WO2021159560A1 - 食管鳞癌原代细胞的培养基及培养方法 - Google Patents

食管鳞癌原代细胞的培养基及培养方法 Download PDF

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WO2021159560A1
WO2021159560A1 PCT/CN2020/076736 CN2020076736W WO2021159560A1 WO 2021159560 A1 WO2021159560 A1 WO 2021159560A1 CN 2020076736 W CN2020076736 W CN 2020076736W WO 2021159560 A1 WO2021159560 A1 WO 2021159560A1
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cells
medium
esophageal squamous
cell carcinoma
squamous cell
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PCT/CN2020/076736
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French (fr)
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刘青松
胡洁
陈程
王文超
王黎
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合肥中科普瑞昇生物医药科技有限公司
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Priority to EP20918987.7A priority Critical patent/EP4105320A4/en
Priority to JP2022548485A priority patent/JP7454290B2/ja
Priority to US17/798,640 priority patent/US20230091960A1/en
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Definitions

  • the invention belongs to the field of biotechnology, and specifically relates to a culture medium and a culture method for rapidly expanding primary cells of esophageal squamous cell carcinoma in vitro.
  • esophageal cancer is one of the most common gastrointestinal malignancies in the world. According to the latest statistics from the National Cancer Center, esophageal cancer ranks fourth among the top ten malignant tumors in men, and esophageal cancer ranks third in women. Many regions in the world have increased endemic morbidity. my country is a region with a high incidence of esophageal cancer. The average annual death toll is about 150,000, accounting for 21.8% of the national tumor mortality rate. It ranks first among the cancers that cause the most deaths. 4 digits (Non-Patent Document 1).
  • Non-Patent Document 2 In the primary cell culture of esophageal cancer, it is known that the tumor cells are directly isolated and then cultured in a medium containing fetal bovine serum (Non-Patent Document 2); in recent years, it has also been reported to use conditional reprogramming Method to culture epithelial-derived cells, which mentions that it can be used to grow and expand primary cells of esophageal cancer (Non-Patent Document 3); subsequently, there are reports in the literature that the intraoperative samples of esophageal cancer are digested with trypsin, and the conditions are reconstituted. Programming method for cultivation method (Non-Patent Document 4). Recently, there has been a literature report on a method of culture using a commercial medium KSFM with added factors (Non-Patent Document 5), and a literature on the use of organoid culture method for culture (Non-Patent Document 6).
  • Non-Patent Document 2 cannot be cultured stably for a long period of time.
  • the methods in Non-Patent Document 3 and Non-Patent Document 4 have a long culture cycle.
  • the method in Non-Patent Document 5 requires a relatively high cost.
  • Non-Patent Document 6 is used for organoids.
  • the culture reagents are too expensive, and the operation during the culture process is complicated, which is not conducive to large-scale popularization and application.
  • the methods in foreign literature are basically based on the research of esophageal adenocarcinoma, so it is not suitable for culturing esophageal squamous cell carcinoma. Therefore, it is necessary to develop a medium and culture method suitable for culturing primary cells of esophageal squamous cell carcinoma.
  • Non-Patent Document 1 Freddie Bray, BSc, MSc, PhD; Jacques Ferlay, ME, et al. Global Cancer Statistics 2018. CA CANCER J CLIN 2018; 68:394-424;
  • Non-Patent Document 2 Karin J. Purdie, Celine Pourreyron, and Andrew P. South. Isolation and Culture of Squamous Cell Culture: Methods and Protocols, Second Edition, 731 Methods, in Molecular Biology, 151-159;
  • Non-Patent Document 3 Xuefeng Liu, Ewa Krawczyk, et al. Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nature Protocols, VOl. 12NO. 2, 2017, 439-451;
  • Non-Patent Document 4 Todd J. Jensen, Christopher Foster, et al. Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation, Journal of Visualized Experiment, March 2017, 121;
  • Non-Patent Document 5 Yuta Kasagi, et al. The Esophageal Organoid System Reveals Functional Interplay Between Notch and Cytokines in Reactive Epithelial Changes. Cellular and Molecular Gastroenterology and Hepatology, 2018, No. 333, Vol. 5, No. 352
  • Non-Patent Document 6 Xiaodun Li, Hayley E. Francies, et al. Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics. NATURE COMMUNICATIONS (2018) 9:
  • the present invention provides a culture medium and a culture method for rapidly expanding primary cells of esophageal squamous cell carcinoma in vitro.
  • One aspect of the present invention is to provide a culture medium for primary cells of esophageal squamous cell carcinoma, the culture medium comprising an initial medium, the following components (1) to (6), and optionally the following components (7) to (9), wherein the initial medium can be, for example, DMEM/F12, DMEM, F12 or RPMI-1640, preferably DMEM/F12.
  • the initial medium can be, for example, DMEM/F12, DMEM, F12 or RPMI-1640, preferably DMEM/F12.
  • Rho protein kinase inhibitor selected from one or more of Y27632, Hydroxyfasudil and GSK429286A, when selected from Y27632, the concentration range is 2.5-40 ⁇ M, preferably 5-20 ⁇ M, more Preferably, it is 10 ⁇ M; when selected from hydroxyfasudil, the concentration range is 2 to 32 ⁇ M, preferably 4 to 16 ⁇ M, more preferably 8 ⁇ M; when selected from GSK429286A, the concentration range is 2 to 32 ⁇ M, preferably 4 to 16 ⁇ M, more Preferably it is 8 ⁇ M;
  • streptomycin/penicillin one or more selected from streptomycin/penicillin, amphotericin B and Primocin.
  • concentration of streptomycin ranges from 25 to 400 ⁇ g/mL, preferably 50 ⁇ 200 ⁇ g/mL, more preferably 200 ⁇ g/m
  • penicillin concentration range is 25 ⁇ 400U/mL, preferably 50 ⁇ 200U/mL, more preferably 200U/mL
  • concentration range is 0.25 ⁇ 4 ⁇ g/mL, preferably 0.5 ⁇ 2 ⁇ g/mL, more preferably 1 ⁇ g/m
  • Primocin the concentration range is 25 ⁇ 400mg/mL, preferably 50 ⁇ 200mg/mL, more preferably 100mg/mL, so
  • the antibiotic is preferably Primocin;
  • the concentration range is 2.5-40 ⁇ g/mL, preferably 10-40 ⁇ g/mL, more preferably 20 ⁇ g/mL;
  • the volume ratio of N2 additive relative to the culture medium is 1:400 to 1:25, preferably 1:100 to 1:25, more preferably 1:50;
  • IGF-1 Insulin-like growth factor 1
  • concentration range is 2.5-40ng/mL, preferably 2.5-10ng/mL, more preferably 5ng/mL;
  • Non-essential amino acids which are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, non-essential amino acids
  • the total concentration of is in the range of 50 ⁇ M to 400 ⁇ M, preferably 100 ⁇ M to 400 ⁇ M, more preferably 400 ⁇ M;
  • Hydrocortisone the concentration range is 0-1.6 ⁇ g/mL, preferably 0.2-0.8 ⁇ g/mL, more preferably 0.4 ⁇ g/mL;
  • Glutamine the concentration range is 0mM-8mM, preferably 1mM-4mM, more preferably 2mM;
  • Bovine pituitary extract the concentration range is 0-56 ⁇ g/mL, preferably 3.5-14 ⁇ g/mL, more preferably 7 ⁇ g/mL.
  • Another aspect of the present invention is to provide a method for culturing primary esophageal squamous cell carcinoma cells.
  • the above-mentioned esophageal squamous carcinoma primary cell culture medium is used for culturing.
  • the above-mentioned cultivation method includes the following steps:
  • tissue samples such as endoscopic specimens
  • tissue cleaning solution for digestion
  • tissue digestive enzymes for digestion Enzyme digestion
  • the digestion temperature range is 4 degrees Celsius to 37 degrees Celsius, preferably 37 degrees Celsius
  • the digestion speed range is 200 rpm to 350 rpm, preferably 300 rpm;
  • the digestion time range is 4-8 hours, preferably 6 hours;
  • the centrifugal speed range is 1200 ⁇ 1600g, preferably 1500rpm; the centrifugation time range is 2 ⁇ 5 minutes, preferably 3 minutes Among them, the serum-containing medium can be, for example, DMEM/F12 containing 5% fetal bovine serum.
  • the primary esophageal squamous cell carcinoma cells obtained in step 1 above are resuspended and counted in the esophageal squamous cell carcinoma primary cell culture medium of the present invention, and placed in a culture dish at a cell density of 5-10 ⁇ 10 4 cells/cm 2 at the same time.
