细胞的培养基及培养方法 Download PDF

Info

Publication number
WO2022227110A1
WO2022227110A1 PCT/CN2021/092587 CN2021092587W WO2022227110A1 WO 2022227110 A1 WO2022227110 A1 WO 2022227110A1 CN 2021092587 W CN2021092587 W CN 2021092587W WO 2022227110 A1 WO2022227110 A1 WO 2022227110A1
Authority
WO
WIPO (PCT)
Prior art keywords
oral cancer
cells
medium
primary cells
cancer primary
Prior art date
Application number
PCT/CN2021/092587
Other languages
English (en)
French (fr)
Inventor
刘青松
汪文亮
陈程
黄涛
任涛
王文超
王黎
Original Assignee
合肥中科普瑞昇生物医药科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 合肥中科普瑞昇生物医药科技有限公司 filed Critical 合肥中科普瑞昇生物医药科技有限公司
Publication of WO2022227110A1 publication Critical patent/WO2022227110A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/73—Hydrolases (EC 3.)
    • C12N2501/734—Proteases (EC 3.4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to the field of biotechnology, in particular to a culture medium and a culture method for rapid expansion of oral cancer primary cells, and its application in the evaluation and screening of drug efficacy.
  • Oral cancer in a broad sense refers to malignant tumors that occur in the oral cavity and its adjacent anatomical structures. Its occurrence covers multiple sub-sites and is the sixth most common cancer in the world. It seriously affects the life and health of the population and causes a huge economic burden. According to a global report published by the International Center for Research on Cancer, about 581,100 new cases of oral cancer were added worldwide in 2018, accounting for 3.2% of all new cancer cases in the world; in the same year, there were about 285,500 oral cancer deaths, accounting for 3% of global cancer deaths (Bray F et al, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185countries. CA Cancer J Clin, 2018, 68(6):394-424).
  • the existing oral cancer cell lines cultured in vitro are mainly obtained by long-term culture of normal cells to spontaneously immortalize or transfect oncogenes that promote the immortalization of normal cells.
  • Cell lines established by traditional methods remain the mainstay of cellular, molecular and cancer biology research. However, these methods change the genetic background of cells, and long-term cultured cell lines are also prone to genomic instability, which may lead to artificial changes in the phenotype of tumor cell lines and tumor cells in vivo.
  • the complex heterogeneity of the primary tumor is often lacking in these cell lines, which limits the utility of these cell lines for predicting tumor cell response, affecting the accuracy of scientific research and drug discovery in oral cancer.
  • Xuefeng Liu et al. used irradiated mouse fibroblasts and a Rho-related kinase inhibitor (Y-27632) to expand epithelial-derived cells, which has the ability to achieve unlimited growth of epithelial-derived cells without genetic manipulation (Xuefeng Liu et al., Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat. Protoc. 2017, 12, 439).
  • the method established by Xuefeng Liu et al. has a long culture period and cannot achieve rapid cell expansion, which limits the application of this technology.
  • the present invention provides a culture medium and a culture method for rapidly expanding oral cancer primary cells in vitro.
  • One aspect of the present invention is to provide a culture medium for oral cancer primary cells, the culture medium comprising initial medium, antibiotics, Rho protease inhibitor, fetal bovine serum (FBS), neuregulin 1 (NRG1), non- Essential amino acids, cholera toxin (CTX), ITS cell culture additive, GlutaMAX, insulin, and an additive selected from at least one of B27 additive and N2 additive.
  • the initial medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640;
  • the antibiotic is selected from one or more of streptomycin/penicillin, amphotericin B and Primocin.
  • the concentration range of streptomycin is preferably 25-400 ⁇ g/mL, and the concentration range of penicillin is preferably 25-400 U/mL, and when amphotericin B is used as antibiotic
  • the concentration range is preferably 0.25-4 ⁇ g/mL; when Primocin is used as an antibiotic, its concentration range is preferably 25-400 ⁇ g/mL;
  • Rho protein kinase inhibitor is selected from Y27632, and its concentration range is preferably 1-30 ⁇ M;
  • the volume ratio of fetal bovine serum to the medium is preferably 0.5% (v/v) to 5% (v/v);
  • the concentration range of Neuregulin 1 is preferably 0.5 to 45 ng/mL;
  • Non-essential amino acids are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the total concentration range of non-essential amino acids is preferably 10-900 ⁇ M ;
  • the concentration range of cholera toxin is preferably 1.6 to 45 ng/mL;
  • the volume ratio of the ITS cell culture additive to the medium is preferably 1:10 to 1:900;
  • the volume ratio of the B27 or N2 additive to the medium is preferably 1:5 to 1:450;
  • the volume ratio of GlutaMAX to the medium is preferably 1:10 to 1:900;
  • the concentration range of insulin is preferably 0.1 to 10 ⁇ g/mL.
  • the invention also provides a method for culturing the oral cancer primary cells.
  • the oral cancer primary cells are cultured using the oral cancer primary cell culture medium of the present invention.
  • trophoblasts are added at a cell density of 2 to 3 ⁇ 10 4 cells/cm 2 .
  • the trophoblasts were irradiated NIH-3T3 cells, the irradiation source was X-ray or ⁇ -ray, and the irradiation dose was 20-50 Gy.
  • the oral cancer primary cell culture method of the present invention further comprises the following steps:
  • tissue samples or endoscopic specimens placed in a 1:3 ratio of basal medium and tissue digestion solution, and digest in a constant temperature shaker, preferably 1 gram of tissue is digested with 8-14 mL of tissue digestion solution; digestion temperature range The temperature is 4 to 37°C, preferably 37°C; the digestion speed range is 200rpm to 350rpm, preferably 300rpm;
  • Digestion is sufficient until the digestion can be terminated if there is no obvious tissue block, and the digestion time ranges from 3 to 6 hours, preferably 4 hours;
  • the centrifugation speed is in the range of 1200-1600 rpm, preferably 1500 rpm; the centrifugation time is in the range of 2-6 minutes, preferably 4 minutes, and the basal medium is added to resuspend.
  • the oral cancer primary cells obtained in the above step 1 were resuspended and counted with the oral cancer primary cell culture medium of the present invention, and were seeded into a culture dish according to the cell density of 1-10 ⁇ 10 4 cells/cm 2 , and the cells were grown according to the cell density. Add trophoblasts at a density of 2-3 ⁇ 10 4 cells/cm 2 until the cells in the culture dish are more than 90% full, and can be digested and passaged.
  • the formula of the basal medium described in step 1 is: DMEM/F12 medium contains 25-400 ⁇ g/mL Primocin; the preparation method of the tissue digestion solution described in step 1 is: 1-2 mg/mL collagenase II, 1 ⁇ 2mg/mL collagenase IV, 50 ⁇ 100U/mL DNA, 0.5 ⁇ 1mg/mL hyaluronidase, 1 ⁇ 5mM calcium chloride, 5 ⁇ 10mg/mL bovine serum albumin dissolved in 1640
  • the trophoblasts described in step 2 can be, for example, irradiated NIH-3T3 cells, the irradiation source is X-rays or ⁇ -rays, preferably ⁇ -rays, and the irradiation dose is 20-50 Gy, preferably 30 Gy.
  • the present invention also provides a drug screening method for oral cancer, comprising the following steps:
  • the cultured primary oral cancer cells are not disturbed by interstitial cells such as fibroblasts and adipocytes;
  • the amplification efficiency is high. As long as there are 10 5 -level cells, 10 6 -level oral cancer primary cells can be successfully amplified within a week or so, and the expanded oral cancer primary cells can also be continuously passaged ;
  • Controllable culture cost the medium does not need to add expensive Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10 and other factors;
  • the oral cancer primary cells cultured by the technology have a large number and a high degree of homogeneity, and are suitable for high-throughput screening of new candidate compounds and high-throughput in vitro drug sensitivity functional tests for patients.
  • 1A-1I are graphs showing the effects of different concentrations of factors added to the oral cancer primary cell culture medium of the present invention on the proliferation of oral cancer primary cells.
  • 2A-2F are photographs of microscopic observation of oral cancer primary cells cultured using the oral cancer primary cell culture medium of the present invention.
  • 3A and 3B show the results of pathological identification of oral cancer primary cells cultured using the oral cancer primary cell culture medium of the present invention.
  • 5A and 5B are graphs showing the results of comparison of the culture of oral cancer primary cells using the oral cancer primary cell culture medium of the present invention and a conventional medium.
  • 6A and 6B are graphs showing the results for drug screening of oral cancer cells of different passages cultured using the oral cancer primary cell culture medium of the present invention.
  • Example 1 Effects of various supplementary factors in the culture medium of oral cancer primary cells on the proliferation of oral cancer primary cells
  • the initial medium can be selected from DMEM/F12, DMEM, F12 or RPMI-1640 commonly used in the art.
  • the formula of the basal medium is: DMEM/F12 medium (purchased from Corning Company) + 100 ⁇ g/mL Primocin (purchased from InvivoGen Company, 0.2% (v/v), commercial product concentration 50mg/ml ).
  • Oral cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical staff in professional medical institutions, and all patients signed informed consent.
  • Tissue digestion solution 1640 medium (Corning, 10-040-CVR), collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75mg) /mL), calcium chloride (3.3 mM), BSA (10 mg/mL).
  • Collagenase II, collagenase IV, DNase and hyaluronidase mentioned above were purchased from Sigma Company; calcium chloride was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; BSA was purchased from Biofroxx Company.
  • the media with different compositions in Table 1 were added to a 48-well plate at a volume of 1 mL/well.
  • Oral cancer primary cells isolated from two cases of oral cancer tissues (numbered OZ(0)008 and OZ(0)010) according to the above step (2) were seeded in 48 cells at a cell density of 3 ⁇ 10 4 cells/well.
  • the culture was carried out at 37°C and 5% CO 2 concentration. After culturing for 7-10 days, the cells grew to 85%, the medium was discarded, rinsed once with 100 ⁇ L of 0.05% trypsin per well (purchased from Gibco), and then added 200 ⁇ L of 0.05% trypsin to each well after aspirating.
  • the cells were placed in a 37°C, 5% CO 2 incubator for 10 minutes, and the cells were completely digested under a microscope (CNOPTEC, BDS400). 300 ⁇ L of serum-containing medium (Excell Bio, FND500) was added to terminate the digestion, and 20 ⁇ L was added to the cell counting plate. (Manufacturer: Countstar, specification: 50 pieces/box), count the total number of cells with a cell counter (Countstar, IC1000). Among them, as an experimental control, a basal medium without any additives was used, and the experimental results are shown in Table 1.
  • “+” indicates that compared with the basal medium, the medium added with the additive has the effect of promoting the proliferation of at least two cases of oral cancer primary cells isolated from oral cancer tissue; “-” indicates that the additive is added
