CULTURE MEDIUM FOR LARYNGEAL CANCER EPITHELIAL CELLS, CULTURE METHOD, AND APPLICATION THEREOF
20230212523 · 2023-07-06
Assignee
Inventors
Cpc classification
C12N2501/999
CHEMISTRY; METALLURGY
C12N5/06
CHEMISTRY; METALLURGY
International classification
Abstract
Provide are a primay cell culture medium that contains an MST1/2 kinase inhibitor and is used for culturing laryngeal cancer epithelial cells, and a culture method using the primary cell culture medium. In the culture method, the primay cell culture medium is used to culture primary cells on a clulture vessel plated with irradiated trophoblasts, so that the primay cells proliferate rapidly. A cell model obtained by the primary cell culture medium and the primay cell culture method can be used for the efficacy evaluation and screening of drugs.
Claims
1. A primary cell culture medium for culturing laryngeal cancer epithelial cells: comprising an MST1/2 kinase inhibitor, wherein the MST1/2 kinase inhibitor comprises a compound of Formula (I) or a pharmaceutically acceptable salt, or a solvate thereof, ##STR00078## wherein, R.sub.1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl optionally independently substituted with 1-2 R.sub.6, aryl C1-C6 alkyl optionally independently substituted with 1-2 R.sub.6 and heteroaryl optionally independently substituted with 1-2 R.sub.6; R.sub.2 and R.sub.3 are each independently selected from C1-C6 alkyl; R.sub.4 and R.sub.5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxyl C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, and C3-C6 heterocyclyl C1-C6 alkyl; and R.sub.6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
2. The primary cell culture medium of claim 1, wherein, R.sub.1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and phenyl optionally independently substituted with 1-2 R.sub.6, naphthyl optionally independently substituted with 1-2 R.sub.6, phenylmethyl optionally independently substituted with 1-2 R.sub.6 and thienyl optionally independently substituted with 1-2 R.sub.6; R.sub.2 and R.sub.3 are each independently selected from C1-C3 alkyl; R.sub.4 and R.sub.5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, hydroxyl C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkylamino C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, piperidyl C1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl; and R.sub.6 is selected from halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
3. The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of Formula (Ia) or a pharmaceutically acceptable salt, or a solvate thereof, ##STR00079## wherein, R.sub.1 is selected from C1-C6 alkyl, phenyl optionally independently substituted with 1-2 R.sub.6, thienyl optionally independently substituted with 1-2 R.sub.6, and phenylmethyl optionally independently substituted with 1-2 R.sub.6; R.sub.5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl; and R.sub.6 is independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
4. The primary cell culture medium of claim 3, wherein R.sub.1 is phenyl optionally independently substituted with 1-2 R.sub.6; R.sub.5 is hydrogen; and R.sub.6 is preferably fluoro, methyl or trifluoromethyl.
5. The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises at least one compound selected from the following compounds or a pharmaceutically acceptable salt thereof: ##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
6. The primary cell culture medium of claim 1, wherein the amount of the MST1/2 kinase inhibitor is 1.25-10 μM.
7. The primary cell culture medium of claim 1, further comprising one or more of the following factors: insulin-like growth factor 1; fibroblast growth factor 7; insulin-transferrin-selenium complex; hepatocyte growth factor; a ROCK kinase inhibitor selected from at least one of Y27632, Fasudil, or H-1152; and a TGFβ type I receptor inhibitor selected from at least one of A83-C1, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, or SJN2511.
8. The primary cell culture medium of claim 7, wherein: the amount of insulin-like growth factor 1 is 10-80 ng/ml; the amount of fibroblast growth factor 7 is 5-80 ng/ml; the respective amounts of insulin/transferrin/sodium selenite in the insulin-transferrin-selenium complex are 5-20 μg/ml, 2.5-10 μg/ml, 2.5-10 ng/ml, respectively; the amount of hepatocyte growth factor is 10-80 ng/ml, more preferably 10 40 ng/ml; the amount of the ROCK kinase inhibitor is 1.25-20 μM, and the amount of the TGFI3 type I receptor inhibitor is 125 -1000 nM.
9. The primary cell culture medium of claim 1, wherein the primary cell culture medium is free of serum, bovine pituitary extract, Wnt agonists, R-spondin family proteins, BMP inhibitors, nicotinamide, or N-acetylcysteine.
10. The primary cell culture medium of claim 1, wherein the laryngeal cancer epithelial cells are selected from laryngeal cancer cells, normal laryngeal cancer epithelial cells, or laryngeal cancer epithelial stem cells.
11. A method for culturing laryngeal cancer epithelial cells, characterized in comprising the following steps: (1) pre-laying a culture vessel with trophoblastic cells irradiated with X-ray or γ-ray; (2) inoculating primary laryngeal cancer epithelial cells isolated from laryngeal cancer tissues in the culture vessel which is pre-laid with trophoblastic cells, and (3) culturing by using the primary cell culture medium of claim 1.
12. A method for evaluating or screening a drug for treating laryngeal cancer diseases, comprising the following steps: (1) culturing laryngeal cancer epithelial cells by the culturing method of claim 11; (2) selecting the drug to be tested and diluting into different drug concentration gradients; and (3) adding the drug which has diluted to gradients to the laryngeal cancer epithelial cells obtained in step (1), and evaluating the cell viability.