  • the formula of the tissue cleaning solution described in step 1 is: DMEM/F12 basal medium containing 100-200 mg/mL Primocin, 2% penicillin/streptomycin solution double antibody; the tissue digestion solution described in step 1
  • the preparation method is: 1 ⁇ 2mg/mL collagenase II, 1 ⁇ 2mg/mL collagenase IV, 50 ⁇ 100U/mL deoxyribonucleic acid I, 0.5 ⁇ 1mg/mL hyaluronidase, 1 ⁇ 3mM calcium chloride, 1 to 2% bovine serum albumin is dissolved in HBSS and RPMI-1640 at a volume ratio of 1:1;
  • the trophoblast cells described in step 2 can be, for example, irradiated NIH-3T3 cells, and the irradiation source is X-ray or
  • the gamma rays are preferably gamma rays, and the irradiation dose is 30-50 Gy, preferably 35 Gy.
  • the improved culture medium of esophageal squamous cell carcinoma primary cells in conditional reprogramming technology, rapid expansion of esophageal squamous cell carcinoma tissue samples in a short time is realized, and sufficient numbers can be expanded in an effective time
  • the cells are used for in vitro high-throughput drug sensitivity experiments and to guide clinical precision medication.
  • the culture medium of the primary esophageal squamous cell carcinoma of the present invention does not need to add expensive factors such as wnt3a, RSPO1, and Noggin, which greatly reduces the culture cost.
  • the digestion process of the organoid is complicated.
  • the culture method of the present invention has a simple processing method, and can quickly obtain a large number of monolayer cells to meet experimental needs.
  • the period of expansion to obtain the same number of cells is shorter, and the expansion of a small number of samples also has a significant proliferation effect.
  • the culture medium and culture method of the primary cells of esophageal squamous cell carcinoma of the present invention can obviously improve the success rate of sustainable expansion of esophageal squamous cell carcinoma tissue samples in vitro, which is above 85% on average.
  • Figure 1 is a graph showing the effect of factor increase in esophageal squamous cell carcinoma primary cell culture medium on the proliferation of esophageal squamous cell carcinoma primary cells.
  • Figure 2 is a graph showing the effect of multi-factor combination in esophageal squamous cell carcinoma primary cell culture medium on the proliferation of esophageal squamous cell carcinoma primary cells.
  • Fig. 3 is a graph showing the effect of adding other factors to the esophageal squamous cell carcinoma primary cell culture medium of the present invention on the proliferation of esophageal squamous carcinoma primary cells.
  • Fig. 4A and Fig. 4B are photographs (bright field) obtained by observing the cultured esophageal squamous cell carcinoma primary cells in vitro using a microscope.
  • Figures 5A and 5B are the results of Wright Giemsa staining identification of esophageal squamous cell carcinoma cells cultured in vitro.
  • 6A and 6B are the results of immunofluorescence staining identification of esophageal squamous cell carcinoma cells cultured in vitro.
  • Figure 7 shows the results of the first expansion cycle and cell number statistics of esophageal squamous cell carcinoma primary cells using the esophageal squamous cell carcinoma primary cell culture medium of the present invention.
  • Figure 8 is an in vitro expansion curve of esophageal squamous cell carcinoma primary cells using the esophageal squamous cell carcinoma primary cell culture medium of the present invention.
  • 9A-9P are graphs showing the effects of different concentrations of different factors in the culture medium of primary esophageal squamous cell carcinoma cells on the proliferation of primary esophageal squamous cell carcinoma cells.
  • Figure 10 is a result of comparing the effects of existing media with media reported in the literature and commercial media KSFM on cell proliferation.
  • Figures 11A to 11D show the results of culturing esophageal squamous cell carcinoma cells using the culture medium of the present invention, and using cells of different generations for drug screening.
  • Example 1 Promoting cell proliferation of esophageal squamous cell carcinoma primary cell culture medium containing different components
  • the medium of different components was prepared to investigate the proliferation-promoting effect of esophageal squamous cell carcinoma cells.
  • DMEM/F12 medium (purchased from Corning) was added with 10 ⁇ M Y27632 (purchased from MCE) and 100mg/mL Primocin as a basal medium (hereinafter sometimes referred to as BM), and different factors were added incrementally on this basis.
  • No.1 medium is obtained by adding insulin (purchased from Gibco) on the basis of BM
  • No.2 medium is obtained by adding hydrocortisone (purchased from Sigma) on the basis of No.1 medium.
  • No.3 medium is obtained by adding non-essential amino acids (NEAA) (purchased from Gibco) on the basis of No.2 medium
  • No.4 medium is obtained by adding glutamine on the basis of No.3 medium (Purchased from Gibco)
  • No.5 medium is obtained by adding bovine pituitary extract (BPE) (purchased from Maichen Technology) on the basis of No.4 medium
  • No.6 medium is obtained from No. Insulin-like growth factor 1 (IGF-1) (purchased from Beijing Yiqiao) was added to the .5 medium.
  • No.7 medium (hereinafter sometimes referred to as EM) was added to the No.6 medium.
  • N2 additive purchasedd from Gibco company
  • No. 8 medium is obtained by adding fetal bovine serum (FBS) (purchased from Gibco company) on the basis of No. 7 medium.
  • Tissue digestive juice composition Add volume (mL) HBSS (purchased from Gibco) 250 RPMI-1640 (purchased from Gibco) 250 Collagenase II (purchased from Sigma company) 1g Collagenase IV (purchased from Sigma) 1g Deoxyribonucleic acid I (purchased from Sigma company) 25000U Hyaluronidase (purchased from Sigma) 250mg Calcium chloride (purchased from Sigma company) 166.5mg Bovine Serum Albumin (purchased from Shanghai Shenggong) 5g Total (mL) 500
  • Tissue cleaning fluid composition Add volume (mL) DMEM/F12 (purchased from Corning) 488 Primocin (purchased from Invitrogen) 2 Penicillin/streptomycin solution double antibody (purchased from Gibco) 10 Total (mL) 500
  • Endoscopic tissue samples for esophageal cancer are derived from the tissue samples obtained by 5 patients with esophageal cancer who have explained and obtained consent during endoscopy. They are sample 50, sample 51, sample 52, sample 53, and sample 54. Aspirate the endoscopic tissue in the ultra-clean table and place it in a 15 mL centrifuge tube, add 5 mL of tissue cleaning solution for mixing, and wash once, and centrifuge at 1500 rpm for 4 minutes.
  • tissue clumps on the filter screen are rinsed with tissue cleaning solution.
  • the remaining cells are flushed into the centrifuge tube and centrifuged at 1500rpm for 4 minutes.
  • 3.3 Cell culture Divide the cell suspensions from each sample counted in 3.2 into 9 equally, centrifuge at 1500rpm for 4 minutes, and resuspend them in 200 microliters of BM and No. 1-8 medium after centrifugation. They were seeded in a 48-well plate (10,000 cells per well) at a living cell density of 1 ⁇ 10 4 cells/cm 2 respectively , and then irradiated by ⁇ -rays were added at a cell density of 2 ⁇ 10 4 cells/cm 2 (irradiation dose 35Gy) NIH-3T3 cells, and finally use the corresponding medium to fill the volume of each well in the 48-well plate to 500 microliters, and mix well. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. Passage is performed until the cells in the 48-well plate are more than 85% full.
  • the No. 1 to No. 7 medium can promote the proliferation of primary esophageal squamous cell carcinoma cells to varying degrees.
  • a medium No. 7 medium
  • the proliferation effect is significantly improved.
  • No. 8 medium obtained by adding 5% fetal bovine serum to No.
  • Example 2 The effect of multi-factor combination in esophageal squamous cell carcinoma primary cell culture medium on the proliferation of esophageal squamous cell carcinoma primary cells
  • Example 1 In the basal medium of Example 1 (DMEM/F12 medium + 10 ⁇ M Y27632+100mg/mL Primocin) were separately added 1:50 ratio of N2 additives, 20 ⁇ g/mL insulin, 7 ⁇ g/mL bovine pituitary extract, 2mM glutamine Amide, 400 ⁇ M non-essential amino acids, 0.4 ⁇ g/mL hydrocortisone, and 5 ng/mL insulin-like growth factor 1, to prepare the medium of this example.
  • the endoscopic tissue sample 57, sample 58, sample 59, sample 60, and sample 61 were separated in the same manner as in Example 1 to obtain esophageal squamous cell carcinoma primary cells, and each medium prepared in this example and the medium prepared in Example 1 were used.
  • the surface After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. On the 7th day of culture, take out the 48-well plate, rinse with 200 microliters of 0.25% trypsin (purchased from Gibco) for 1 minute, and then add 500 microliters of 0.05% trypsin (purchased from Gibco) to each well after aspirating.
  • a 37°C, 5% CO 2 incubator purchased from Thermo Fisher
  • the basic medium (DMEM/F12 medium + 10 ⁇ M Y27632+100mg/mL Primocin) is further added selected from N2 additives, insulin, bovine pituitary extract, glutamine, non-essential amino acids, hydrogenated
  • the cell proliferation efficiency is better than that of the basal medium, but not as good as the No. 7 medium (EM) in Example 1.