  • the medium with the additive showed an inhibitory effect on at least one of the oral cancer primary cells isolated from the oral cancer tissue;
  • “ ⁇ ” indicates that the medium supplemented with the additive inhibited the growth of the oral cancer primary cells isolated from the oral cancer tissue. Proliferation was not significantly affected in at least two of the cases.
  • Y27632 (10 ⁇ M), fetal bovine serum FBS (5%), neuregulin 1NRG1 (5ng/mL), ITS cell culture additive (1:100), non-essential amino acids (100 ⁇ M), cholera toxin CTX (5ng/mL), B27 or N2 (1:50), GlutaMAX (1:100), insulin (1.5 ⁇ g/mL) and other factors for further culture experiments.
  • Example 2 The effect of different concentrations of factors added to the medium on the proliferation of oral cancer primary cells
  • Oral cancer primary cells were obtained from intraoperative tissue samples (numbered OZ(0)013, OZ(0)014) according to the method of step (2)-3 of Example 1, and OC-1 in Table 2 below was used culture medium.
  • the obtained primary oral cancer cells were seeded in a 6-well plate according to the viable cell density of 1 ⁇ 10 4 cells/cm 2 (100,000 cells per well), and added with ⁇ -rays according to the cell density of 2 ⁇ 10 4 cells/cm 2 .
  • NIH-3T3 cells purchased from ATCC, resuspended in basal medium (BM)
  • irradiated with radiation irradiation dose of 30 Gy
  • the surfaces were sterilized and cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
  • Formula 1 Y27632 is not contained in the medium OC-1 medium;
  • the medium OC-1 medium does not contain fetal bovine serum
  • Formulation 4 the medium OC-1 medium does not contain non-essential amino acids
  • Formulation 8 GlutaMAX is not contained in the medium OC-1 medium;
  • Formulation 9 Medium OC-1 medium without insulin.
  • the above-mentioned digested cell suspensions were diluted using the above formulas 1 to 9, respectively, and 10 ⁇ L of the cell suspensions (1 ⁇ 10 4 cells) were seeded in a 48-well plate pre-plated with ⁇ -irradiated NIH-3T3 cells.
  • the ratio is the ratio of the number of cells obtained by culturing each medium for one generation to the number of cells obtained by culturing one generation of the corresponding control wells. If the ratio is greater than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds has a better effect on promoting proliferation than the control well medium; if the ratio is less than 1, it means that the prepared medium containing different concentrations of factors or small molecule compounds can promote proliferation. The effect was weaker than that of the control well medium in promoting proliferation.
  • the content of Y27632 is preferably 1 ⁇ M to 30 ⁇ M; the volume concentration of FBS is preferably 0.5% (v/v) to 5% (v/v); the content of NRG1 is preferably 0.5 ng/ml to 45 ng /ml; the content of non-essential amino acids is preferably 10 ⁇ M ⁇ 900 ⁇ M; the content of CTX is preferably 1.6ng/ml ⁇ 45ng/ml; the volume concentration of ITS is preferably 1:10 ⁇ 1:900; the volume concentration of B27 is preferably 1:1: 5 to 1:450; the volume concentration of GlutaMAX is preferably 1:10 to 1:900; the content of insulin is preferably 0.1 ⁇ g/ml to 10 ⁇ g/ml.
  • the obtained oral cancer primary cells are at a viable cell density of 1 ⁇ 10 4 cells/cm 2 It was seeded in a 6-well plate (100,000 cells per well), and NIH-3T3 cells irradiated with ⁇ -rays (irradiation dose 30 Gy) were added according to the cell density of 2 ⁇ 10 4 cells/cm 2 , and mixed well. The surfaces were sterilized and cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher Scientific).
  • Figures 2A-2F are photos taken under a 10x objective lens. irregular.
  • Example 2 Use the OC-1 medium in Example 2 to culture the samples OZ(0)018 and OZ(0)021 until the cells grow to more than 85%, add 500 ⁇ L of 0.05% trypsin (purchased from Gibco) to rinse for 1 minute, and aspirate the cells. After removal, 500 ⁇ L of 0.05% trypsin was added to each well, and the cells were placed in a 37°C, 5% CO 2 incubator to react for 2 to 10 minutes, and the digestion was terminated until the cells were completely digested.
  • trypsin purchased from Gibco
  • Figures 3A and 3B are the results of pathological and immunohistochemical identification of oral cancer primary cells cultured in samples OZ(0)018 and OZ(0)021 in vitro, which are pictures taken under a 20x objective lens, respectively.
  • the expression of CK, 34 ⁇ E12, P63, CK5/6, and Ki67 indicates that the sample is oral cancer cells, and the primary cells cultured in the OC-1 medium of the present invention and the diagnosis results of oral cancer tissue Consistent.
  • Example 4 Primary culture period and cell number statistics of oral cancer primary cells and calculation of Population Doubling (PD) value
  • Oral cancer was obtained from 4 samples of oral cancer tissue samples (numbered as OZ(0)018, OZ(0)019, OZ(0)020, OZ(0)021) according to the method of step (2)-3 of Example 1 Primary cells.
  • the obtained primary oral cancer cells were cultured in the OC-1 medium in Example 2, and the cells were inoculated in T25 flasks at a viable cell density of 2 ⁇ 10 4 cells/cm 2 and cultured until the cells were expanded. After reaching 95%, it was digested and counted, and the number of days of culture until digestion was recorded at the same time, and the number of days of culture during digestion was regarded as a culture cycle.
  • the amplified cells are amplified in different generations, each generation is counted after digestion and the corresponding culture cycle is recorded. /Initial seeded cells) to calculate PD, see the formula (Chapman et al., Stem Cell Research & Therapy 2014, 5:60).
  • the growth curves of 4 cases of primary cells cultured using the oral cancer primary cells of the present invention were drawn using Graphpad Prism software.
  • the fold of cell expansion in the culture cycle The larger the value, the more times the cells are expanded in a certain cycle, that is, the more cells are amplified.
  • the slope represents the rate of cell expansion. It can be confirmed from FIG. 4 that the oral cancer primary cells cultured in the OC-1 medium of the present invention can be continuously cultured and expanded, and the cell expansion rate remains basically unchanged for at least 70 days, and still has the ability to continue expansion.
  • the medium used in the preparation literature (Xuefeng Liu et al., Nat Protoc. 2017, 12(2): 439-451.), its formula is DMEM/F12 medium+250ng/ml amphotericin B (purchased from Selleck company) +10 ⁇ g/ml gentamicin (purchased from MCE company)+0.1nM cholera toxin+0.125ng/ml EGF+25ng/ml hydrocortisone+10 ⁇ M Y27632+10%FBS.
  • LXF medium LXF medium.
  • Oral cancer primary cells were obtained from intraoperative tissue samples (OZ(0)020, OZ(0)021) according to the method of step (2)-3 of Example 1, and were cultured with trophoblast conditions and without trophoblasts, respectively. conditioned cultivation.
  • the oral cancer primary cells were seeded in 48-well plates at a viable cell density of 3 ⁇ 10 4 cells/cm 2 using the above-mentioned formula LXF, Article1 medium and OC-1 medium in Example 2, respectively. (30,000 cells per well), then NIH-3T3 cells irradiated with ⁇ -rays (irradiation dose 30 Gy) were added at a cell density of 2 ⁇ 10 4 cells/cm 2 , and 48 wells were filled with the corresponding medium at last. The volume of each well in the plate was reduced to 500 ⁇ l and mixed well. The surfaces were sterilized and cultured in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher Scientific). Passage until the cells in the 48-well plate grow to more than 85%.
  • the 48-well plate was taken out, the medium was discarded, rinsed once with 100 ⁇ L of 0.05% trypsin (purchased from Gibco), and then 200 ⁇ L of 0.05% trypsin was added to each well.
  • 0.05% trypsin purchased from Gibco
  • OC-1 medium can significantly promote the expansion of oral cancer primary cells, and its effect is better than the prior art LXF medium and Article medium used.
  • the OC-1 medium has a more obvious effect of promoting the expansion of oral cancer primary cells.
  • Example 6 Oral cancer primary cells amplified using the culture medium of the present invention are used for drug screening
  • Oral cancer primary cells were isolated from the obtained intraoperative samples of oral cancer (OZ(0)020, OZ(0)021) in the same manner as in Example 1, and cultured in OC-1 medium, and the cells were expanded to 85% were digested and passaged as one generation. The 1st, 2nd, 3rd, 4th, and 5th passages were cultured for drug screening.
  • the cells were digested and counted according to the steps in Example 1. Using OC-1 medium, the cells were fully mixed in the sample addition tank (purchased from Corning) according to the viable cell density of 5.76 ⁇ 10 4 cells/mL, and then placed at 384 Well opaque white cell culture plate (purchased from Corning) was used for culture, the volume of each well was 50 ⁇ L, and the number of cells was 3000 cells/well. OC-1 medium was added from the edge of the well plate to seal the plate, and the sample name and the detection time of CellTiter-Glo (purchased from Promega) were marked on the plate. The surface was disinfected with 75% alcohol (purchased from Lierkang), placed in a 37° C., 5% CO 2 incubator for cultivation, and added medicine after 24 hours.
  • 75% alcohol purchasedd from Lierkang
  • the prepared drug plate was taken out, placed at room temperature, centrifuged in a centrifuge (Beckman) at room temperature at 1000 rpm for 1 minute and taken out. High-throughput dosing was performed using a high-throughput automated dosing system (JANUS, Perkin Elmer). To the 384-well plate cultured with oral cancer cells, 0.1 ⁇ L of the corresponding concentration of the screening drug was added to each well. After the addition of the drug, the surface of the 384-well plate was sterilized and moved to an incubator to continue culturing, and cell viability was measured 72 hours later.
  • cell inhibition rate (%) 100% - the chemiluminescence value of the dosing well/the chemiluminescence value of the control well * 100%, calculate the cytostatic rate of cells treated with different drugs, and use graphpad prism software to calculate the effect of drugs on cells.
  • the oral cancer cells cultured with the oral cancer primary cell culture medium of the present invention are used for drug screening, and the inhibitory effect of the same drug on the cultured cells of different generations is basically the same (the inhibition curve is basically the same).
  • the cells of the same patient have different sensitivities to different drugs at the maximum plasma concentration in the human body. According to the results, the effectiveness of the drug in clinical use of the oral cancer patient can be judged, and it can be shown that the sensitivity of the tumor cells of different generations obtained according to the patented culture method to the drug is stable.
  • the invention provides a culture medium and a culture method for culturing or amplifying oral cancer primary cells in vitro, and the cultured cells can be used for the evaluation and screening of the curative effect of drugs.
  • the present invention is suitable for industrial applications.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • General Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种实现口腔癌原代细胞体外快速扩增的培养基和培养方法。口腔癌原代细胞培养基包含初始培养基、抗生素、Rho蛋白酶抑制剂、胎牛血清、神经调节蛋白1、非必需氨基酸、霍乱毒素、GlutaMAX、ITS细胞培养添加剂、胰岛素、以及选自B27添加剂和N2添加剂中的至少一种的添加剂。通过使用口腔癌原代细胞培养基，能够实现口腔癌原代细胞的有效快速扩增，由此扩增得到的细胞保持了病人的病理特性，提高了口腔癌原代细胞的培养成功率和细胞扩增速率，可以为患者的个性化治疗提供研究基础。