13. The primary cell culture medium of claim 1, wherein the amount of the MST1/2 kinase inhibitor is 2.5-10 μM.
14. The primary cell culture medium of claim 8, wherein: the amount of insulin-like growth factor 1 is 10-40 ng/ml; the amount of fibroblast growth factor 7 is 20-80 ng/ml; the amounts of insulin, transferrin and sodium selenite in the insulin-transferrin-selenium complex are 10-20 μg/ml, 5-10 μg/ml, 5-10 ng/ml, respectively; the amount of hepatocyte growth factor is 10-40 ng/ml; the amount of the ROCK kinase inhibitor is 2.5-10 μM; and the amount of the TGFβ type I receptor inhibitor is 125 -500 nM.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0067] In the specification, the epithelial cells include differentiated epithelial cells and epithelial stem cells obtained from epithelial tissues. “Epithelial stem cells” refer to the cells with long-term self-renewal ability that may differentiate into epithelial cells, and the stem cells originated from epithelial tissues. Examples of epithelial tissues include cornea, oral mucosa, skin, conjunctiva, bladder, renal tubule, kidney, digestive organs (esophagus, stomach, duodenum, small intestine (including jejunum and ileum), large intestine (including colon)), liver, pancreas, mammary gland, salivary gland, lacrimal gland, prostate, hair root, trachea, lung, etc. Among others, the cell culture medium of the embodiment is preferably the culture medium for laryngeal cancer originated epithelial cells.
[0068] In addition, in this specification, “epithelial tumor cells” refer to the cells obtained by tumorigenesis of cells originated from the aforementioned epithelial tissues.
[0069] In the specification, “organoid” refers to a three-dimensional, organ-like cellular tissue formed by spontaneously organizing and aggregating cells within a controlled space in high density.
[0070] [Preparation Examples of MST1/2 Kinase Inhibitors]
[0071] In the specification, MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1/2 signaling. Generally, MST1/2 kinase inhibitors reduce the activity of MST1/2 kinase by, for example, binding to the same. Since MST1 and MST2 have similar structures, MST1/2 kinase inhibitors may be, for example, compounds that bind to MST1 or MST2 and reduce the activity thereof.
[0072] 1. Preparation of MST1/2 kinase inhibitor Compound 1 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino)benzsulfamide 1
##STR00018##
[0073] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): 2-amino-2-(2,6-difluorophenyl)acetic acid (2.0 g) and then methanol (30 ml) were added into a round bottom flask, followed by addition of thionyl chloride (1.2 ml) dropwise under an ice bath. The reaction system was reacted overnight at 85° C. After the completion of the reaction, the system was evaporated under reduced pressure to dry the solvent, and the obtained white solid was directly used in the next step.
[0074] Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl) acetate (A3): methyl 2-amino-2-(2,6-difluorophenyl)acetate (2 g) and then acetone (30 ml) and potassium carbonate (2.2 g) were added into a round bottom flask, and then the system was cooled to -10° C. with an ice salt bath, and then a solution of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone was slowly added. The reaction system was stirred overnight at room temperature. After the completion of the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressurized silica gel column chromatography to obtain compound A3. LC/MS: M+H 359.0.
[0075] 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (A4): methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) and then acetic acid (50 ml) and iron powder (3.9 g) were added into a round bottom flask. The reaction system was stirred at 60° C. for two hours. After the completion of the reaction, the reaction system was evaporated under reduced pressure to dry the solvent, and the resultant was neutralized to be alkaline with saturated sodium bicarbonate solution and was extracted with ethyl acetate. The organic phase was washed with water and saturated brine and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with diethyl ether to obtain compound A4. LC/MS: M+H 297.0.
[0076] 2-chloro-7-(2,6-difluorophenyI)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (A5): 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridin-6(5H)-one (2 g) and N,N-dimethylacetamide (10 ml) were added into a round bottom flask, and cooled to −35° C., followed by addition of iodomethane (0.9 ml) and then sodium hydride (615 mg), and the reaction system was stirred for two hours. After the completion of the reaction, the reaction mixture was quenched with water, and extracted with ethyl acetate. The organic phase was washed with water and saturated brine, respectively, and dried with anhydrous sodium sulfate. The organic phase was filtered and evaporated to dryness under reduced pressure to obtain a crude product. The crude product was washed with diethyl ether to obtain compound A5. LC/MS: M+H 325.0.
[0077] 4-((7-(2,6-difluorophenyI)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino)benzsulfamide (1): 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfanilamide (53 mg), p-toluenesulfonic acid (53 mg) and sec-butanol (5 ml) were added into a round bottom flask. The reaction system was stirred at 120° C. overnight. After the completion of the reaction, the reaction mixture was filtered, and washed with methanol and diethyl ether to obtain compound 1. LC/MS: M+H 461.1.
[0078] 2. Preparation of Other MST1/2 Inhibitor Compounds of the Invention
[0079] Other MST1/2 inhibitor compounds of the invention were synthesized via the method similar to that of Compound 1, and their structures and mass spectrum data are shown in the following table.
TABLE-US-00001 MS(ESI) No. Compound m/z(M + 1)+ 1
Example 1
[0080] Isolation of Human Primary Laryngeal Cancer Epithelial Cells
[0081] Laryngeal cancer tissue samples were obtained from the cancer tissues of three informed and consenting laryngeal cancer patients by surgical resection, namely Sample Nos. 0S0020, 0S0021, and 0S0022. One of the samples (No. 0S0020) will be described below.
[0082] The aforementioned tissue samples were collected within half an hour after the surgical excision or the biopsy. More specifically, in a sterile environment, tissue samples from non-necrotic sites were cut with a volume of more than 0.5 cm.sup.3 and were placed in 4 mL of pre-cooled tissue transport fluid (specific formulation shown in Table 1). The transport fluid was placed in a 5 mL plastic sterile cryopreservation tube with a lid (purchased from Guangzhou Jet Bio-Filtration Co., Ltd.) and cold chain (0-10° C.) transported to the laboratory.