  • Example 3 The effect of adding other factors to the esophageal squamous cell carcinoma primary cell culture medium of the present invention on the proliferation of esophageal squamous cell carcinoma primary cells
  • Example 1 In the No. 7 medium (EM) of Example 1, separately added 10ng/mL fibroblast growth factor 2 (FGF2), 10ng/mL fibroblast growth factor 10 (FGF10), 10ng/mL fibroblast growth Factor 7 (FGF7) or add 10ng/mL FGF2, 10ng/mL FGF10, and 10ng/mL FGF7 together to prepare the medium of this example.
  • FGF2 fibroblast growth factor 2
  • FGF10 10ng/mL fibroblast growth factor 10
  • FGF7 10ng/mL fibroblast growth Factor 7
  • Example 2 In the same manner as in Example 1, the endoscopic tissue sample 62 and the sample 63 were separated to obtain esophageal squamous cell carcinoma primary cells, and each medium prepared in this example and the No. 7 medium (EM) in Example 1 were used.
  • EM No. 7 medium
  • esophageal squamous cell cultured primary cells obtained according to viable cell density of 1 ⁇ 10 4 pieces / cm 2 were seeded in 48-well plates (10,000 cells per well), in accordance with a cell density of 2 ⁇ 10 4 pieces / cm 2 was added NIH-3T3 cells irradiated with gamma rays (irradiation dose 35Gy), and mixed.
  • the surface After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. On the 7th day of culture, take out the 48-well plate, rinse with 200 microliters of 0.25% trypsin (purchased from Gibco) for 1 minute, and then add 500 microliters of 0.05% trypsin (purchased from Gibco) to each well after aspirating. , Placed in a 37°C, 5% CO 2 incubator for 10 minutes, until the cells have been completely digested can be observed under the microscope (Invitrogen's EVOS M500), stop the digestion, centrifuge at 1500 rpm for 4 minutes, discard the supernatant, and add 1 ml No.
  • a 37°C, 5% CO 2 incubator purchased from Thermo Fisher
  • the ordinate ratio is the ratio of the number of cells obtained by culturing for 7 days in the medium with different factors added to the EM medium and the number of cells obtained by culturing for 7 days in the EM medium. Adding different factors to the culture medium can play a better role in promoting proliferation. A ratio of less than 1 means that adding different factors does not have a better effect than EM medium.
  • Example 4 Primary cell culture and identification of esophageal squamous cell carcinoma
  • the endoscopic tissue sample 64 was separated in the same manner as in Example 1 to obtain esophageal squamous cell carcinoma primary cells, and the obtained esophageal squamous cell carcinoma primary cells were cultured using the No. 7 medium (EM) in Example 1, according to the viable cell density 1 ⁇ 10 4 cells/cm 2 were seeded in a 12-well plate (45,000 cells per well), and NIH-3T3 irradiated with gamma rays (irradiation dose 35Gy) was added according to the cell density of 2 ⁇ 10 4 cells/cm 2 Cells, mix well. After the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation.
  • EM No. 7 medium
  • Figure 4A and Figure 4B are photos taken under a 4x objective lens and a 10x objective lens, respectively. The cells are closely arranged under the microscope. The shape is slightly irregular.
  • the dyeing method is as follows.
  • Figures 5A and 5B show the results of Wright Giemsa staining identification of esophageal squamous cell carcinoma cells cultured in vitro. They are pictures taken under a 10x objective lens in different fields of view. The nucleus is large and deeply stained, which is consistent with the characteristics of squamous cell carcinoma cells.
  • the cultured esophageal squamous cell carcinoma cells were seeded on a cell slide (purchased from Thermo Fisher Scientific) and placed in a 37°C, 5% CO 2 incubator for culture, until the cells adhered.
  • PBS+0.3% Triton X-100 was used to prepare 5% BSA (purchased from Shanghai Shenggong) for blocking, and blocking at 37°C for 30 minutes.
  • Dilute DAPI (purchased from Sigma) in 1:1000 PBS, stain for 5 minutes at room temperature and avoid light, and wash with PBS for 5 minutes x 3 times.
  • Image under microscope Invitrogen EVOS M500, take pictures and record.
  • Figure 6A and Figure 6B are the results of immunofluorescence staining identification of esophageal squamous cell carcinoma cells cultured in vitro, respectively, are pictures taken by fluorescence in different fields of view under a 10x objective lens. As shown in the figure, the cells in the field of view showed green under 488 excitation light, indicating that the cultured cells are squamous carcinoma cells, which is consistent with the clinicopathological diagnosis.
  • Example 6 The primary culture cycle and cell number statistics of esophageal squamous cell carcinoma and the calculation of Population Doubling (PD) value
  • the endoscopic tissue samples of esophageal cancer were digested according to the method of Example 1 to obtain primary cells of esophageal squamous cell carcinoma.
  • the No. 7 medium in Example 1 was used, and the cells were seeded in a 12-well plate and cultured at a viable cell density of 1 ⁇ 10 4 cells/cm 2, and the cells were expanded. After increasing to 85%, digest and count. At the same time, record the number of days of culture until digestion, and the number of days of culture until digestion is regarded as a culture cycle. As shown in Figure 7, the average culture period obtained by culturing 40 samples is 10 days, and the average number of cells obtained by expansion is 700,000.
  • sample 2 sample 5, sample 6, sample 8, sample 9, sample 11, sample 27, sample 39, and sample 40 were continuously cultured, and a total of 9 samples were cultured, and the amplified cells were amplified by different generations. After digestion for each generation, count and record the corresponding culture period.
  • the abscissa represents cell culture
  • the ordinate is the cumulative multiple of cell proliferation, which indicates the multiple of cell expansion during the culture cycle. The larger the value, the more the cells are expanded in a certain cycle, that is, the more cells can be expanded.
  • the slope represents the rate of cell expansion.
  • Example 7 The influence of the concentration of the added factor on the proliferation of esophageal squamous cell carcinoma
  • the endoscopic tissue sample 65, sample 66, sample 67, sample 68, and sample 69 were similarly separated according to the method of Example 1 to obtain primary esophageal squamous cell carcinoma cells, and the obtained cells were cultured using the No. 7 medium in Example 1.
  • Primary esophageal squamous cell carcinoma cells were seeded in a 12-well plate at a viable cell density of 1 ⁇ 10 4 cells/cm 2 (45,000 cells per well), and added at a cell density of 2 ⁇ 10 4 cells/cm 2 after ⁇ -ray irradiation. Irradiate NIH-3T3 cells (irradiation dose 35Gy) and mix.
  • the surface is disinfected, it is placed in a 37°C, 5% CO 2 incubator (purchased from Thermo Fisher) for cultivation. After the cells are expanded to 85%, take out the 12-well plate, rinse with 200 microliters of 0.25% trypsin (purchased from Gibco) for 1 minute, and then add 500 microliters of 0.05% trypsin (purchased from Gibco) to each well. Company), placed in a 37°C, 5% CO 2 incubator for 10 minutes until the cells have been completely digested can be observed under the microscope (Invitrogen’s EVOS M500). Resuspend 1 ml of basal medium, and use flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL) for counting to obtain the total number of cells. The obtained cells were used in the following culture experiments.
  • Formulation 1 No. 7 medium components in Example 1 do not contain N2 additives;
  • Formulation 4 No. 7 medium component in Example 1 does not contain insulin
  • Formulation 5 No. 7 medium component in Example 1 does not contain IGF1;
  • Formulation 6 No. 7 medium components in Example 1 do not contain bovine pituitary extract
  • Formulation 8 The No. 7 medium component in Example 1 does not contain hydrocortisone.
  • Example 1 The above-mentioned formulas 1-8 and No. 7 medium in Example 1 were used to dilute the above-mentioned digested cell suspension, and 20,000 cells per well and 500 microliters of volume were seeded into a 24-well plate.
  • IGF1 insulin-like growth factor 1
  • a 24-well plate inoculated with primary cells was added with 500 microliters of prepared insulin-like growth factor (IGF1) per well; and a control well (BC) was set up with the medium of formula 5.
  • bovine pituitary extracts were prepared. 500 microliters of bovine pituitary extract prepared per well was added to the 24-well plate of cells; and the control well (BC) was set up with the medium of formula 6.
  • the No. 7 medium in the example was used to prepare 5 concentration gradients of 8 small molecule compounds (LDN193189, IWP2, A83-01, Forskolin, Forskolin, SB431542, CHIR99021, DMH-1, DAPT; all purchased from MCE company), add prepared 8 small molecule compounds (LDN193189, IWP2, A83-01, Forskolin, SB431542) to the 24-well plate seeded with primary cells. , CHIR99021, DMH-1, DAPT) 500 microliters per well; and use No. 7 medium to set up control wells (BC).