Description

口腔癌原代细胞的培养基及培养方法 技术领域
本发明涉及生物技术领域，具体涉及一种用于口腔癌原代细胞的快速扩增的培养基及培养方法，及其在药物的疗效评估和筛选中的应用。
背景技术
口腔癌在广义上是指发生于口腔及其邻近解剖结构的恶性肿瘤，其发生涵盖了多个亚部位，是全球第6大常见的癌症，严重影响人群的生命健康，造成巨大的经济负担。据国际癌症研究中心发表的全球报告显示，2018年在世界范围内增加约58.11万口腔癌新发病例，占全球所有新发癌症病例的3.2％；同年约有28.55万口腔癌死亡病例，占当年全球癌症死亡人数的3％(Bray F等，Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185countries.CA Cancer J Clin,2018,68(6):394-424)。发病率最高的地域包括南亚、欧洲及拉丁美洲，其中印度、孟加拉国等国口腔癌高度流行。虽然中国口腔癌发病率相对较低，但由于嚼食槟榔、吸烟酗酒人数众多，且人口老年化严重，口腔癌造成的长期经济负担值得社会关注。
过去20年，虽然传统治疗方法包括手术、放疗、化疗都有不同程度的发展，但是口腔癌的预后仍然难以让人满意，其5年生存率只有50％～60％左右，大约1/3的患者会复发。因此，新的治疗方法如靶向肿瘤治疗等生物治疗手段正受到越来越多的关注，未来的发展方向是如何合理的选择靶向药物和制定个体化治疗方案。而药敏试验技术的不断革新为靶向药物、化疗药物以及靶向药物联合的疗效预测提供了有力的技术支持，为实现口腔癌患者的个体化治疗奠定坚实的基础。
现有的体外培养的口腔癌细胞系主要通过将正常细胞长期培养而自发永生化或者转染促使正常细胞永生化的癌基因获得。传统方法建立的细胞系依然是细胞、分子和癌生物学研究的主要支柱。但是，这些方法改变了细胞的遗传背景，长期培养的细胞系，也容易造成基因组不稳定，可能导致肿瘤细胞系的表型和体内肿瘤细胞发生人为的改 变。这些细胞系中通常缺乏原发肿瘤的复杂异质性，从而限制了这些细胞系对于预测肿瘤细胞反应的应用，影响口腔癌的科学研究和药物研发的准确性。另外，从口腔癌组织获得的细胞培养成癌细胞的过程中，常规培养方法较难得到癌细胞，培养过程中存在被成纤维细胞干扰，形成的克隆无法传代等问题，限制了人口腔癌原代细胞的应用。
2017年Xuefeng Liu等人使用辐照的小鼠成纤维细胞和Rho相关激酶抑制剂(Y-27632)来扩增上皮来源的细胞，该体系具有无需基因操作就能实现上皮来源细胞无限增长的能力(Xuefeng Liu等，Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nat.Protoc.2017,12,439)。但是，Xuefeng Liu等人建立的方法的培养周期较长，不能实现细胞的快速扩增，限制了该技术的应用。
发明内容
为了解决上述技术问题，本发明提供了一种用于在体外快速扩增口腔癌原代细胞的培养基及培养方法。
本发明的一个方面在于提供一种口腔癌原代细胞的培养基，所述培养基包含初始培养基、抗生素、Rho蛋白酶抑制剂、胎牛血清(FBS)、神经调节蛋白1(NRG1)、非必需氨基酸、霍乱毒素(CTX)、ITS细胞培养添加剂、GlutaMAX、胰岛素、以及选自B27添加剂和N2添加剂中的至少一种的添加剂。其中，所述初始培养基选自DMEM/F12、DMEM、F12或RPMI-1640；抗生素选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种。
在优选的实施方式中，当链霉素/青霉素作为抗生素时，链霉素的浓度范围优选为25～400μg/mL，青霉素的浓度范围优选为25～400U/mL，当两性霉素B作为抗生素时，其浓度范围优选为0.25～4μg/mL，当Primocin作为抗生素时，其浓度范围优选为25～400μg/mL；
Rho蛋白激酶抑制剂选自Y27632，其浓度范围优选为1～30μM；
胎牛血清相对于培养基的体积比优选为0.5％(v/v)～5％(v/v)；
神经调节蛋白1的浓度范围优选为0.5～45ng/mL；
非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷 氨酸、脯氨酸和丝氨酸中的一种或多种，非必需氨基酸的总浓度范围优选为10～900μM；
霍乱毒素的浓度范围优选为1.6～45ng/mL；
ITS细胞培养添加剂相对于培养基的体积比优选为1:10～1:900；
B27或N2添加剂相对于培养基的体积比优选为1:5～1:450；
GlutaMAX相对于培养基的体积比优选为1:10～1:900；
胰岛素的浓度范围优选为0.1～10μg/mL。
本发明还提供一种口腔癌原代细胞的培养方法。本发明的口腔癌原代细胞的培养方法中，使用本发明的口腔癌原代细胞培养基对口腔癌原代细胞进行培养。
本发明的口腔癌原代细胞的培养方法中，按照细胞密度2～3×10 4个/cm 2加入滋养细胞。滋养细胞为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，辐照剂量为20～50Gy。
本发明的口腔癌原代细胞培养方法还包括以下步骤：
1.口腔癌原代细胞的分离
(1)将组织样本或内镜标本置于1:3比例的基础培养基和组织消化液中，于恒温摇床进行消化，优选1克组织用8～14mL组织消化液进行消化；消化温度范围为4～37℃，优选为37℃；消化转速范围为200rpm～350rpm，优选为300rpm；
(2)消化充分直至若未见明显组织块即可终止消化，消化时间范围为3～6小时，优选为4小时；
(3)离心后弃去上清液，离心转速范围为1200～1600rpm，优选为1500rpm；离心时间范围为2～6分钟，优选为4分钟，加入基础培养基重悬。
2.使用本发明的口腔癌原代细胞培养基进行培养
将上述步骤1中获得的口腔癌原代细胞用本发明的口腔癌原代细胞培养基重悬并计数，按照细胞密度1～10×10 4个/cm 2种入培养皿中，同时按照细胞密度2～3×10 4个/cm 2加入滋养细胞，直至培养皿中细胞长满90％以上可进行消化传代。
其中，步骤1中所述的基础培养基的配方为：DMEM/F12培养基含25～400μg/mL Primocin；步骤1中所述的组织消化液的配制方法为： 将1～2mg/mL胶原酶Ⅱ、1～2mg/mL胶原酶Ⅳ、50～100U/mL脱氧核糖核酸、0.5～1mg/mL透明质酸酶、1～5mM氯化钙、5～10mg/mL牛血清白蛋白溶于1640培养基中；步骤2中所述的滋养细胞例如可以为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，优选为γ射线，辐照剂量为20～50Gy，优选为30Gy。
本发明还提供一种口腔癌疾病的药物筛选方法，其包括以下步骤：
(1)使用本发明的口腔癌原代细胞的培养方法培养口腔癌原代细胞，用于药物筛选；
(2)选定需要检测的药物并按照所需浓度梯度进行稀释；
(3)对(1)中培养得到的细胞添加稀释后的所述药物；
(4)进行细胞活性测试。
本发明的技术方案能够取得以下技术效果：
(1)提高口腔癌原代细胞培养的成功率，成功率达到80％以上；
(2)保证体外原代培养的口腔癌原代细胞保持病人的病理特性；
(3)所培养的原代口腔癌细胞不受成纤维细胞、脂肪细胞等间质细胞的干扰；
(4)扩增效率高，只要有10 5级别的细胞数量就可在一周左右时间内成功扩增出10 6数量级的口腔癌原代细胞，扩增出的口腔癌原代细胞还可以连续传代；