TABLE-US-00002 TABLE 1 Formulation of tissue transport fluid Components of tissue transport fluid Supplier Final concentration DMEM/F12 Corning 97.8 vol. % Primocin Invivogen 0.2 vol. % (concentration of commercial product: 50 mg/ml) penicillin/streptomycin Corning 2 vol. % (concentration of solution double antibody commercial product: 10000 U/ml penicillin, 10 mg/ml streptomycin)
TABLE-US-00003 TABLE 2 Fumulation of tissue digestive solution Components of tissue Final digestive solution Supplier concentration HBSS Gibco 50 vol. % RPMI-1640 Corning 50 vol. % collagenase II Sigma 2 mg/mL collagenase IV Sigma 2 mg/mL deoxyribonucleic acid I Sigma 50 U/mL hyaluronidase Sigma 0.5 mg/mL calcium chloride Sangon Biotech 0.33 mg/mL bovine serum albumin Sangon Biotech 10 mg/mL
[0083] In the biological safety cabinet, the tissue sample (No. 0S0020) was transferred to a 100 mm cell culture dish (purchased from NEST). The tissue sample was rinsed with the tissue transport fluid. The residual blood on the surface of the tissue sample was washed away. Excess tissues such as fat on the surface of the tissue sample were removed. The rinsed tissue sample was transferred to another new 100 mm culture dish; 2 mL of transport fluid was added, and a sterile scalpel blade and a forceps were used to divide the tissue sample into tissue fragments less than 3 mm.sup.3 in volume.
[0084] The tissue sample fragments were transferred to a 15 mL centrifuge tube, and centrifuged at 1500 rpm for 4 minutes in a tabletop centrifuge (Sigma, 3-18K); after discarding the supernatant, the tissue transport fluid and the tissue digestive solution were added in a ratio of 1:1 (the dosage is about 5 mL of tissue digestive solution per 10 mg of tissue; specific formulation was shown in Table 2); then the sample was numbered and sealed with sealing film, and was then digested in a constant-temperature shaker (Zhichu Instrument ZQLY-180N) at 37° C., 300 revolutions; whether the digestion was completed was determined via observation every 1 hour.
[0085] After digestion, undigested tissue blocks were filtered through a 70 μm filter screen; the tissue blocks on the filter screen were rinsed with the tissue transport fluid; the residual cells were rinsed into a centrifuge tube and centrifuged at 1500 rpm for 4 minutes.
[0086] After discarding the supernatant, the remaining cell clusters were observed to determine whether blood cells were remained; if there were blood cells, 3 mL blood cell lysate (purchased from Sigma) was added, which was then mixed well, lysed at 4° C. for 15 minutes, with shaking and mixing well once every 5 minutes; after lysis, the resultant was take out and centrifuged at 1500 rpm for 4 minutes. The supernatant was discarded to provide digested and isolated primary laryngeal cancer cells, which were added with basic medium (BM) for resuspension. The basic medium was prepared by adding 0.2 vol. % of Primocin (purchased from Invivogen, with a concentration of 50 mg/mL) to the commercial DMEM/F-12 medium to provide a final concentration of 100 μg/mL. The total number of cells was 2,080,000, which was obtained by counting with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.).
[0087] Other two laryngeal cancer tissue samples were isolated according to the same method above, and the total number of cells were 1,970,000 (0S0021) and 2,320,000 (0F0022), respectively.
Example 2
[0088] Optimization of Culture Medium for Primary Laryngeal Cancer Epithelial Cell
[0089] (1) Effects of Different Factors
[0090] The cultured NIH-3T3 cells (purchased from ATCC, and cultured in DMEM culture solution containing 10% fetal bovine serum) were digested with 0.25% trypsin (purchased from Thermo Fisher), and the digestion was terminated by using DMEM culture solution (purchased from Corning) containing 5%(v/v) fetal bovine serum (purchased from ExCell Biotech Co., Ltd.), 100 U/mL penicillin and 100 μg/mL streptomycin; the resultant was collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was discarded. The centrifuged cell sediments were resuspended in the above DMEM culture solution containing 10% fetal bovine serum and were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.). The cells were irradiated with γ-ray of 35 Gy irradiation dose, and then were inoculated in a culture vessel at a density of 2×10.sup.4 cells/cm.sup.2. The cells were incubated in a 37° C. incubator until the cells adhere to the wall. Before the inoculation of primary cells, the culture medium was removed from the culture vessel.
[0091] Preparation of basic medium (abbreviated as BM): BM was prepared by adding 0.2 vol. % of Primocin (purchased from Invivogen, with a concentration of 50 mg/mL) to the commercial DMEM/F-12 medium to provide a final concentration of 100 μg/mL.
[0092] Next, different kinds and concentrations of additive factors (Table 3) were added to the basic medium (BM), so as to prepare culture mediums for laryngeal cancer epithelial cells containing different additive components.