  • 8 small molecule compounds LDN193189, IWP2, A83-01, Forskolin, Forskolin, SB431542
  • CHIR99021, DMH-1, DAPT 500 microliters per well
  • BC No. 7 medium to set up control wells
  • the ratio is the ratio of the number of cells obtained by culturing for one generation using each medium to the number of cells obtained by culturing for one generation in the corresponding control wells.
  • a ratio greater than 1 indicates that the proliferation-promoting effect of the formulated medium containing different concentrations of factors or small molecule compounds is better than that of the control well.
  • the ratio is less than 1, indicating that the formulated medium containing different concentrations of factors or small molecule compounds has a better proliferation effect.
  • the medium of the control wells had a weak proliferation promotion effect.
  • the concentration of N2 additive ranges from 1:400 to 1:25, and the concentration of 1:50 is the most effective when added to cell proliferation; the concentration of non-essential amino acids ranges from 25 to 400 ⁇ M, and the concentration of 400 ⁇ M is added to cell proliferation.
  • the effect is the most obvious; the concentration range of glutamine is 0.5-8mM, and the cell proliferation effect is most obvious when the concentration is 2mM; the concentration range of insulin-like growth factor 1 is 2.5-40ng/mL, and the cell proliferation effect is 5ng/mL
  • the concentration range of bovine pituitary extract is 3.5 ⁇ 56 ⁇ g/mL, the cell proliferation effect is the most obvious when the concentration is 7 ⁇ g/mL; the concentration range of Y27632 is 2.5 ⁇ 40 ⁇ M, and the cell proliferation effect is the most obvious when the concentration is 10 ⁇ M; the concentration range of insulin
  • the cell proliferation effect is most obvious when the concentration is 2.5-40ng/mL, and the concentration is 20ng/mL; the concentration range of hydrocortisone is 0.1-1.6 ⁇ g/mL, and the cell proliferation effect is the most obvious when the concentration is 0.4 ⁇ g/mL.
  • the other 8 small molecule compounds did not show significant proliferation under the concentration gradients of 10 ⁇ M, 3 ⁇ M, 1 ⁇ M, 0.3 ⁇ M, and 0.1 ⁇ M effect.
  • Example 8 Comparison of the effect of the culture medium of the present invention and the existing culture medium on cell proliferation
  • the KM (Keratinocyte medium) medium in Non-Patent Document 2 was prepared.
  • the FM medium in Non-Patent Document 3 was prepared.
  • the endoscopic tissue sample 70, sample 71, and sample 72 were separated in the same manner as in Example 1 to obtain esophageal squamous cell carcinoma primary cells, which were cultured using EM medium, KSFM medium, FM medium, and KM medium, respectively.
  • sample 70 The obtained primary esophageal squamous cell carcinoma cells were resuspended in 1 ml of EM medium, KSFM medium, FM medium, and KM medium, respectively, and counted using a flow image counter (Jiangsu Zhuo Microbiology Technology Co., Ltd. JIMBIO FIL).
  • the living cell density of 1 ⁇ 10 4 cells/cm 2 they were respectively seeded in two 24-well plates (20,000 cells per well).
  • One of the 24-well plates was cultured without ⁇ -ray-irradiated NIH-3T3 cells.
  • EM medium has the largest number of expanded cells when inoculated with the same number of cells (20,000/well) and cultured for the same time. , Has a significant effect on the proliferation of esophageal squamous cell carcinoma cells.
  • the EM medium is better when cultured with trophoblasts than without trophoblasts.
  • the primary cell culture medium for esophageal squamous cell carcinoma of the present invention uses trophoblasts for culturing. It has a significant effect on the proliferation of esophageal squamous cell carcinoma cells and shortens the period required for culture.
  • Example 9 Use of the primary esophageal squamous cell carcinoma cells amplified by the medium of the present invention for drug screening
  • the primary cells of esophageal squamous cell carcinoma were isolated in the same manner as in Example 1, and cultured using the No. 7 medium in Example 1. After the cells were expanded to 85%, they were digested and passaged. As a generation. The first, third, fifth, seventh, and ninth generation cells were cultured for drug screening.
  • the cells were digested and counted.
  • the cells were thoroughly mixed in the sample tank (purchased from Corning) at a viable cell density of 4 ⁇ 10 4 cells/mL. After homogenization, the cells were cultured in a 384-well opaque white cell culture plate (purchased from Corning) with a volume of 50 ⁇ L per well and the number of cells was 2000 cells/well.
  • the No. 7 medium in Example 1 was added from the edge of the well plate to seal the plate, and the plate was marked with the name of the sample and the detection time of CellTiter-Glo (purchased from Promega).