(5)培养成本可控：培养基无需加入价格昂贵的Wnt激动剂、R-spondin家族蛋白、BMP抑制剂、FGF10等因子；
(6)所述技术培养获得的口腔癌原代细胞数量大，均一化程度高，适合高通量筛选新候选化合物和为病人提供高通量药物体外敏感性功能测试。
附图说明
图1A-1I为显示本发明口腔癌原代细胞培养基所添加因子的不同浓度对口腔癌原代细胞增殖的影响的图。
图2A-2F为利用显微镜观察使用本发明的口腔癌原代细胞培养基培养得到的口腔癌原代细胞的照片。
图3A和3B为对使用本发明的口腔癌原代细胞培养基培养得到的 口腔癌原代细胞进行病理鉴定的结果。
图4为使用本发明的口腔癌原代细胞培养基对口腔癌原代细胞进行培养的细胞生长曲线。
图5A和5B为显示使用本发明的口腔癌原代细胞培养基和现有培养基对口腔癌原代细胞进行培养的比较结果的图。
图6A和6B为显示使用本发明的口腔癌原代细胞培养基培养得到的不同代数的口腔癌细胞用于药物筛选的结果的图。
具体实施方式
为更好理解本发明，下面结合实施例及附图对本发明作进一步描述，以下实施例仅是对本发明进行说明而非对其加以限定。
实施例1口腔癌原代细胞培养基中各添加因子对口腔癌原代细胞增殖的影响
(1)口腔癌原代细胞培养基的配制
首先配制含有初始培养基的基础培养基。初始培养基可选自本领域常用的DMEM/F12、DMEM、F12或RPMI-1640。在本实施例中，基础培养基的配方为：DMEM/F12培养基(购自Corning公司)+100μg/mL Primocin(购自InvivoGen公司，0.2％(v/v)，市售产品浓度50mg/ml)。
在基础培养基内分别加入不同种类的添加剂(参见表1)配制成含有不同添加成分的口腔癌原代细胞培养基。
(2)口腔癌原代细胞的分离和处理
1样品选择
口腔癌实体瘤组织样品(术中)由专业医疗机构的专业医务人员从患者获取，患者均签署了知情同意书。术中样本0.25cm 3，采用商品化组织保存液(生产厂家：Miltenyi Biotec)存储运输。
2材料准备
15mL无菌离心管、移液枪、10mL移液管、无菌枪头等表面消毒后放入超净工作台中紫外照射30分钟。提前30分钟从4℃冰箱取出基础培养基，提前30分钟从-20℃冰箱取出组织消化液。
组织消化液：1640培养基(Corning，10-040-CVR)、胶原酶Ⅱ(2mg/mL)、胶原酶Ⅳ(2mg/mL)、DNA酶(50U/mL)、透明质酸酶(0.75mg/mL)、氯化钙(3.3mM)、BSA(10mg/mL)。
以上提及的胶原酶Ⅱ、胶原酶Ⅳ、DNA酶、透明质酸酶均购自Sigma公司；氯化钙购自生工生物工程(上海)股份有限公司；BSA购自Biofroxx公司。
3样品分离
3.1超净台中取组织样品于培养皿中，去除带血液的组织，用基础培养基冲洗2次，将组织转移至另一培养皿中用无菌手术刀进行机械分离，将组织块分割为1*1*1mm 3大小；
3.2将切割后的术中组织吸至15mL离心管中，加入5mL基础培养基，混匀，于1500rpm离心4分钟；
3.3弃上清，按1:3比例加入基础培养基和组织消化液(注：组织消化液的加入量是1g肿瘤组织使用约10mL组织消化液)，标记样品名称及编号，用封口膜密封，在37℃下于300rpm摇床中进行消化，期间每30分钟观察消化是否完成，判断依据为无肉眼可见的颗粒物；
3.4消化完成后，经100μm滤网过滤掉未消化的组织团块，滤网上的组织团块用基础培养基冲洗入离心管中以减少细胞损失，于25℃下1500rpm离心4分钟；
3.5弃上清，观察是否有血细胞，若有血细胞，加8mL血细胞裂解液(购自Sigma公司)，混匀，4℃裂解20分钟，期间颠倒混匀一次，25℃下1500rpm离心4分钟；
3.6弃上清，加入2mL基础培养基重悬细胞，备用。
4细胞计数及处理
4.1镜下观察：移取少量重悬细胞平铺于培养皿中，显微镜(CNOPTEC，BDS400)下观察癌细胞密度和形态；
4.2活细胞计数：取重悬的细胞悬液12μL，12μL台盼蓝染液(生产厂家：生工生物工程(上海)股份有限公司)充分混合后，取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)下计算出活的大细胞(细胞粒径>10μm)百分率＝活细胞数/总细胞数*100％。
(3)口腔癌原代细胞的培养
将表1中不同成分的培养基按1mL/孔体积加入48孔板内。将按照上述步骤(2)从两例口腔癌组织(编号为OZ(0)008、OZ(0)010)分离得到的口腔癌原代细胞以3×10 4个/孔的细胞密度接种在48孔培养板内，以37℃、5％CO 2浓度的条件进行培养。培养7～10天后，细胞长至85％，弃培养基，使用每孔100μL 0.05％胰蛋白酶(购自Gibco公司)润洗1遍，吸去后再每孔加入200μL 0.05％胰蛋白酶。置于37℃、5％CO 2培养箱中反应10分钟，显微镜(CNOPTEC，BDS400)下观察细胞已完全消化，加入300μL含血清培养基(Excell Bio，FND500)终止消化，取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)计出细胞总数。其中，作为实验对照，使用未添加任何添加剂的基础培养基，将实验结果示于表1。
表1培养基中的添加成分及促细胞增殖效果
序号 培养基添加剂种类 供应商 终浓度 促细胞增殖程度分级
1 N2 Gibco 1:50 +
2 EGF R&D 5ng/mL ○
3 R-spondin1 R&D 20ng/mL ○
4 前列腺素E2 Tocris 0.5μM ○
5 胰岛素 Peprotech 1.5μg/mL +
6 B27 Gibco 1:50 +
7 A8301 MCE 100nM -
8 SB202190 MCE 200nM ○
9 霍乱毒素 Sigma 5ng/mL +
10 氢化可的松 Sigma 10ng/mL ○
11 Noggin R&D 30ng/mL ○
12 FBS Excell 5％ +
13 IGF-1 R&D 45ng/mL ○
14 KGF R&D 5ng/mL ○
15 GlutaMAX Gibco 1:100 +
16 非必需氨基酸 Corning 100μM +
17 FGF10 sino biological 1ng/mL ○
18 NRG1 sino biological 5ng/mL +
19 Y27632 MCE 10μM +
20 ITS Gibco 1:100 +
其中，“+”表示与基础培养基相比，加入该添加剂的培养基对 从口腔癌组织分离出的口腔癌原代细胞中的至少两例有促进增殖的作用；“－”表示添加该添加剂的培养基对从口腔癌组织分离出的口腔癌原代细胞中的至少一例显示有抑制增殖的作用；“○”表示添加该添加剂的培养基对从口腔癌组织分离出的口腔癌原代细胞中的至少两例的增殖没有明显的影响。
根据以上结果，拟选择Y27632(10μM)、胎牛血清FBS(5％)、神经调节蛋白1NRG1(5ng/mL)、ITS细胞培养添加剂(1:100)、非必需氨基酸(100μM)、霍乱毒素CTX(5ng/mL)、B27或N2(1:50)、GlutaMAX(1:100)、胰岛素(1.5μg/mL)等因子进行进一步培养实验。
实施例2培养基添加因子的不同浓度对口腔癌原代细胞的增殖作用
按照实施例1的步骤(2)之3的方法从术中组织样本(编号为OZ(0)013、OZ(0)014)获得口腔癌原代细胞，并使用下表2中的OC-1培养基进行培养。
表2培养基组分(浓度为终浓度)