TABLE-US-00004 TABLE 3 Preparation of culture mediums containing different components (final concentrations are shown) Sources of additive Culture medium factors Composition Basic medium/BM DMEM/F12 + 100 μg/mL Primocin BM + epidermal Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 growth factor (EGF) ng/ml EGF BM + hepatocyte Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 growth factor (HGF) ng/ml HGF BM + insulin-like Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 growth factor 1 ng/ml IGF-1 (IGF-1) BM + fibroblast growth Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 factor (FGF) ng/ml FGF BM + fibroblast growth Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 factor 7 (FGF-7) ng/ml FGF-7 BM + fibroblast growth Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 factor 10 (FGF-10) ng/ml FGF-10 BM + Neuregulin-1 Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 ng/ml Neuregulin-1 BM + Human Periostin Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 ng/ml Human Periostin BM + bovine pituitary Sino Biological BM + 80, 40, 20, 10, 5, 2.5, 1.25 extract ng/ml bovine pituitary extract BM + N2 additive Gibco BM + 1/25, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600 diluting ratio of N2 BM + B27 additive Gibco BM + 1/25, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600 diluting ratio of B27 BM + insulin- Insulin, transferrin, BM + 1/25, 1/50, 1/100, 1/200, 1/400, transferrin-selenium sodium selenite all 1/800, 1/1600 diluting ratio of ITS complex (ITS) purchased from stock solution (the stock solution Sigma contains 500 μg/ml insulin, 250 μg/ml transferrin, and 250 ng/ml sodium selenite in DMEM/F-12) BM + non-essential Gibco BM + 1/25, 1/50, 1/100, 1/200, 1/400, amino acids 1/800, 1/1600 diluting ratio of non-essential amino acids BM + glutamine Gibco BM + 1/25, 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600 diluting ratio of glutamine BM + Y27632 MCE BM + 40, 20, 10, 5, 2.5, 1.25, 0.625 μM Y27632 BM + Compound 1 Preparation BM + 40, 20, 10, 5, 2.5, 1.25, 0.625 Example μM Compound 1 BM + A83-01 MCE BM + 4000, 2000, 1000, 500, 250, 125, 62.5 nM A83-01 BM + SB202190 MCE BM + 4000, 2000, 1000, 500, 250, 125, 62.5 nM SB202190 BM + hydrocortisone MCE BM + 800, 400, 200, 100, 50, 25, 12.5 ng/ml hydrocortisone BM + R-spondin1 Sino Biological BM + 800, 400, 200, 100, 50, 25, 12.5 ng/ml RSPO1 BM + Noggin Sino Biological BM + 800, 400, 200, 100, 50, 25, 12.5 ng/ml Noggin
[0093] The culture mediums with different components were added at a volume of 500 μl/well to 48-well plates which were pre-laid with γ-ray irradiated NIH-3T3 cells. Laryngeal cancer cells (No. 0S0064) isolated from laryngeal cancer tissue according to the same method as described in Example 1 were inoculated at a cell count of 4×10.sup.4cells/well in the above-mentioned 48-well culture plates which were pre-laid with γ-ray irradiated NIH-3T3 cells. After surface disinfection, the plates were placed in a 37° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher), such that equal numbers of freshly isolated laryngeal cancer cells (No. 0S0064) were cultured under different medium formulations. The culture mediums were replaced and the trophoblastic cells were supplemented every 4 days. after the start of culture. After 10 days of culture, cell counts were performed. The basic medium (BM) without addition of any additive was used as the experimental control. The results were shown in
[0094] The ordinate in the figures shows the ratio of the number of cells obtained after culture in different mediums to the number of cells obtained after culture in basic medium BM. As shown in the figures, adding different concentrations of different factors according to Table 3 to BM would result in different effects on cell proliferation. Among others, B27 additive, N2 additive, insulin-transferrin-selenium complex, hepatocyte growth factor, insulin-like growth factor 1, fibroblast growth factor 7, Compound 1, Y27632 and A83-C1 provided certain promoting effects on cell proliferation in specific concentration ranges.
[0095] (2) Effects of Increasing Factors in ihe Culture Mediums on the Proliferation of Primary Laryngeal Cancer Cells Obtained by the Method of the Invention
[0096] Different small molecules, additives and growth factors (Table 4) were sequentially added to the basic medium BM, respectively, so as to prepare culture mediums for laryngeal cancer epithelial cells containing different additive components.
TABLE-US-00005 TABLE 4 Preparation of culture mediums containing different components (final concentrations are shown) Medium Composition basic medium/BM DMEM/F12 + 100 μg/mL Primocin NO.1 BM + 5 μM Compound 1 NO.2 NO.1 + 1:50 insulin-transferrin-selenium complex NO.3 NO.2 + 10 ng/mL hepatocyte growth factor NO.4 NO.3 + 10 μM Y27632 NO.5 NO.4 + 10 ng/mL insulin-like growth factor 1 NO.6 NO.5 + 40 ng/mL fibroblast growth factor 7 NO.7 NO.6 + 250 nM A83-01 NO.8 NO.7 + 10 ng/mL epidermal growth factor NO.9 NO.8 + 500 nM SB202190 NO.10 NO.9 + 10 μg/mL bovine pituitary extract NO.11 NO.10 + 1:50 B27 additive NO.12 NO.11 +1:100 N2 additive NO.13 NO.12 + 5% (volume ratio) fetal bovine serum
[0097] The culture mediums with different components were added at a volume of 500 μl/well to 48-well plates which were pre-laid with γ-ray irradiated NIH-3T3 cells, and simultaneously, the BM medium was used as the experiment control. Laryngeal cancer cells (No. 0S0065) isolated from laryngeal cancer tissue according to the method described in Example 1 were inoculated at a cell count of 4×10.sup.4 cells/well in the above-mentioned 48-well culture plates which were pre-laid with γ-ray irradiated NIH-3T3 cells. After surface disinfection, the plate was placed in a 37° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher), such that equal numbers of freshly isolated laryngeal cancer cells (No. 0S0065) were cultured under different medium formulations. After 7 days of culture, cell counts were performed. The results were shown in
[0098] As shown in the figure, it was determined that No. 7 is the most preferred culture medium in this patent for culturing and expanding primary laryngeal cancer cells (hereinafter abbreviated as SCM). On this basis, further addition of some factors, small molecule inhibitors, or certain concentration of serum and substitute of serum, such as N2 additive and B27 additive, did not provide significant effect on promoting the cell proliferation.