  • the surface was disinfected with 75% alcohol (purchased from Lierkang), placed in a 37°C, 5% CO 2 incubator, and added 24 hours later.
  • cell survival rate (%) chemiluminescence value of dosing hole/ chemiluminescence value of control hole*100%, calculate the cell survival rate after different drugs are applied to cells, and use graphpad prism software to calculate the half inhibition rate of drugs on cells (IC50). The results are shown in Figs. 11A to 11D, respectively.
  • the invention provides a culture medium and a culture method for rapidly amplifying primary cells of esophageal squamous cell carcinoma in vitro, which can realize rapid expansion of esophageal squamous cell carcinoma tissue samples in a short time, and can be expanded to obtain a sufficient number in an effective time
  • the cells are used for in vitro high-throughput drug sensitivity experiments and to guide clinical precision medication.

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Abstract

提供了一种用于体外快速扩增食管鳞癌原代细胞的培养基及培养方法及其在筛选药物中的应用。该培养基含有选自DMEM/F12、DMEM、F12或RPMI-1640的初始培养基、Rho蛋白酶抑制剂、抗生素、胰岛素、N2添加剂、胰岛素样生长因子1、非必需氨基酸、以及任选的氢化可的松、任选的谷氨酰胺、和任选的牛垂体提取物。

Description

食管鳞癌原代细胞的培养基及培养方法 技术领域
本发明属于生物技术领域,具体涉及一种用于体外快速扩增食管鳞癌原代细胞的培养基及培养方法。
背景技术
当今中国,不论城市还是农村,肿瘤都是中国居民的主要死亡原因。其中食管癌是目前世界上最常见的胃肠道恶性肿瘤之一。根据国家癌症中心统计最新数据显示,男性死亡前十位恶性肿瘤中食管癌占第四位,女性食管癌占第三位。世界上多个地区呈地方性发病率升高,而我国是属于食管癌高发地区,年平均死亡人数约15万人,占全国肿瘤死亡率21.8%,在导致死亡最多的癌症中死亡率排名第4位(非专利文献1)。在食管癌的病理分型中,国外和国内有明显差别,国外90%以上食管癌是腺癌,而国内90%以上是鳞癌,由于NCCN指南是基于国外病例研究后给出的指导,导致用NCCN来指导国内食管癌病人用药存在一定的差异性,因此亟需建立中国人自己的原代细胞样本库,用来体外研究食管癌发病机制以及开发新型的治疗食管癌的药物。在这过程中,食管癌原代细胞培养就显得尤为重要。
目前食管癌原代细胞培养中,已知有直接将肿瘤细胞分离后培养在含有胎牛血清的培养基中进行培养的方法(非专利文献2);近些年还报道了使用条件重编程的方法来培养上皮来源的细胞的方法,其中提及可以用来培养扩增食管癌原代细胞(非专利文献3);随后有文献报道将食管癌术中样本通过胰酶消化后,利用条件重编程方法进行培养的方法(非专利文献4)。最近有文献报道使用商品化培养基KSFM中添加因子进行培养的方法(非专利文献5),也有文献使用类器官培养的方法来进行培养(非专利文献6)。
但是,上述非专利文献2中方法不能长期稳定培养,非专利文献3和非专利文献4方法培养周期较长,非专利文献5中方法则需要成本比较高,非专利文献6中用于类器官培养的试剂太昂贵,培养过程中操作复杂,不利于大范围推广应用。
并且由于国外食管癌病理类型主要是食管腺癌,国外文献中方法基本都是基于食管腺癌进行的研究,所以不适于用来培养食管鳞癌。因此,需要开发一种适于食管鳞癌原代细胞培养的培养基以及培养方法。
现有技术文献
非专利文献
非专利文献1:Freddie Bray,BSc,MSc,PhD;Jacques Ferlay,ME,et al.Global Cancer Statistics 2018.CA CANCER J CLIN 2018;68:394-424;
非专利文献2:Karin J.Purdie,Celine Pourreyron,and Andrew P.South.Isolation and Culture of Squamous Cell Carcinoma lines.Cancer Cell Culture:Methods and Protocols,Second Edition,Methods in Molecular Biology,vol.731,2011,151-159;
非专利文献3:Xuefeng Liu,Ewa Krawczyk,et al.Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens.Nature Protocols,VOl.12NO.2,2017,439-451;
非专利文献4:Todd J.Jensen,Christopher Foster,et al.Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation.Journal of Visualized Experiments,March 2017,121,e55243;
非专利文献5:Yuta Kasagi,et al.The Esophageal Organoid System Reveals Functional Interplay Between Notch and Cytokines in Reactive Epithelial Changes.Cellular and Molecular Gastroenterology and Hepatology Vol.5,No.3,2018,333-352;
非专利文献6:Xiaodun Li,Hayley E.Francies,et al.Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics.NATURE COMMUNICATIONS(2018)9:2983-2995。
发明内容
为了解决上述现有技术中的问题,本发明提供了一种用于体外快速扩增食管鳞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食管鳞癌原代细胞的培养基,所述培养基包括初始培养基、以下组分(1)~(6)、以及任选的以下组分(7)~(9),其中,初始培养基例如可以为DMEM/F12、DMEM、F12或RPMI-1640,优选为DMEM/F12。
(1)Rho蛋白激酶抑制剂,选自Y27632、羟基法舒地尔(Hydroxyfasudil)和GSK429286A中的一种或多种,当选自Y27632时,浓度范围为2.5~40μM,优选为5~20μM,更优选为10μM;当选自羟基法舒地尔时,浓度范围为2~32μM,优选为4~16μM,更优选为8μM;当选自GSK429286A时,浓度范围为2~32μM,优选为4~16μM,更优选为8μM;
(2)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,当选自链霉素/青霉素时,链霉素浓度范围为25~400μg/mL,优选为50~200μg/mL,更优选为200μg/m,青霉素浓度范围为25~400U/mL,优选为50~200U/mL,更优选为200U/mL;当选自两性霉素B时,浓度范围为0.25~4μg/mL,优选为0.5~2μg/mL,更优选为1μg/m;当选自Primocin时,浓度范围为25~400mg/mL,优选为50~200mg/mL,更优选为100mg/mL,所述抗生素优选为Primocin;
(3)胰岛素,浓度范围为2.5~40μg/mL,优选为10~40μg/mL,更优选为20μg/mL;
(4)N2添加剂,相对于培养基的体积比1:400~1:25,优选1:100~1:25,更优选1:50;
(5)胰岛素样生长因子1(IGF-1),浓度范围为2.5~40ng/mL,优选为2.5~10ng/mL,更优选为5ng/mL;
(6)非必需氨基酸,所述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为50μM~400μM,优选为100μM~400μM,更优选为400μM;
(7)氢化可的松,浓度范围为0~1.6μg/mL,优选为0.2~0.8μg/mL,更优选为0.4μg/mL;
(8)谷氨酰胺,浓度范围为0mM~8mM,优选为1mM~4 mM,更优选为2mM;
(9)牛垂体提取物,浓度范围为0~56μg/mL,优选为3.5~14μg/mL,更优选为7μg/mL。
本发明的另一个方面在于提供一种食管鳞癌原代细胞的培养方法,该培养方法中,使用上述食管鳞癌原代细胞培养基进行培养。
上述培养方法包括以下步骤:
1、食管鳞癌原代细胞的分离
1.1对组织样本、例如内镜标本,用组织清洗液冲洗后加入组织消化酶置于恒温摇床(知楚仪器ZQLY-180N)中进行消化,例如,利用8~14毫升、优选12毫升组织消化酶进行消化;消化温度范围为4摄氏度~37摄氏度,优选为37摄氏度;消化转速范围为200rpm~350rpm,优选为300rpm;
1.2消化后取出观察,若未见明显组织块即可终止消化,否则继续消化,直至消化充分,消化时间范围为4~8小时,优选为6小时;
1.3消化完成后取出,离心后弃去上清液,加入含血清初始培养基重悬终止消化,离心转速范围为1200~1600g,优选为1500rpm;离心时间范围为2~5分钟,优选为3分钟,其中,含血清培养基例如可以使用含5%胎牛血清的DMEM/F12。
2.使用本发明的食管鳞癌原代细胞培养基进行培养
将上述步骤1中获得的食管鳞癌原代细胞用本发明的食管鳞癌原代细胞培养基重悬并计数,按照细胞密度5~10×10 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,培养5~7天后按照细胞密度0.5~1×10 4个/cm 2补充加入滋养细胞,直至培养皿中细胞长满85%以上可进行消化传代。
其中,步骤1中所述的组织清洗液的配方为:DMEM/F12基础培养基含100~200mg/mL Primocin、2%青霉素/链霉素溶液双抗;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Ⅰ、0.5~1mg/mL透明质酸酶、1~3mM氯化钙、1~2%牛血清白蛋白溶于体积比1:1的HBSS和RPMI-1640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30~50Gy,优选为35Gy。