Figure PCTCN2021092587-appb-000001
所获得的口腔癌原代细胞，按照活细胞密度1×10 4个/cm 2接种于6孔板中(每孔10万细胞数)，按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞(购自ATCC，使用基础 培养基(BM)进行重悬)，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。在OC-1培养基的基础之上培养扩增，至细胞长至85％以上，加入500μL 0.05％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500μL 0.05％胰蛋白酶，置于37℃、5％CO 2培养箱中反应2～10分钟，直至细胞已经消化完全即终止消化，1500rpm离心4分钟后，弃上清。DMEM/F12重悬细胞沉淀。取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)计出细胞总数。所得细胞用于以下培养实验。
接着，配制以下9种配方培养基进行实验：
配方1：培养基OC-1培养基中不含Y27632；
配方2：培养基OC-1培养基中不含胎牛血清；
配方3：培养基OC-1培养基中不含NRG1；
配方4：培养基OC-1培养基中不含非必需氨基酸；
配方5：培养基OC-1培养基中不含CTX；
配方6：培养基OC-1培养基中不含ITS；
配方7：培养基OC-1培养基中不含B27；
配方8：培养基OC-1培养基中不含GlutaMAX；
配方9：培养基OC-1培养基中不含胰岛素。
分别使用上述配方1～9来稀释上述消化后的细胞悬液，在预铺有γ射线辐照过后的NIH-3T3细胞的48孔板中种入10μL细胞悬液(1x10 4个细胞)。
在使用配方1的培养基时，在接种有原代细胞的48孔板中在配方1的基础上分别添加配制好的Y27632每孔1mL，Y27632的终浓度分别为1μM、3μM、10μM、30μM、90μM；并使用配方1的培养基设置对照孔(BC)。该系列的培养基中其他添加因子的终浓度与OC-1培养基相同。以下配方1-9的实验也以同样的方式进行，不再赘述。
在使用配方2的培养基时，在接种有原代细胞的48孔板中在配方2的基础上分别添加配制好的胎牛血清FBS每孔1mL，胎牛血清的终浓度分别为0.5％(v/v)、1.6％(v/v)、5％(v/v)、15％(v/v)、45％(v/v)；并使用配方2的培养基设置对照孔(BC)。
在使用配方3的培养基时，在接种有原代细胞的48孔板中在配方 3的基础上分别添加配制好的NRG1每孔1mL，NRG1终浓度分别为0.5ng/ml、1.6ng/ml、5ng/ml、15ng/ml、45ng/ml；并使用配方3的培养基设置对照孔(BC)。
在使用配方4的培养基时，在接种有原代细胞的48孔板中在配方4的基础上分别添加配制好的非必需氨基酸每孔1mL，非必需氨基酸的终浓度分别为10μM、30μM、100μM、300μM、900μM；并使用配方4的培养基设置对照孔(BC)。
在使用配方5的培养基时，在接种有原代细胞的48孔板中在配方5的基础上分别添加配制好的CTX每孔1mL，霍乱毒素的终浓度分别为0.5ng/ml、1.6ng/ml、5ng/ml、15ng/ml、45ng/ml；并使用配方5的培养基设置对照孔(BC)。
在使用配方6的培养基时，在接种有原代细胞的48孔板中在配方6的基础上分别添加配制好的ITS每孔1mL，ITS终浓度分别为1:900、1:300、1:100、1:30、1:10；并使用配方6的培养基设置对照孔(BC)。
在使用配方7的培养基时，在接种有原代细胞的48孔板中在配方7的基础上分别添加配制好的B27每孔1mL，B27的终浓度分别为1:450、1:150、1:50、1:16、1:5；并使用配方7的培养基设置对照孔(BC)。
在使用配方8的培养基时，在接种有原代细胞的48孔板中在配方8的基础上分别添加配制好的GlutaMAX每孔1mL，GlutaMAX的终浓度分别为1:900、1:300、1:100、1:30、1:10；并使用配方8的培养基设置对照孔(BC)。
在使用配方9的培养基时，在接种有原代细胞的48孔板中在配方9的基础上分别添加配制好的胰岛素每孔1mL，胰岛素的终浓度分别为0.1μg/ml、0.3μg/ml、1μg/ml、3μg/ml、10μg/ml；并使用配方9的培养基设置对照孔(BC)。
待细胞扩增至48孔的85％左右消化计数，分别参比对照孔(BC)细胞数计算增殖倍数，将2例样本收集的数据汇总示于图1A～1I。图1A～1I中，比值为使用各培养基培养一代得到的细胞数与对应的对照孔培养一代得到的细胞数的比。比值大于1说明配制的含不同浓度的因子或小分子化合物的培养基促增殖效果优于对照孔培养基；比值小于1，则说明配制的含不同浓度的因子或小分子化合物的培养基促增殖 效果较对照孔培养基促增殖效果弱。
根据图1A～1I的结果，Y27632的含量优选为1μM～30μM；FBS的体积浓度优选为0.5％(v/v)～5％(v/v)；NRG1的含量优选为0.5ng/ml～45ng/ml；非必需氨基酸的含量优选为10μM～900μM；CTX的含量优选为1.6ng/ml～45ng/ml；ITS的体积浓度优选为1:10～1:900；B27的体积浓度优选为1:5～1:450；GlutaMAX的体积浓度优选为1:10～1:900；胰岛素的含量优选为0.1μg/ml～10μg/ml。
实施例3口腔癌原代细胞培养及鉴定
按照实施例1的步骤(2)之3的方法从术中组织样本(编号为OZ(0)013、OZ(0)014、OZ(0)018、OZ(0)019、OZ(0)020、OZ(0)021)获得口腔癌原代细胞，并使用实施例2中的OC-1培养基进行培养，所获得的口腔癌原代细胞，按照活细胞密度1×10 4个/cm 2接种于6孔板中(每孔10万细胞数)，按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞，混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。
在第3-14天，使用显微镜(Invitrogen公司EVOS M500)观察培养得到的口腔癌原代细胞，图2A-2F的是10倍物镜下拍摄得到的照片，细胞在镜下呈紧密排列，形态略不规则。
使用实施例2中的OC-1培养基培养样本OZ(0)018、OZ(0)021直至细胞长至85％以上，加入500μL 0.05％胰蛋白酶(购自Gibco公司)润洗1分钟，吸去后再每孔加入500μL 0.05％胰蛋白酶，置于37℃、5％CO 2培养箱中反应2～10分钟，直至细胞已经消化完全即终止消化。1500rpm离心4分钟后，弃上清，加入500μL OC-1培养基重悬，对培养得到的口腔癌原代细胞送去金域进行病理和免疫组化鉴定。
图3A和3B为样本OZ(0)018、OZ(0)021体外培养后的口腔癌原代细胞进行病理和免疫组化鉴定的结果，分别为20倍物镜下拍照的图片。如图3A和3B所示，CK、34βE12、P63、CK5/6、Ki67表达，提示该样本为口腔癌细胞，且使用本发明的OC-1培养基培养的原代细胞与口腔癌组织诊断结果一致。
实施例4口腔癌原代细胞初次培养周期和细胞数统计及Population Doubling(PD)值计算