[0099] (3) Effects of Different Concentrations of the Additive Factors on the Proliferation of Primary Laryngeal Cancer Cells Obtained in the Invention
[0100] Preparation of the culture medium for primary laryngeal cancer epithelial cells of the invention (abbreviated as SCM): to the basic medium (BM), was added fibroblast growth factor 7 (FGF-7) at the final concentration of 40 ng/ml, insulin-like growth factor 1 (IGF-1) at the final concentration of 10 ng/ml, hepatocyte growth factor (HGF) at the final concentration of 10 ng/ml, insulin-transferrin-selenium complex (ITS) stock solution at the diluting ratio of 1:50 (insulin at the final concentration of 10 μg/ml, transferrin at the final concentration of 5 μg/ml, and sodium selenite at the final concentration of 5 ng/ml in SCM medium), Compound 1 at the final concentration of 5 μM, Y27632 at the final concentration of 10 μM, and TGFβ1 inhibitor A83-C1 at the final concentration of 250 nM, to prepare the culture medium for primary laryngeal cancer epithelial cells.
[0101] Laryngeal cancer epithelial cells originated from cancer tissue were isolated and obtained from the cancer tissue of a laryngeal cancer patient (Sample No. 0S0005) using the same method as in Example 1. Next, laryngeal cancer cells originated from cancer tissue were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells. Then, the cells were inoculated in a 12-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells at a density of 4×10.sup.4 cells/cm.sup.2. 2 mL of the prepared culture medium for primary laryngeal cancer epithelial cells SCM was added to the 12-well plate, which was then placed in a 37 ° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher) for culture. When the cells in the culture plate grew to cover about 80% of the bottom area, the medium supernatant in the 12-well plate was discarded and 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) was added for digestion for 1 minute; after removing 0.25% trypsin, 0.5 mL of 0.05% trypsin was added again for cell digestion, which was then incubated at room temperature for 5-20 min, until the cells were completely digested as observed under the microscope (EVOS M500, Invitrogen); then the digestion was terminated by using 1 ml of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL penicillin; the resultant was collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was discarded. The centrifuged cell sediments were resuspended in the basic medium BM and were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells. The obtained cells were used in the following cultivation experiments.
[0102] Next, the following 7 culture mediums with different formulations were prepared for experiments:
[0103] Formulation 1: SCM medium composition without fibroblast growth factor 7;
[0104] Formulation 2: SCM medium composition without insulin-transferrin-selenium complex;
[0105] Formulation 3: SCM medium composition without insulin-like growth factor 1;
[0106] Formulation 4: SCM medium composition without hepatocyte growth factor;
[0107] Formulation 5: SCM medium composition without Y27632;
[0108] Formulation 6: SCM medium composition without Compound 1;
[0109] Formulation 7: SCM medium composition without A83-C1.
[0110] The digested cell suspension was diluted with the above Formulations 1-7 respectively, and were seeded into a 48-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells with 10,000 cells and 250 μL volume per well.
[0111] When using the medium of Formulation 1, to a 48-well plate inoculated with primary cells was added the prepared fibroblast growth factor 7, with 250 μL per well, such that the final concentrations of fibroblast growth factor 7 were 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, and 1.25 ng/ml, respectively; the medium of Formulation 1 was used as the control well (BC).
[0112] When using the medium of Formulation 2, to a 48-well plate inoculated with primary cells was added the prepared insulin-transferrin-selenium complex, with 250 μL per well, such that the final concentrations of insulin-transferrin-selenium complex stock solution were 1:1600, 1:800, 1:400, 1:200, 1:100, 1:50, and 1:25, respectively (corresponding to the final concentrations of insulin/transferrin/sodium selenite of 0.3125 μg/ml, 0.15625 μg/ml, 0.15625 ng/ml; 0.625 μg/ml, 0.3125 μg/ml, 0.3125 ng/ml; 1.25 μg/ml, 0.625 μg/ml, 0.625 ng/ml; 2.5 μg/ml, 1.25 μg/ml, 1.25 ng/ml; 5 μg/ml, 2.5 μg/ml, 2.5 ng/ml; 10 μg/ml, 5 μg/ml, 5 ng/ml; and 20 μg/ml, 10 μg/ml, 10 ng/ml, respecrtively); the medium of Formulation 2 was used as the control well (BC).
[0113] When using the medium of Formulation 3, to a 48-well plate inoculated with primary cells was added the prepared insulin-like growth factor 1, with 250 μL per well, such that the final concentrations of insulin-like growth factor 1 were 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, and 1.25 ng/ml, respectively; the medium of Formulation 3 was used as the control well (BC).
[0114] When using the medium of Formulation 4, to a 48-well plate inoculated with primary cells was added the prepared hepatocyte growth factor, with 250 μL per well, such that the final concentrations of hepatocyte growth factor were 80 ng/ml, 40 ng/ml, 20 ng/ml, 10 ng/ml, 5 ng/ml, 2.5 ng/ml, and 1.25 ng/ml, respectively; the medium of Formulation 4 was used as the control well (BC).
[0115] When using the medium of Formulation 5, to a 48-well plate inoculated with primary cells was added the prepared Y27632, with 250 μL per well, such that the final concentrations of Y27632 were 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM, respectively; the medium of Formulation 5 was used as the control well (BC).
[0116] When using the medium of Formulation 6, to a 48-well plate inoculated with primary cells was added the prepared Compound 1, with 250 μL per well, such that the final concentrations of Compound 1 were 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625 μM, respectively; the medium of Formulation 6 was used as the control well (BC).
[0117] When using the medium of Formulation 7, to a 48-well plate inoculated with primary cells was added the prepared A83-C1, with 250 μL per well, such that the final concentrations of A83-C1 were 4000 nM, 2000 nM, 1000 nM, 500 nM, 250 nM, 125 nM, and 62.5 nM, respectively; the medium of Formulation 7 was used as the control well (BC).