发明效果
根据本发明,通过在条件重编程技术利用改良后的食管鳞癌原代细胞的培养基,实现了食管鳞癌组织样本在短时间内快速扩增,能够在有效的时间内扩增得到足够数量的细胞,用于体外高通量药物敏感实验和指导临床精准用药。
与类器官培养基相比,本发明的食管鳞癌原代细胞的培养基中不需要加入wnt3a、RSPO1以及Noggin等昂贵因子,大大降低了培养成本。另外,类器官培养中,类器官消化过程复杂,与此相比,本发明的培养方法处理方式简单,能够迅速地得到大量单层细胞,满足实验需求。与传统的条件重编程技术中所使用的培养基相比,扩增得到相同细胞数目的周期要短,并且对于少量样本扩增也具有明显促增殖效果。本发明的食管鳞癌原代细胞的培养基和培养方法可以明显提高食管鳞癌组织样本体外可持续扩增的成功率,平均在85%以上。
附图说明
图1为表示食管鳞癌原代细胞培养基中因子递增对食管鳞癌原代细胞增殖的影响的图。
图2为表示食管鳞癌原代细胞培养基中多因子组合对食管鳞癌原代细胞增殖的影响的图。
图3为表示在本发明的食管鳞癌原代细胞培养基中添加其它因子对食管鳞癌原代细胞增殖的影响的图。
图4A和图4B为利用显微镜观察体外培养的食管鳞癌原代细胞得到的照片(明场)。
图5A和图5B为对体外培养后的食管鳞癌细胞进行瑞氏吉姆萨染色鉴定的结果。
图6A和图6B为对体外培养后的食管鳞癌细胞进行免疫荧光染色鉴定的结果。
图7为使用本发明的食管鳞癌原代细胞培养基的食管鳞癌原代细胞首次扩增周期和细胞数统计的结果。
图8为使用本发明的食管鳞癌原代细胞培养基的食管鳞癌原代细胞体外扩增曲线。
图9A~图9P为表示食管鳞癌原代细胞培养基中不同因子的不同浓度对食管鳞癌原代细胞增殖的影响的图。
图10为比较现有培养基与文献中报道培养基以及商品化培养基KSFM对细胞增殖的影响的结果。
图11A~图11D为使用本发明培养基培养食管鳞癌细胞,并将不同代数细胞用于药物筛选的结果。
具体实施方式
以下通过具体实施方式的描述并结合附图对本发明作进一步说明,这些实施例仅用于说明本发明,本发明的范围不限定于这些实施例。
实施例1含不同成分的食管鳞癌原代细胞培养基的促细胞增殖作用
(1)不同组分培养基的配制
根据表1中的成分配制不同组分培养基,考察对食管鳞癌细胞的促增殖作用。
在DMEM/F12培养基(购自康宁公司)上添加10μM Y27632(购自MCE公司)和100mg/mL Primocin作为基础培养基(以下有时简称为BM),在此基础上递增加入不同的因子。No.1培养基是在BM基础上加入胰岛素(购自Gibco公司)得到的,No.2培养基是在No.1培养基基础上再加入氢化可的松(购自Sigma公司)得到的,No.3培养基是在No.2培养基基础上再加入非必需氨基酸(NEAA)(购自Gibco公司)得到的,No.4培养基是在No.3培养基基础上再加入谷氨酰胺(购自Gibco公司)得到的,No.5培养基是在No.4培养基基础上再加入牛垂体提取物(BPE)(购自迈晨科技)得到的,No.6培养基是在No.5培养基基础上再加入胰岛素样生长因子1(IGF-1)(购自北京义翘)得到的,No.7培养基(以下有时简称EM)是在No.6培养基基础上再加入N2添加剂(购自Gibco公司)得到的,No.8培养基是在No.7培养 基基础上再加入胎牛血清(FBS)(购自Gibco公司)得到的。
表1不同组分培养基的配制(浓度为终浓度)
培养基 组分
BM DMEM/F12+10μM Y27632+100mg/mL Primocin
No.1 BM+20μg/mL胰岛素
No.2 No.1+0.4μg/mL氢化可的松
No.3 No.2+400μM非必需氨基酸
No.4 No.3+2mM谷氨酰胺
No.5 No.4+7μg/mL牛垂体提取物
No.6 No.5+5ng/mL胰岛素样生长因子1
No.7 No.6+1:50N2添加剂
No.8 No.7+5%胎牛血清
(2)食管鳞癌原代细胞的获取和培养
1准备工作
将15mL无菌离心管、移液器、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℃冰箱取出根据表1配制好的各培养基、根据表2配制好的组织消化液、根据表3配制好的组织清洗液,平衡至室温。
表2组织消化液配方
组织消化液成分 添加体积(mL)
HBSS(购自Gibco公司) 250
RPMI-1640(购自Gibco公司) 250
胶原酶Ⅱ(购自Sigma公司) 1g
胶原酶Ⅳ(购自Sigma公司) 1g
脱氧核糖核酸Ⅰ(购自Sigma公司) 25000U
透明质酸酶(购自Sigma公司) 250mg
氯化钙(购自Sigma公司) 166.5mg
牛血清白蛋白(购自上海生工) 5g
总量(mL) 500
表3组织清洗液配方
组织清洗液成分 添加体积(mL)
DMEM/F12(购自康宁公司) 488
Primocin(购自Invitrogen公司) 2
青霉素/链霉素溶液双抗(购自Gibco公司) 10
总量(mL) 500
2食管鳞癌原代细胞的分离
2.1食管癌内镜组织样本来源于5例进行过说明并获得同意的食管癌患者在进行内镜检查时获取的组织样本,它们分别是样本50、样本51、样本52、样本53、样本54。在超净台中将内镜组织吸出并置于15mL离心管中,加入5mL组织清洗液进行混匀,并清洗一次,以1500rpm离心4分钟。
2.2弃上清,按1:1比例加入基础培养基和组织消化液(使用量约为每10mg组织使用5mL组织消化液),标记样品名称及编号,封口膜密封,以37℃、300rpm摇床消化,每间隔1h观察消化是否完成。
2.3消化完成后,40μm滤网过滤掉未消化的组织团块,滤网上的组织团块用组织清洗液冲洗,将残留细胞冲入离心管中,1500rpm离心4分钟。
2.4弃上清,观察剩余细胞团是否含有血细胞,若有血细胞,加3mL血细胞裂解液(购自Sigma公司),混匀,4℃裂解15分钟,5分钟摇晃混匀一次,裂解结束后取出1500rpm离心4分钟。
2.5弃上清,加入2mL基础培养基重悬细胞,备用。
3.食管鳞癌原代细胞的培养
3.1镜下观察:吸取10μL重悬细胞平铺于载玻片上中,在显微镜(Invitrogen公司EVOS M500)下用10倍物镜观察癌细胞状态。
3.2活细胞计数:取重悬的细胞悬液10μL,与10μL台盼兰染液(购自Invitrogen公司)充分混合后,取10μL加入流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL),可以得出细胞浓度,细胞粒径以及细胞活率。选取细胞粒径大于10微米的细胞进行计数。
3.3细胞培养:将上述3.2中计数后的分别来源于各个样本的细胞悬液平均分成9份,1500rpm离心4分钟,离心后分别使用200微升BM以及No.1~8号培养基重悬,分别按照活细胞密度1×10 4个/cm 2接种于48孔板中(每孔1万细胞数),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35Gy)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孔板中细胞长满85%以上,进行传代。
(3)培养结果
在培养第7天,取出48孔板,使用200微升0.25%胰蛋白酶(购自Gibco公司)润洗1分钟,吸去后再每孔加入500微升0.05%胰蛋白酶(购自Gibco公司),置于37℃、5%CO 2培养箱中反应10分钟,直至显微镜(Invitrogen公司EVOS M500)下能观察到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1毫升基础培养基重悬,使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数,得到细胞总数。将由分离自内镜组织样本50的食管鳞癌原代细胞得到的结果示于图1。
根据图1的结果可知,与基础培养基相比,在使用上述No.1~No.7培养基时,均能够不同程度地促进食管鳞癌原代细胞的增殖。在使用含有Y27632和Primocin、胰岛素、氢化可的松、非必需氨基酸、谷氨酰胺、牛垂体提取物、IGF1、N2添加剂的培养基(No.7培养基)培养食管鳞癌原代细胞时,增殖效果明显提高。另外,使用No.7的培养基的基础上加入5%胎牛血清得到的No.8培养基进行食管鳞癌原代细胞的培养时,增殖效果与没有添加5%胎牛血清的No.7培养基相比没有明显提高,因此,可以认为本发明的食管鳞癌原代细胞在不添加胎牛血清时也能够使用。
实施例2食管鳞癌原代细胞培养基中多因子组合对食管鳞癌原代细胞的增殖的影响
在实施例1的基础培养基(DMEM/F12培养基+10μM Y27632+100mg/mL Primocin)中分别单独添加1:50比例N2添加剂、20μg/mL胰岛素、7μg/mL牛垂体提取物、2mM谷氨酰胺、400μM非必需氨基酸、0.4μg/mL氢化可的松、5ng/mL胰岛素样生长因子1,配制本实施例的培养基。
与实施例1同样地分离内镜组织样本57、样本58、样本59、样本60和样本61获得食管鳞癌原代细胞,并使用本实施例中所配制的各培养基以及实施例1中的BM培养基、No.7培养基(EM)分别培养所获得的食管鳞癌原代细胞,按照活细胞密度1×10 4个/cm 2接种于48孔板中(每孔1万细胞数),按照细胞密度2×10 4个/cm 2加入经γ射线辐照 (辐照剂量35Gy)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。在培养第7天,取出48孔板,使用200微升0.25%胰蛋白酶(购自Gibco公司)润洗1分钟,吸去后再每孔加入500微升0.05%胰蛋白酶(购自Gibco公司),置于37℃、5%CO 2培养箱中反应10分钟,直至显微镜(Invitrogen公司EVOS M500)下能观察到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1毫升基础培养基重悬,使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数,得到细胞总数,评价食管鳞癌原代细胞培养基中双因子组合对食管鳞癌原代细胞的增殖的影响。将由分离自内镜组织样本57的食管鳞癌原代细胞得到的结果示于图2。
如图2所示,在基础培养基(DMEM/F12培养基+10μM Y27632+100mg/mL Primocin)中进一步添加选自N2添加剂、胰岛素、牛垂体提取物、谷氨酰胺、非必需氨基酸、氢化可的松、胰岛素样生长因子1中的一种添加因子时,细胞增殖效率优于基础培养基,但是不及实施例1中的No.7培养基(EM)。
实施例3在本发明的食管鳞癌原代细胞培养基中添加其它因子对食管鳞癌原代细胞的增殖的影响
在实施例1的No.7培养基(EM)中分别单独添加10ng/mL成纤维细胞生长因子2(FGF2)、10ng/mL成纤维细胞生长因子10(FGF10)、10ng/mL成纤维细胞生长因子7(FGF7)或一并添加10ng/mL FGF2、10ng/mL FGF10和10ng/mL FGF7,配制本实施例的培养基。