按照实施例1步骤(2)之3的方法从4例样本口腔癌组织样本(编号为OZ(0)018、OZ(0)019、OZ(0)020、OZ(0)021)获得口腔癌原代细胞。对于所获得的口腔癌原代细胞，使用实施例2中的OC-1培养基培养，按照活细胞密度2×10 4个/cm 2将细胞接种在T25瓶中并进行培养，待细胞扩增至95％后消化并计数，同时记录直至消化时培养的天数，将消化时培养的天数作为一个培养周期。在该实验条件下持续培养，将扩增所得的细胞进行不同代数扩增，每一代进行消化后计数并记录相应培养的周期，根据公式Population Doubling(PD)＝3.32*log 10(消化后细胞总数/初始种入细胞数)计算PD，公式参见(Chapman等，Stem Cell Research&Therapy 2014,5:60)。
如图4所示，采用Graphpad Prism软件绘制使用本发明的口腔癌原代细胞培养的4例原代细胞的生长曲线，横坐标表示细胞培养的天数，纵坐标是累计的细胞增殖倍数，其表示细胞在培养周期内扩增的倍数，数值越大表示细胞在一定周期内扩增的次数越多，即扩增得到的细胞数也就越多，斜率代表的是细胞扩增的速率。从图4中可以确认，本发明的OC-1培养基培养的口腔癌原代细胞可进行持续培养扩增，且至少70天时细胞扩增速率基本保持不变，仍具有继续扩增的能力。
实施例5与现有培养基培养效果的比较
(1)对照培养基的配制
配制文献(Xuefeng Liu等，Nat Protoc.2017,12(2):439-451.)中使用的培养基，其配方为DMEM/F12培养基+250ng/ml两性霉素B(购自Selleck公司)+10μg/ml庆大霉素(购自MCE公司)+0.1nM霍乱毒素+0.125ng/ml EGF+25ng/ml氢化可的松+10μM Y27632+10％FBS。以下简称为LXF培养基。
配置另一文献(Meysam Ganjibakhsh等，J Dent(Tehran).2017,14(4):191-202.)中使用的培养基，其配方为DF12(购自Corning公司)+10％FBS(购自Excell公司)+100U/ml青霉素(购自corning公司)+100 μg/ml链霉素(购自corning公司)+6mM L-谷氨酰胺(购自赛默飞公司)+10μg/ml胰岛素+20ng/ml EGF+0.5μg/ml氢化可的松+100ng/ml霍乱毒素+2mM非必需氨基酸(购自corning公司)。以下简称为Article1培养基。
(2)口腔癌原代细胞的获取和培养
按照实施例1的步骤(2)之3的方法从术中组织样本(OZ(0)020、OZ(0)021)获得口腔癌原代细胞，分别进行有滋养细胞条件的培养和无滋养细胞条件的培养。
在有滋养细胞时，分别使用上述配方LXF、Article1培养基和实施例2中的OC-1培养基按照活细胞密度3×10 4个/cm 2将口腔癌原代细胞接种于48孔板中(每孔3万细胞数)，随后按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量30Gy)的NIH-3T3细胞，最后分别使用对应的培养基补齐48孔板中各孔体积至500微升，充分混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。直至48孔板中细胞长至85％以上，进行传代。
在无滋养细胞时，分别使用上述LXF、Article1培养基和实施例2中的OC-1培养基按照活细胞密度3×10 4个/cm 2将口腔癌原代细胞接种于48孔板中(每孔3万细胞数)，再分别使用对应的培养基补齐48孔板中各孔体积至500微升，充分混匀。表面消毒后置于37℃、5％CO 2培养箱(购自赛默飞)培养。直至48孔板中细胞长至85％以上，进行传代。
在培养第7天，取出48孔板，弃培养基，使用100μL 0.05％胰蛋白酶(购自Gibco公司)润洗1遍，吸去后再每孔加入200μL 0.05％胰蛋白酶。置于37℃、5％CO 2培养箱中反应10分钟，显微镜(CNOPTEC，BDS400)下观察细胞已完全消化，加入300μL含10％血清DF12终止消化，取20μL加入细胞计数板(生产厂家：Countstar，规格：50片/盒)，细胞计数仪(Countstar，IC1000)计出细胞总数，将计数结果示于图5A和5B。
根据图5A和5B的结果可知，与LXF培养基和Article培养基相比，不管有无滋养细胞，OC-1培养基均能显著促进口腔癌原代细胞扩增，其效果优于现有技术采用的LXF培养基和Article培养基。并且， 在有滋养细胞的条件下，OC-1培养基对于口腔癌原代细胞的扩增促进效果更为明显。
实施例6使用本发明培养基扩增得到的口腔癌原代细胞用于药物筛选
1、细胞培养和铺板
从得到的口腔癌术中样本(OZ(0)020、OZ(0)021)与实施例1同样地分离得到口腔癌原代细胞，并使用OC-1培养基进行培养，待细胞扩增至85％，进行消化传代，作为一代。分别取培养第1代、第2代、第3代、第4代、第5代细胞进行药物筛选。
按照实施例1中步骤将细胞消化计数，使用OC-1培养基，将细胞按照活细胞密度5.76×10 4个/mL细胞于加样槽(购自康宁公司)中充分混匀后，在384孔不透明白色细胞培养板(购自康宁公司)进行培养，每孔体积50μL，细胞数目为3000个/孔。从孔板边缘加入OC-1培养基封板，板上标注样品名称及CellTiter-Glo(购自Promega公司)检测时间。表面75％酒精(购自利尔康)消毒，置37℃、5％CO 2培养箱培养，24小时后加药。
2、筛选药物配制
按照下表配制6个浓度梯度的2种药物(柔红霉素、硼替佐米；均购自MCE公司)，在384孔药板(购自赛默飞公司)每孔中添加30μL，保存待用。
表3柔红霉素、硼替佐米药物添加液的配制
Figure PCTCN2021092587-appb-000002
3、高通量加药
取出配制好的药板，置于室温，于离心机(贝克曼)中室温1000rpm 离心1分钟后取出。采用高通量自动化加样系统(Perkin Elmer公司JANUS)进行高通量加药。对培养有口腔癌细胞的384孔板在每孔加入0.1μL对应浓度的筛选药物，加药结束后，384孔板表面消毒后移至培养箱中继续培养，72小时后测定细胞活性。
4、细胞活性测试
4℃冰箱取出CellTiter-Glo发光试剂(购自Promega公司)，取10毫升试剂于加样槽中，培养箱中取出待检测384孔板，每孔加入10μL CellTiter-Glo发光试剂，静置10分钟后混匀，使用多功能酶标仪(Perkin Elmer公司Envision)检测。
5、数据处理
按照公式细胞抑制率(％)＝100％-加药孔化学发光数值/对照孔化学发光数值*100％，计算得到不同药物作用细胞后的细胞抑制率，使用graphpad prism软件计算药物对细胞作用的半数抑制率(IC 50)。将结果示于图6A和6B。
由图6A和6B可以确认，使用本发明的口腔癌原代细胞培养基培养得到的口腔癌细胞进行药物筛选，相同药物对于培养的不同代数细胞抑制效果基本保持一致(抑制曲线基本保持一致)。同一病人的细胞对不同药物在人体内最大血药浓度时的敏感性不同。根据结果可以判断口腔癌病人在临床使用该种药物时的有效性，同时可以说明根据本专利培养方法得到不同代数的肿瘤细胞对药物的敏感性是稳定的。
工业应用性
本发明提供一种用于在体外培养或扩增口腔癌原代细胞的培养基及培养方法，可将培养得到的细胞应用于药物的疗效评估和筛选。因而，本发明适于工业应用。
尽管本文对本发明作了详细说明，但本发明不限于此，本技术领域的技术人员可以根据本发明的原理进行修改，因此，凡按照本发明的原理进行的各种修改都应当理解为落入本发明的保护范围。