[0118] After the cells were expanded to about 85% of the 48 wells, the cells were digested and counted, the ratios were calculated by referring to the cell numbers in the control well (BC), and the results were shown in
[0119] According to the results of
Example 3
[0120] Culture of Primary Laryngeal Cancer Cells Derived from Human Laryngeal Cancer Tissue
[0121] Laryngeal cancer epithelial cells originated from cancer tissue were isolated and obtained from the cancer tissue of a laryngeal cancer patient (Sample No. 0S0003) using the same method as in Example 1. Next, laryngeal cancer cells orginated from cancer tissue were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells. Then, the cells were inoculated in a 12-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells at a density of 4×10.sup.4 cells/cm.sup.2. 2 mL of the prepared culture medium for primary laryngeal cancer epithelial cells SCM was added to the 12-well plate, which was then placed in a 37° C. , 5% CO.sub.2 incubator (purchased from Thermo Fisher) for culture.
[0122]
Example 4
[0123] Effect of Different Culture Mediums on Promoting Proliferation of Laryngeal Cancer Tissue-Derived Primary Laryngeal Cancer Cells
[0124] (1) Comparison of the Influences of Different Culture Mediums on Clone Formation of Primary Cells and Proliferation Effects of Primary Cells
[0125] Culture medium for primary laryngeal cancer epithelial cell SCM was prepared in the same method of Example 2, and basic medium BM was prepared as control. As another control, a culture medium FM used in the cell conditional reprogramming technology was prepared additionally. For the preparation steps, please refer to Liu et al., Nat Protoc., 12(2): 439-451, 2017. The formulation of the culture medium is shown in Table 5. Simultaneously, as further controls, culture mediums for primary laryngeal cancer cell SCM-1 and SCM-2 were prepared, and the formulations of the culture mediums were obtained by replacing insulin-transferrin-selenium complex in medium SCM wth 1:50 volume ratio of B27 additive and 1:100 volume ratio of N2 additive, respectively. Moreover, as an additional control, a commercial medium Defined Keratinocyte SFM (hereinafter also referred to as “KSFM medium”) was purchased from Gibco, and the formulation of the culture medium is shown in Table 6.
TABLE-US-00006 TABLE 5 Composition of culture medium FM used in the cell conditional reprogramming technology Medium composition Supplier Final concentration DMEM medium Corning 65 vol. % Fetal bovine serum Gibico 10 vol. % Ham’s F12 Nutrient Mixture Gibico 25 vol. % Hydrocortisone Sigma-Aldrich 25 ng/ml Epidermal growth factor R&D 0.125 ng/ml Insulin Sigma-Aldrich 5 μg/ml Amphotericin B Sigma-Aldrich 250 ng/ml Gentamicin Gibico 10 μg/ml Cholera Toxin Sigma-Aldrich 0.1 nM Y27632 Enzo 10 μM
TABLE-US-00007 TABLE 6 Composition of commercial medium Defined Keratinocyte SFM (KSFM) Medium composition Supplier Final concentration Defined Keratinocyte- Gibco 99 vol. % SFM Basal Medium Defined Keratinocyte- Gibco 1 vol. % SFM Growth Supplement
[0126] By using the same method of Example 1, the primary laryngeal cancer cells (No. 0S0006) derived from laryngeal cancer tissues were obtained. Next, the cells were cultured at the same density (4×10.sup.4 cells/cm.sup.2) under the following 6 culture conditions:
[0127] A. The technology of the invention: the primary laryngeal cancer cells were inoculated into a 24-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells (purchased from ATCC) at a density of 4×10.sup.4 cells/cm.sup.2, cultured using 1 mL of the culture medium for primary laryngeal cancer epithelial cells SCM of the invention;
[0128] B. Cell conditional reprogramming technology: the primary laryngeal cancer cells were inoculated into a 24-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells (purchased from ATCC) at a density of 4×10.sup.4 cells/cm.sup.2, cultured in the 24-well plate using 1 mL of cell conditional reprogramming medium FM (the detailed steps, see Liu et al., Nat. Protoc., 12(2): 439-451, 2017);
[0129] C. The primary laryngeal cancer cells were inoculated into a 24-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells (purchased from ATCC) at a density of 4×10.sup.4 cells/cm.sup.2, and cultured in the 24-well plate using 1 mL of the culture medium SCM-1;
[0130] D. The primary laryngeal cancer cells were inoculated into a 24-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells (purchased from ATCC) at a density of 4×10.sup.4 cells/cm.sup.2, and cultured in the 24-well plate using 1 mL of the culture medium SCM-2;
[0131] E. The primary laryngeal cancer cells were inoculated into a 24-well plate at a density of 4×10.sup.4 cells/cm.sup.2, and cultured in the 24-well plate using 1 mL of the commercial medium KSFM;
[0132] F. The primary laryngeal cancer cells were inoculated into a 24-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells (purchased from ATCC) at a density of 4×10.sup.4 cells/cm.sup.2, and cultured in the 24-well plate using 2 mL of the basic medium BM.
[0133] In the above six cultures, the cells cultured under the six culture conditions wherein the mediums were renewed every 5 days. At the same time, the cell clone formation and the cell proliferation status under the cultivation of each medium in the 24-well plate was observed, and the cell growth status was recorded by taking pictures with a microscope (EVOS M500, Invitrogen).
[0134] To the primary cancer cells of laryngeal cancer (No. 0S0006) cultured with the technology of the invention, when the cells in the culture plate grew to cover about 80% of the bottom area, the medium supernatant in the 24-well plate was discarded and 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) was added for digestion for 1 minute; after removing 0.25% trypsin, 0.5 mL of 0.05% trypsin was added again for cell digestion, which was then incubated at 37° C. for 10 minutes until the cells were completely digested as observed under the microscope (EVOS M500, Invitrogen); then the digestion was terminated by using 1 ml of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL penicillin; the resultant was collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was discarded. The centrifuged cell sediments were resuspended in the culture medium of the invention and were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells, which was 317,000. The cells cultured under the other five culture conditions were digested and counted in the same operation process as above. The total number of cells cultured by using the mediums FM, SCM-1, SCM-2, KSFM and BM were 166,700, 266,100, 277,200, 95,600 and 35,900, respectively.