与实施例1同样地分离内镜组织样本62和样本63获得食管鳞癌原代细胞,并使用本实施例中所配制的各培养基以及实施例1中的No.7培养基(EM)分别培养所获得的食管鳞癌原代细胞,按照活细胞密度1×10 4个/cm 2接种于48孔板中(每孔1万细胞数),按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量35Gy)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。在培养第7天,取出48孔板,使用200微升0.25%胰蛋白酶(购自Gibco公司)润洗1分钟,吸去后再每孔加入500微升0.05%胰蛋白酶(购自 Gibco公司),置于37℃、5%CO 2培养箱中反应10分钟,直至显微镜(Invitrogen公司EVOS M500)下能观察到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1毫升No.7培养基(EM)重悬,使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数,得到细胞总数,评价在本发明的食管鳞癌原代细胞培养基中添加其它因子对食管鳞癌原代细胞的增殖的影响。将由分离自内镜组织样本62的食管鳞癌原代细胞得到的结果示于图3。
图3中,纵坐标比值为使用在EM培养基基础上加入不同因子得到的培养基培养7天得到的细胞数与使用EM培养基培养7天得到的细胞数的比,比值大于1表示在EM培养基基础上加入不同因子可以起到更好的促增殖作用,比值小于1则表示加入不同因子没有更优于EM培养基的作用。
如图3所示,在使用在实施例1的No.7培养基(EM)中进一步添加FGF2或FGF7得到的培养基进行培养时,与使用EM培养基时相比,没有进一步的增殖促进作用。而在使用在实施例1的No.7培养基(EM)中进一步添加FGF7得到的培养基进行培养时,与使用EM培养基时相比,食管鳞癌原代细胞的增殖反而被抑制。
实施例4食管鳞癌原代细胞培养及鉴定
与实施例1同样地分离内镜组织样本64获得食管鳞癌原代细胞,并使用实施例1中的No.7培养基(EM)培养所获得的食管鳞癌原代细胞,按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数),按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量35Gy)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12天,使用显微镜(Invitrogen公司EVOS M500)观察培养得到的食管鳞癌细胞,图4A和图4B分别是4倍物镜和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。
接着,对所培养的细胞,使用200微升0.25%胰蛋白酶(购自Gibco公司)润洗1分钟,吸去后再每孔加入500微升0.05%胰蛋白酶(购自Gibco公司),置于37℃、5%CO 2培养箱中反应10分钟,直至显微镜 (Invitrogen公司EVOS M500)下能观察到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500微升No.7培养基(EM)重悬,对培养得到的食管鳞癌细胞进行鉴定。
取100微升上述食管鳞癌细胞悬液涂片进行瑞氏-吉姆萨染色鉴定。染色方法如下所述。
(1)细胞悬液涂片晾干,滴入1滴瑞氏-吉姆萨A液(购自贝索公司),随后滴入3滴瑞氏-吉姆萨B液(购自贝索公司),混匀后染色3分钟;
(2)流水冲洗,冲洗时不能先倒掉染液,应以流水冲去,以防有沉渣沉淀在标本上;
(3)干燥,显微镜(奥林巴斯公司CX41)下观察拍照。
图5A和图5B表示体外培养后的食管鳞癌细胞进行瑞氏吉姆萨染色鉴定的结果,分别为10倍物镜下不同视野拍照的图片,细胞核大且深染,符合鳞癌细胞特征。
将该样本培养后的食管鳞癌细胞进行免疫荧光染色。染色方法如下所述。
(1)爬片
将培养后的食管鳞癌细胞细胞种于细胞玻片(购自赛默飞公司)上,置于37℃、5%CO 2培养箱培养,待细胞贴壁。
(2)固定
①PBS(购自上海生工)配制4%甲醛(购自Sigma公司),4℃冰箱保存备用。
②细胞贴壁后,弃培养液,4%甲醛冰上固定细胞30分钟。PBS(购自上海生工)洗5分钟x 3次。
(3)透明(避光)
①配制透明液:PBS+0.3%H 2O 2(购自上海生工)+0.3%Triton X-100(购自上海生工)。
②透明:弃PBS,加入透明液,避光下,摇床(100rpm左右)透明30分钟,PBS洗5分钟x 3次。
(4)封闭
使用PBS+0.3%Triton X-100配制5%BSA(购自上海生工)用于 封闭,37℃封闭30分钟。
(5)一抗孵育
①配制PBS+0.3%Triton X-100用于稀释抗体,按照1:50比例稀释鳞癌特异性抗体p63(购自CST公司),弃封闭液,加入配制好的一抗稀释液,至4℃冰箱孵育过夜。
②4℃取出,平衡至室温,37℃继续孵育1小时,PBS洗5分钟x 3次。
(6)二抗孵育(避光)
配制PBS+0.3%Triton X-100用于二抗稀释,按照1:1000比例稀释激发光为488且种属为兔的荧光二抗(购自赛默飞公司),常温避光孵育1h,PBS洗5分钟x 3次。
(7)DAPI染色(避光)
1:1000PBS稀释DAPI(购自Sigma公司),常温避光染色5分钟,PBS洗5分钟x 3次。显微镜(Invitrogen公司EVOS M500)下成像,拍照记录。
图6A和图6B为体外培养后的食管鳞癌细胞进行免疫荧光染色鉴定的结果,分别为10倍物镜下不同视野荧光拍照的图片。如图所示,视野内细胞在488激发光下均显示绿色,表明培养后的细胞为鳞癌细胞,与临床病理诊断一致。
实施例6食管鳞癌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的方法消化食管癌内镜组织样本(样本1~40号)获得食管鳞癌原代细胞。对于所获得的食管鳞癌原代细胞,使用实施例1中的No.7培养基,按照活细胞密度1×10 4个/cm 2将细胞接种在12孔板中并进行培养,待细胞扩增至85%后消化并计数,同时记录直至消化时培养的天数,将该直至消化时培养的天数作为一个培养周期。如图7所示,培养40例样本所得平均培养周期是10天,扩增得到的平均细胞数是70万。
在该实验条件下持续培养样本2、样本5、样本6、样本8、样本9、样本11、样本27、样本39和样本40总共9例样本,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*log10(消化后细胞总数/初始种入细胞数)计算PD,如图8所示,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,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8可以看出,使用本发明的食管鳞癌原代细胞培养基对上述9例样本进行培养时,扩增56天后的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7所添加的因子的浓度对食管鳞癌细胞增殖的影响
按照实施例1的方法同样地分离内镜组织样本65、样本66、样本67、样本68和样本69获得食管鳞癌原代细胞,并使用实施例1中的No.7培养基培养所获得的食管鳞癌原代细胞,按照活细胞密度1×10 4个/cm 2接种于12孔板中(每孔4.5万细胞数),按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量35Gy)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。待细胞扩增至85%,取出12孔板,使用200微升0.25%胰蛋白酶(购自Gibco公司)润洗1分钟,吸去后再每孔加入500微升0.05%胰蛋白酶(购自Gibco公司),置于37℃、5%CO 2培养箱中反应10分钟,直至显微镜(Invitrogen公司EVOS M500)下能观察到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1毫升基础培养基重悬,使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数,得到细胞总数。所得细胞用于以下培养实验。
接着,配制以下8种配方培养基进行实验:
配方1:实施例1中的No.7培养基组分中不含N2添加剂;
配方2:实施例1中的No.7培养基组分中不含非必需氨基酸;
配方3:实施例1中的No.7培养基组分中不含谷氨酰胺;
配方4:实施例1中的No.7培养基组分中不含胰岛素;
配方5:实施例1中的No.7培养基组分中不含IGF1;
配方6:实施例1中的No.7培养基组分中不含牛垂体提取物;
配方7:实施例1中的No.7培养基组分中不含Y27632;
配方8:实施例1中的No.7培养基组分中不含氢化可的松。
分别使用上述配方1~8和实施例1中No.7培养基来稀释上述消化后的细胞悬液,按照每孔2万细胞,500微升体积种入24孔板中。
在使用配方1的培养基时,按照终浓度分别为1:400、1:200、1:100、1:50、1:25配制5个浓度梯度的N2添加剂,在接种有原代细胞的24孔板中分别添加配制好的N2添加剂每孔500微升;并使用配方1的培养基设置对照孔(BC)。
在使用配方2的培养基时,按照终浓度分别为400μM、200μM、100μM、50μM、25μM配制5个浓度梯度的非必需氨基酸,在接种有原代细胞的24孔板中分别添加配制好的非必需氨基酸每孔500微升;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,按照终浓度分别为8mM、4mM、2mM、1mM、0.5mM配制5个浓度梯度的谷氨酰胺,在接种有原代细胞的24孔板中分别添加配制好的谷氨酰胺每孔500微升;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,按照终浓度分别为40μg/mL、20μg/mL、10μg/mL、5μg/mL、2.5μg/mL配制5个浓度梯度的胰岛素,在接种有原代细胞的24孔板中分别添加配制好的胰岛素每孔500微升;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,按照终浓度分别为40ng/mL、20ng/mL、10ng/mL、5ng/mL、2.5ng/mL配制5个浓度梯度的胰岛素样生长因子1(IGF1),在接种有原代细胞的24孔板中分别添加配制好的胰岛素样生长因子(IGF1)每孔500微升;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,按照终浓度分别为56μg/mL、28μg/mL、14μg/mL、7μg/mL、3.5μg/mL配制5个浓度梯度的牛垂体提取物,在接种有原代细胞的24孔板中分别添加配制好的牛垂体提取物每孔500微升;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,按照终浓度分别为40μM、20μM、10μM、5μM、2.5μM配制5个浓度梯度的Y27632,在接种有原代细胞 的24孔板中分别添加配制好的Y27632每孔500微升;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,按照终浓度分别为1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL配制5个浓度梯度的氢化可的松,在接种有原代细胞的24孔板中分别添加配制好的氢化可的松每孔500微升;并使用配方8的培养基设置对照孔(BC)。