Claims (6)

  1. 一种口腔癌原代细胞培养基，其特征在于，包含：
    初始培养基、抗生素、Rho蛋白酶抑制剂、胎牛血清、神经调节蛋白1、非必需氨基酸、霍乱毒素、GlutaMAX、ITS细胞培养添加剂、胰岛素、以及选自B27添加剂和N2添加剂中的至少一种的添加剂，
    其中所述初始培养基选自DMEM/F12、DMEM、F12或RPMI-1640；
    所述抗生素选自链霉素/青霉素、两性霉素B和Primocin中的一种或多种。
  2. 如权利要求1所述的口腔癌原代细胞培养基，其特征在于：
    当抗生素选自链霉素/青霉素时，链霉素浓度范围为25～400μg/mL，青霉素浓度范围为25～400U/mL，当抗生素选自两性霉素B时，浓度范围为0.25～4μg/mL，当抗生素选自Primocin时，浓度范围为25～400μg/mL；
    Rho蛋白激酶抑制剂选自Y27632，其浓度范围为1～30μM；
    胎牛血清相对于培养基的体积比为0.5％(v/v)～5％(v/v)；
    神经调节蛋白1的浓度范围为0.5～45ng/mL；
    非必需氨基酸为选自甘氨酸、丙氨酸、天冬酰胺、天冬氨酸、谷氨酸、脯氨酸和丝氨酸中的一种或多种，总浓度为10～900μM；
    霍乱毒素的浓度范围为1.6～45ng/mL；
    ITS细胞培养添加剂相对于培养基的体积比为1:10～1:900；
    B27或N2添加剂相对于培养基的体积比为1:5～1:450；
    GlutaMAX相对于培养基的体积比为1:10～1:900；
    胰岛素的浓度范围为0.1～10μg/mL。
  3. 一种口腔癌原代细胞的培养方法，其特征在于：
    使用权利要求1或2所述的口腔癌原代细胞培养基对口腔癌原代细胞进行培养。
  4. 如权利要求3所述的培养方法，其特征在于：
    将口腔癌原代细胞按照细胞密度1～10×10 4个/cm 2种入培养皿中，并按照细胞密度2～3×10 4个/cm 2加入滋养细胞，然后使用如权利要求1或2所述的口腔癌原代细胞培养基进行培养。
  5. 如权利要求4所述的培养方法，其特征在于：
    所述滋养细胞为辐照后的NIH-3T3细胞，辐照源为X射线或者γ射线，辐照剂量为30～50Gy。
  6. 一种口腔癌疾病的药物筛选方法，其特征在于，包括以下步骤：
    (1)使用权利要求3～5中任一项所述的口腔癌原代细胞的培养方法培养口腔癌原代细胞；
    (2)选定需要检测的药物并按照所需浓度梯度进行稀释；
    (3)对(1)中培养得到的细胞添加稀释后的所述药物；
    (4)进行细胞活性测试。
PCT/CN2021/092587 2021-04-29 2021-05-10 口腔癌原代细胞的培养基及培养方法 WO2022227110A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110472721.2A CN115261325B (zh) 2021-04-29 2021-04-29 口腔癌原代细胞的培养基及培养方法
CN202110472721.2 2021-04-29