[0135]
[0136]
[0137] (2) Continuous Cultivation and Growth Curve of Primary Laryngeal Cancer Cells in Different Culture Mediums
[0138] The culture medium for primary laryngeal cancer epithelial cell SCM and the culture mediums FM, SCM-1, SCM-2, KSFM and BM as controls were obtained by using the same method as in (1) of this Example.
[0139] The laryngeal cancer tissue originated primary laryngeal cancer cells (No. 0S0004) were cultured in six culture mediums, and then digested, passaged and counted by using the same method as in (1) of this Example.
[0140] When the passaged cells grew in the culture plate to cover about 80% of the bottom area of the plate again, the cultured cells were digested, collected and counted according to the above operating method. The cells were inoculated at a density of 4×10.sup.4 cells/well again and cultured continuously.
[0141] The following is the formula for calculating the cell population doubling number of primary laryngeal cancer epithelial cells under different culture conditions:
[0142] Population Doubling (PD)=3.32*log.sub.10 (total number of digested cells/the number of cells at initial inoculation), see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.
[0143]
Example 5
[0144] Identification of Primary Laryngeal Cancer Cells Derived from Cancer Tissues
[0145] (1) Immunofluorescence Identification of Primary Laryngeal Cancer Tissues and Laryngeal Cancer Cells After Passage Culture
[0146] Laryngeal cancer epithelial cells originated from cancer tissue were isolated and obtained from the cancer tissue of a laryngeal cancer patient (Sample No. 0S0015) using the same method as in Example 1. Next, laryngeal cancer cells originated from cancer tissue were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells.
[0147] Then, the cells were inoculated in a 24-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells at a density of 4×10.sup.4cells/cm.sup.2. At the same time, round cell slides (purchased from Thermo Fisher) for immunofluorescence staining were pre-placed in the 24-well plate. 1 mL of the prepared culture medium for primary laryngeal cancer epithelial cells SCM was added to the 24-well plate, which was then placed in a 37° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher) for culture.
[0148] When the cells in the 24-well plate were expanded to cover 80% of the bottom area, the culture solution was discarded and 4% formaldehyde was used to fix the cells on ice for 30 minutes. The resultant was washed with PBS (purchased from Shanghai Sangon Biotech) for 5 minutes x 3 times. After discarding PBS, penetrating liquid was added, and the resultant was shaked (about 100 rpm) without exposure to light to break membrane for 30 minutes, and then was wash with PBS for 5 minutes×3 times. Thereafter, PBS +0.3% Triton X-100 (purchased from Shanghai Sangong Biotech) was used to prepare 5 vol. % BSA (purchased from Shanghai Sangong Biotech) solution for blocking, and the blocking was conducted at 37° C. for 30 minutes.
[0149] PBS+0.3% Triton X-100 was prepared in advance to dilute the antibody, wherein the squamous carcinoma specific antibody p63 (purchased from CST) was diluted in a ratio of 1:50. The blocking solution was discarded, and the prepared primary antibody diluent was added. The resultant was incubated in a refrigerator at 4° C. overnight. The resultant was taken out at 4° C., balanced to room temperature, continued for incubation at 37° C. for 1 hour, and then washed with PBS for 5 minutes×3 times.
[0150] PBS+0.3% Triton X-100 was prepared in advance for secondary antibody dilution, wherein the rabbit fluorescent secondary antibody (purchased from Thermo Fisher) with excitation light of 488 nm was diluted in a ratio of 1:1000. The resultant was incubated at room temperature in the dark for 1 hour, and then was washed with PBS for 5 minutes×3 times.
[0151] Non-specific fluorescent dye DAPI (purchased from Sigma) was diluted with PBS in volume ratio of 1:1000, which was used for staining at room temperature in the dark for 5 minutes. The resultant was washed with PBS for 5 minutes×3 times. Pictures were taken under microscope (EVOS M500, Invitrogen) for record
[0152]
[0153] (2) Immunohistochemical Identification of Primary Laryngeal Cancer Tissues and Laryngeal Cancer Cells After Passage Culture
[0154] Cancer tissue (Sample No. 0S0001) about the size of a mung bean was taken from a surgical resection sample of a laryngeal cancer patient, and immersed in 1 mL of 4% paraformaldehyde. Laryngeal cancer epithelial cells (Sample No. 0S0001) were obtained from the remaining cancer tissue in the same method of Example 1. Sample 0S0001 was continuously cultured to the sixth passage using the culture medium SCM of the invention according to the method of Example 3.
[0155] Immunohistochemistry assay was used to detect the expression of important laryngeal cancer-related biomarkers in the original tissue of Sample 0S0001 and the primary cells obtained by continuous culture to the sixth passage. The tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into tissue sections of 4 μm thickness with a microtome. Routine immunohistochemical detection was then performed (for detailed steps, see Li et al., Nature Communication, (2018) 9: 2983). The primary antibodies used were Cytokeratin (pCK) (purchased from CST), p63 antibody (purchased from CST), and Ki67 antibody (purchased from R&D).