另外,使用实施例中的No.7培养基按照终浓度分别为10μM、3μM、1μM、0.3μM、0.1μM配制5个浓度梯度的8种小分子化合物(LDN193189、IWP2、A83-01、Forskolin、SB431542、CHIR99021、DMH-1、DAPT;均购自MCE公司),在接种有原代细胞的24孔板中分别添加配制好的8种小分子化合物(LDN193189、IWP2、A83-01、Forskolin、SB431542、CHIR99021、DMH-1、DAPT)每孔500微升;并分别使用No.7培养基设置对照孔(BC)。
同时上述各孔加入1万个辐照后的NIH-3T3细胞作为滋养细胞。
待细胞扩增至24孔的85%左右消化计数,分别参比对照孔(BC)细胞数计算比值,将结果分别示于图9A~9P。图9A~图9P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。
如图9所示,N2添加剂的浓度范围为1:400~1:25,浓度为1:50比例加入细胞增殖效果最明显;非必需氨基酸的浓度范围为25~400μM,浓度为400μM加入细胞增殖效果最明显;谷氨酰胺的浓度范围为0.5~8mM,浓度为2mM加入时细胞增殖效果最明显;胰岛素样生长因子1的浓度范围为2.5~40ng/mL,浓度为5ng/mL时细胞增殖效果最明显;牛垂体提取物的浓度范围为3.5~56μg/mL,浓度为7μg/mL时细胞增殖效果最明显;Y27632浓度范围为2.5~40μM,浓度为10μM时细胞增殖效果最明显;胰岛素浓度范围为2.5~40ng/mL,浓度为20ng/mL时细胞增殖效果最明显;氢化可的松浓度范围为0.1~1.6μg/mL,浓度为0.4μg/mL时细胞增殖效果最明显。其他8种小分 子化合物(LDN193189、IWP2、A83-01、Forskolin、SB431542、CHIR99021、DMH-1、DAPT)在10μM、3μM、1μM、0.3μM、0.1μM浓度梯度下均未表现出明显的促增殖作用。
实施例8本发明培养基与现有培养基对细胞增殖作用的比较
如表4所示,配制非专利文献2中的KM(Keratinocyte medium)培养基。
表4KM培养基的配制
培养基成分 添加体积(mL)
DMEM(购自康宁公司) 329.25
F12(购自康宁公司) 109.75
胰岛素(购自Gibco公司) 5
表皮生长因子(购自北京义翘) 0.5
碘塞罗宁(购自mce) 0.005
转铁蛋白(购自北京义翘) 0.5
霍乱毒素(购自sigma公司) 0.005
氢化可的松(购自mce) 5
胎牛血清(购自依科赛公司) 50
总量(mL) 500
如表5所示,配制非专利文献3中的FM培养基。
表5FM培养基的配制
培养基成分 添加体积(mL)
DMEM(购自康宁公司) 373
F12(购自康宁公司) 125
Y27632(购自mce) 0.5
胰岛素(购自Gibco公司) 1.25
表皮生长因子(购自北京义翘) 0.02
霍乱毒素(购自sigma公司) 0.0043
两性霉素B(购自sigma公司) 0.5
氢化可的松(购自mce) 0.03
总量(mL) 500
从Stem Cell公司购入KSFM培养基。
与实施例1同样地分离内镜组织样本70、样本71和样本72得到食管鳞癌原代细胞,分别使用EM培养基、KSFM培养基、FM培养基 和KM培养基进行培养。
下面以其中一例样本(样本70)进行说明。将得到的食管鳞癌原代细胞分别使用1毫升EM培养基、KSFM培养基、FM培养基和KM培养基重悬,使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数。
按照活细胞密度1×10 4个/cm 2分别接种于2块24孔板中(每孔2万细胞数),其中1块24孔板中不加入γ射线辐照的NIH-3T3细胞培养,另外一块24孔中按照细胞密度2×10 4个/cm 2加入经γ射线辐照(辐照剂量35Gy)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。在培养第5天,取出24孔板,使用200微升0.25%胰蛋白酶(购自Gibco公司)润洗1分钟,吸去后再每孔加入500微升0.05%胰蛋白酶(购自Gibco公司),置于37℃、5%CO 2培养箱中反应10分钟,直至显微镜(Invitrogen公司EVOS M500)下能观察到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1毫升基础培养基重悬,使用流式图像计数仪(江苏卓微生物科技有限公司JIMBIO FIL)进行计数,得到不同培养基培养的细胞总数。将结果示于图11。
从图10中可以看出,EM培养基与KM培养基、FM培养基和KSFM培养基相比,在接种同样细胞数(2万/孔)培养相同时间条件下,扩增得到的细胞数最多,对食管鳞癌细胞促增殖作用有显著效果。并且,从图10中还可以得出EM培养基在使用滋养细胞培养时效果优于没有滋养细胞进行培养的情况,由此可知,本发明的食管鳞癌原代细胞培养基在使用滋养细胞培养时对食管鳞癌细胞促增殖左右有显著效果,缩短培养所需周期。
实施例9使用本发明培养基扩增得到的食管鳞癌原代细胞用于药物筛选
1、细胞培养和铺板
从得到的食管鳞癌内镜样本与实施例1同样地分离得到食管鳞癌原代细胞并使用实施例1中的No.7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3代、第5 代、第7代、第9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,使用实施例1中的No.7培养基,将细胞按照活细胞密度4×10 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2000个/孔。从孔板边缘加入实施例1中的No.7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7个浓度梯度的3种药物(硼替佐米、丝裂霉素、羟基喜树碱;均购自MCE公司),在384孔药板(购自赛默飞公司)每孔中添加50μL,-20℃保存待用。
表6硼替佐米、丝裂霉素、羟基喜树碱药物添加液的配制
终浓度(μM) 配制浓度(μM)
10 5000
3.33 1666.7
1.11 555.6
0.37 185.2
0.12 61.7
0.04 20.6
0.01 6.9
按照下表配制7个浓度梯度的厄洛替尼(购自MCE公司),在384孔药板(购自赛默飞公司)每孔中添加50μL,-20℃保存待用。
表7厄洛替尼添加液的配制
终浓度(μM) 配制浓度(μM)
40 20000
13.33 6666.7
4.44 2222.2
1.48 740.7
0.49 246.9
0.16 82.3
0.05 27.4
3、高通量加药
取出配制好的药板,置于室温,待完全融化后置于离心机(Sigma公司3-18K)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。对培养有食管鳞癌细胞的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10min后使用多功能酶标仪(Perkin Elmer公司Envision)检测。
5、数据处理
按照公式细胞存活率(%)=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存活率,使用graphpad prism软件计算药物对细胞作用的半数抑制率(IC50)。将结果分别示于图11A~图11D。
由图11A~图11D可以确认,使用本发明的食管鳞癌原代细胞培养基培养得到的食管鳞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)。
工业上的可利用性
本发明提供一种用于体外快速扩增食管鳞癌原代细胞的培养基及培养方法,能够实现食管鳞癌组织样本在短时间内快速扩增,能够在有效的时间内扩增得到足够数量的细胞,用于体外高通量药物敏感实验和指导临床精准用药。

Claims (6)

  1. 一种食管鳞癌原代细胞培养基,其特征在于,包括:
    初始培养基、Rho蛋白酶抑制剂、抗生素、胰岛素、N2添加剂、胰岛素样生长因子1、非必需氨基酸、以及任选的氢化可的松、任选的谷氨酰胺和任选的牛垂体提取物,
    所述初始培养基选自DMEM/F12、DMEM、F12或RPMI-1640。
  2. 如权利要求1所述的食管鳞癌原代细胞培养基,其特征在于:
    Rho蛋白激酶抑制剂选自Y27632、羟基法舒地尔和GSK429286A中的一种或多种,当选自Y27632时,浓度范围为2.5~40μM,优选为5~20μM;当选自羟基法舒地尔时,浓度范围为2~32μM,优选为4~16μM;当选自GSK429286A时,浓度范围为2~32μM,优选为4~16μM;
    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,当选自链霉素/青霉素时,链霉素浓度范围为25~400μg/mL,优选为50~200μg/mL,更优选为200μg/m,青霉素浓度范围为25~400U/mL,优选为50~200U/mL,更优选为200U/mL;当选自两性霉素B时,浓度范围为0.25~4μg/mL,优选为0.5~2μg/mL;当选自Primocin时,浓度范围为25~400mg/mL,优选为50~200mg/mL;
    胰岛素的浓度范围为2.5~40μg/mL,优选为10~40μg/mL;
    N2添加剂相对于培养基的体积比1:400~1:25,优选1:100~1:25;
    胰岛素样生长因子1的浓度范围为2.5~40ng/mL,优选为2.5~10ng/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为50μM~400μM,优选为100μM~400μM;
    氢化可的松的浓度范围为0~1.6μg/mL,优选为0.2~0.8μg/mL;
    谷氨酰胺的浓度范围为0mM~8mM,优选为1mM~4mM;
    牛垂体提取物的浓度范围为0~56μg/mL,优选为3.5~14μg/mL。
  3. 一种食管鳞癌原代细胞的培养方法,其特征在于:
    使用权利要求1或2所述的食管鳞癌原代细胞培养基对食管鳞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:
    在所述培养中,按照细胞密度2~3×10 4个/cm 2加入滋养细胞。
  5. 如权利要求4所述的培养方法,其特征在于:
    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. 一种食管鳞癌疾病的药物筛选的方法,其特征在于,包括以下步骤:
    (1)使用权利要求3~5中任一项所述的食管鳞癌原代细胞的培养方法培养食管鳞癌原代细胞,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。
PCT/CN2020/076736 2020-02-11 2020-02-26 食管鳞癌原代细胞的培养基及培养方法 WO2021159560A1 (zh)

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