Publications (1)

Publication Number Publication Date
WO2022227110A1 true WO2022227110A1 (zh) 2022-11-03

Family

ID=83745007

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/092587 WO2022227110A1 (zh) 2021-04-29 2021-05-10 口腔癌原代细胞的培养基及培养方法

Country Status (2)

Country Link
CN (1) CN115261325B (zh)
WO (1) WO2022227110A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117467616B (zh) * 2023-12-26 2024-03-26 北京基石生命科技有限公司 用于制备口腔癌微肿瘤模型的专用培养基以及相关的方法产品和应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110912A1 (en) * 2000-12-07 2002-08-15 Ranju Ralhan Stable human oral cancer cell carcinoma cell line
CN107142237A (zh) * 2011-05-17 2017-09-08 李晖 培养基、细胞培养用试剂盒及细胞培养方法
CN108624561A (zh) * 2018-05-26 2018-10-09 复旦大学 原代肿瘤细胞培养基、培养方法以及应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012131733A2 (en) * 2011-03-31 2012-10-04 Godavari Biorefineries Limited Media compositions, method of initiating and enriching cultures of stem cells and/or cancer stem-like cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110912A1 (en) * 2000-12-07 2002-08-15 Ranju Ralhan Stable human oral cancer cell carcinoma cell line
CN107142237A (zh) * 2011-05-17 2017-09-08 李晖 培养基、细胞培养用试剂盒及细胞培养方法
CN108624561A (zh) * 2018-05-26 2018-10-09 复旦大学 原代肿瘤细胞培养基、培养方法以及应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HANXIAO XU, XIAODONG LYU, MING YI, WEIHENG ZHAO, YONGPING SONG, KONGMING WU: "Organoid technology and applications in cancer research", JOURNAL OF HEMATOLOGY & ONCOLOGY, BIOMED CENTRAL LTD, LONDON UK, vol. 11, no. 1, 15 September 2018 (2018-09-15), London UK , pages 1 - 15, XP009530295, ISSN: 1756-8722, DOI: 10.1186/s13045-018-0662-9 *
LIU, XUEFENG ET AL.: "Conditional Reprogramming and Long-term Expansion of Normal and Tumor Cells from Human Biospecimens", NATURE PROTOCOLS, vol. 12, no. 2, 26 January 2017 (2017-01-26), XP037547631, DOI: 10.1038/nprot.2016.174 *
YANG MIN, WANG SHENGNAN;LI MENGQIAN;LIU ZHAOCEN;LIU LINGLING;ZHANG MANJIN;WANG FU: "The expansion of the Oral Mucosa Epithelial Cells in Vitro in Conditioned Culture Media Supplemented with Y27632", ORAL BIOMEDICINE, vol. 6, no. 2, 30 June 2015 (2015-06-30), XP055982502, ISSN: 1674-8603, DOI: 10.3969/j.issn.1674-8603.2015.02.008 *

Also Published As

Publication number Publication date
CN115261325A (zh) 2022-11-01
CN115261325B (zh) 2023-12-12

Similar Documents

Publication Publication Date Title
CN113528444B (zh) 一种用于食管鳞癌上皮细胞的培养基、培养方法及其应用
CN112779209B (zh) 原代乳腺上皮细胞培养基、培养方法及其应用
WO2021184408A1 (zh) 胃癌原代细胞的培养基及培养方法
WO2021179354A1 (zh) 原代肝癌细胞培养基、原代肝癌细胞培养方法及其应用
WO2021159560A1 (zh) 食管鳞癌原代细胞的培养基及培养方法
WO2023060774A1 (zh) 宫颈癌类器官的培养基、及其培养方法和应用
WO2022227110A1 (zh) 口腔癌原代细胞的培养基及培养方法
WO2022241845A1 (zh) 肝癌原代细胞的培养基及培养方法
WO2023060764A1 (zh) 胃癌原代细胞的培养基和培养方法
WO2022160368A1 (zh) 肠癌原代细胞的培养基及培养方法
WO2023035299A1 (zh) 一种用于肝癌类器官培养的培养基、及其培养方法和应用
WO2023004888A1 (zh) 一种用于口腔癌类器官培养的培养基、及其培养方法和应用
WO2024065883A1 (zh) 一种用于鼻咽癌类器官培养的培养基、及其培养方法和应用
EP4389875A1 (en) Culture medium and culture method for human primary acute myeloid leukemia cells
WO2023060695A1 (zh) 肝癌悬浮类器官的培养基和培养方法
WO2022151520A1 (zh) 原代胃肠间质瘤细胞培养基、培养方法及其应用
CN113969262B (zh) 一种用于肺癌上皮细胞的培养基、培养方法及其应用
WO2023060642A1 (zh) 一种肠癌原代细胞的培养基及体外培养方法和用途
WO2023019637A1 (zh) 人原代急性淋巴白血病细胞的培养基及培养方法
WO2023060696A1 (zh) 卵巢癌原代细胞的培养基、培养方法及其应用
WO2023060710A1 (zh) 胃癌类器官的培养基及培养方法
CN115975932A (zh) 卵巢癌原代细胞的培养基、培养方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21938583

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21938583

Country of ref document: EP

Kind code of ref document: A1