[0156]
Example 6
[0157] Xenograft Tumorigenesis Experiments of Cancer Tissue-Derived Primary Laryngeal Cancer Cells in Mice
[0158] Laryngeal cancer cells (No. 0S0003) were isolated and obtained from the cancer tissues of one pathologically diagnosed laryngeal cancer patient by using the same method as in Example 1. 0S0003 was cultured using the culture medium SCM of the invention according to the method of Example 3, and when the number of laryngeal cancer cells reached 1 ×10.sup.7, the laryngeal cancer cells were digested and collected by using the same method of Example 4. The culture medium for laryngeal cancer cells of the invention SCM and Matrigel® (purchased from BD Biosciences) were mixed at a ratio of 1:1, and 100 μL of the culture medium mixed with Matrigel was used to resuspend 5×10.sup.6 laryngeal cancer cells, and the resultant was injected into the laryngeal cancer fat pad and the axilla of the right forelimb of 6-week-old female highly immunodeficient mice (NCG) (purchased from Nanjing Model Animal Research Center), respectively. The volume and growth rate of tumors in mice generated from the laryngeal cancer cells were observed and photographed every three days.
[0159] Tumor formation can be observed in both of the two tumor cell inoculation sites of the mice on day 21 after tumor cell inoculation. From day 21 to day 40, the tumor proliferation in mice was obvious. This indicates that the cancer tissue-derived laryngeal cancer cells cultured by the culture method of the invention have tumorigenicity in mice.
Example 7
[0160] Drug sensitivity Functional Test of Cancer Tissue-Derived Laryngeal Cancer Cells
[0161] Taking a surgical resection sample from a laryngeal cancer patient as an example, it is verified that the laryngeal cancer cells cultured from the patient-derived laryngeal tumor samples can be used to test the sensitivity of the tumor cells of the patient to different drugs.
[0162] 1. Plating of primary laryngeal cancer cells: Cell suspensions of isolated laryngeal cancer cells (No. 0S0020 and No. 0S0022) obtained according to the method of Example 1, were inoculated in a 12-well plate which was pre-laid with γ-ray irradiated NIH-3T3 cells at a density of 4×10.sup.4 cells/cm.sup.2. 2 mL of the prepared culture medium for primary laryngeal cancer epithelial cells SCM was added to the 12-well plate, which was then placed in a 37° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher) for culture. When the cells in the culture plate grew to cover about 80% of the bottom area, the medium supernatant in the 12-well plate was discarded and 0.5 mL of 0.25% trypsin (purchased from Thermo Fisher) was added for digestion for 1 minute; after removing 0.25% trypsin, 0.5 mL of 0.05% trypsin was added again for cell digestion; the cells were incubated at 37° C. for 10 minutes until the cells were completely digested as observed under the microscope (EVOS M500, Invitrogen); then the digestion was terminated by using 1 mL of DMEM/F12 culture solution containing 5% (v/v) fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL penicillin; the resultant was collected into a 15 mL centrifuge tube and centrifuged at 1500 rpm for 4 minutes, and then the supernatant was discarded. The centrifuged cell sediments were resuspended in the culture medium SCM and were counted with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.) to obtain the total number of cells, which were 830,000 and 768,000, respectively. The cells were incubated into a 384-well plate at a density of 2,000-4,000 cells/well, and the cells were adhered overnight.
[0163] 2. Drug Gradient Experiments:
[0164] (1) The drug storage plates were prepared by gradient dilution method: 40 μL of 10 μM drug stock solutions to be tested were respectively taken as the maximum concentrations, and 10 μL of solutions were respectively pipetted therefrom and added into 0.5 mL EP tubes containing 20 μL of DMSO; 10 μL of solutions from the above EP tubes were pipetted again into second 0.5 mL EP tubes loaded with 20 μL of DMSO, that is, diluting the drugs in a ratio of 1:3. The above method was repeated for gradually dilution and 7 concentrations required for dosing were obtained. Different concentrations of drugs were added to the 384-well drug storage plates. Equal volume of DMSO was added to each well of the solvent control group as a control. In this example, the drugs to be tested were Bortezomib (purchased from MCE), Disulfiram (purchased from MCE), Gefitinib (purchased from MCE), and Erlotinib (purchased from MCE).
[0165] (2) Using a high-throughput automated workstation (JANUS, Perkin Elmer), different concentrations of drugs and solvent controls in the 384-well drug storage plates were added to 384-well cell culture plates plated with the laryngeal cancer cells. The drug groups and the solvent control groups were each arranged with 3 duplicate wells. The volume of drugs added to each well was 100 nL.
[0166] (3) Test of cell viability: 72 hours after administration, Cell Titer-Glo assay kit (purchased from Promega) was used to detect the chemiluminescence value of the cultured cells after drug administration. The magnitude of the chemiluminescence value reflects the cell viability and the effect of the drug on the cell viability. 10 μL of the prepared Cell Titer-Glo detection solution was added to each well, and a microplate reader (Envision, Perkin Elmer) was used to detect the chemiluminescence value after mixing.
[0167] (4) Test of cell viability: According to the formula, Cell survival rate (%)=Chemiluminescence value of drug well/Chemiluminescence value of control well*100%, the cell survival rates of cells treated with different drugs were calculated. Graphs were made by using Graphpad Prism software and the half-inhibition rates IC.sub.50 were calculated, and simultaneously, the cell survival rates under the maximum blood concentrations C.sub.max of different drugs in human body were calculated.
[0168] (5) The drug sensitivity testing results were shown in
INDUSTRIAL APPLICABILITY
[0169] The invention provides a culture medium and a culture method for culturing or expanding primary laryngeal cancer epithelial cells in vitro. The cell progeny and organoid cultured using the culture medium and culture method of the invention can be used for the efficacy evaluation and screening of drugs, toxicity determination, and regenerative medicine. Therefore, the invention is applicable in the industry.
[0170] Although the present invention has been described in details herein, the present invention is not limited thereto, and those skilled in the art can make modification according to the principle of the invention. Therefore, all modifications made according to the principle of the invention should be interpreted as falling within the protection scope of the invention.