CULTURE MEDIUM FOR ESOPHAGEAL SQUAMOUS CELL CARCINOMA EPITHELIAL CELLS, CULTURE METHOD, AND APPLICATION THEREOF
20230235283 · 2023-07-27
Assignee
Inventors
- Qingsong LIU (Hefei, Anhui, CN)
- Jie HU (Hefei, Anhui, CN)
- Wenchao WANG (Hefei, Anhui, CN)
- Cheng CHEN (Hefei, Anhui, CN)
- Tao REN (Hefei, Anhui, CN)
- Li WANG (Hefei, Anhui, CN)
Cpc classification
C12N5/0625
CHEMISTRY; METALLURGY
C12N2533/90
CHEMISTRY; METALLURGY
C40B30/06
CHEMISTRY; METALLURGY
C12N2501/115
CHEMISTRY; METALLURGY
International classification
Abstract
Provided are a primary cell culture medium that contains a combination of an MST1/2 kinase inhibitor and a ROCK kinase inhibitor and is used for culturing primary esophageal squamous cell carcinoma epithelial cells, and a culture method using the primay cell culture medium. In the culture method, the primary cell culture medium is used to culture primay cells on a culture vessel coated with an extracellular matrix glue, so that the primary cells prolilferate rapidly. A cell model obtained by using the primary cell culture medium and the primary cell culture method of the present invention can be used for the efficacy evaluation and screening of drugs.
Claims
1. A primary cell culture medium for culturing primary esophageal squamous cell carcinoma (ESCC) epithelial cells, characterized in that: comprising an MST1/2 kinase inhibitor and a ROCK kinase inhibitor, wherein the MST1/2 kinase inhibitor comprises a compound of Formula (I) or a pharmaceutically acceptable salt, or a solvate thereof, and the ROCK kinase inhibitor is at least one selected from the group consisting of Y27632, Fasudil, and H-1152, ##STR00079## 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; 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; 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, ##STR00080## 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; 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; R.sub.6 is preferably fluoro, methyl or trifluoromethyl.
5. The primary cell culture medium of claim 1, wherein the MST1/2 kinase inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt thereof: ##STR00081## ##STR00082## ##STR00083## ##STR00084## ##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089## ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## ##STR00095## ##STR00096##
6. The primary cell culture medium of claim 1, characterized in that: the amount of the MST1/2 kinase inhibitor is 0.3 μM-10 μM, preferably 0.75 μM-6 μM, more preferably 2 μM-6 μM.
7. The primary cell culture medium of claim 1, characterized in that: the amount of the ROCK kinase inhibitor in the culture medium is 0.3 μM-20 μM, preferably 2.5 μM-15 μM, more preferably 7.5 μM-12.5 μM.
8. The primary cell culture medium of claim 1, characterized in that: further containing one or more of the following factors: epidermal growth factor; insulin-transferrin-selenium complex; B27 additive or N2 additive; hepatocyte growth factor; a TGFβ type I receptor inhibitor selected from at least one of A83-01, SB431542, Repsox, SB505124, SB525334, SD208, LY36494, and SJN2511; and a P38/MAPK inhibitor selected from at least one of SB202190, SB203580, VX-702, VX-745, PD169316, RO4402247, and BIRB796.
9. The primary cell culture medium of claim 8, characterized in that: the amount of epidermal growth factor is 12.5 ng/ml-100 ng/ml, more preferably 50 ng/ml-100 ng/ml; the respective amounts of insulin/transferrin/sodium selenite in the insulin-transferrin-selenium complex are 2.5-20 μg/ml, 1.25-10 μg/ml, 1.25-10 ng/ml, respectively, and more preferably 5-20 μg/ml, 2.5-10 μg/ml, 2.5-10 ng/ml, respectively; the volume concentration of the B27 additive or N2 additive is 1:25-1:200, more preferably 1:25-1:50; the amount of hepatocyte growth factor is 2.5 ng/ml-20 ng/ml, more preferably 10 ng/ml-20 ng/ml; the amount of the TGFβ type I receptor inhibitor is 125 nM-500 nM, preferably 250 nM-500 nM; and the amount of the P38/MAPK inhibitor is 125 nM-500 nM, preferably 250 nM-500 nM.
10. The primary cell culture medium of claim 1, characterized in that: being free of serum, bovine pituitary extract, Wnt agonists, R-spondin family proteins, BMP inhibitors, nicotinamide, or N-acetylcysteine.
11. The primary cell culture medium of claim 1, characterized in that: the primary ESCC epithelial cells are selected from ESCC tumor cells, normal ESCC epithelial cells, and ESCC epithelial stem cells.
12. A method for culturing primary ESCC epithelial cells, characterized in comprising the following steps: (1) preparing the primary cell culture medium according to claim 1; (2) coating a culture vessel with extracellular matrix gel diluent; (3) inoculating primary ESCC epithelial cells isolated from ESCC tissues in the coated culture vessel, and culturing by using the primary cell culture medium of step (1).
13. A method for evaluating or screening a drug for treating ESCC diseases, characterized in that, comprising the following steps: (1) culturing the ESCC epithelial cells by the culturing method of claim 12; (2) selecting the drug to be tested and diluting into different drug concentration gradients; (3) adding the drug which has diluted to gradients to the ESCC epithelial cells obtained in step (1), and detecting the cell viability.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
[0072] 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 epithelial cells derived from esophagus.
[0073] In addition, in this specification, “epithelial tumor cells” refer to the cells obtained by tumorigenesis of cells originated from the aforementioned epithelial tissues.
[0074] 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.
[0075] [Preparation Examples of MST1/2 Kinase Inhibitors]
[0076] 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.
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
[0077] ##STR00019##
[0078] 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.
[0079] 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.
[0080] 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.
[0081] 2-chloro-7-(2,6-difluorophenyl)-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.
[0082] 4-((7-(2,6-difluorophenyl)-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.
2. Preparation of Other MST1/2 Inhibitor Compounds of the Invention
[0083] 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) m/z No. Compound (M + 1)+ 1
Example 1
[0084] Isolation of Human Primary ESCC Epithelial Cells
[0085] ESCC tissue samples were obtained from the cancer tissues of three informed and consenting ESCC cancer patients by surgical resection, namely Sample Nos. 0F0060, 0F0061, and 0F0062. One of the samples (No. 0F0060) will be described below.
[0086] 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
[0087] In the biological safety cabinet, the tissue sample (No. 0F0060) 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.
[0088] 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.
[0089] 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.
[0090] 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 ESCC 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 2,280,000, which was obtained by counting with a flow imaging counter (JIMBIO FIL, Jiangsu Jimbio Technology Co., Ltd.).
[0091] Other two ESCC tumor tissue samples were isolated according to the same method above, and the total number of cells were 1,470,000 (0F0061) and 2,680,000 (0F0062), respectively.
Example 2
[0092] Optimization of Culture Medium for Primary ESCC Epithelial Cell
[0093] (1) Effects of Different Factors
[0094] Extracellular matrix gel (Matrigel®) (manufactured by BD Biosciences) was diluted at 1:100 in serum-free DMEM/F12 medium to prepare an extracellular matrix dilution, which was added to a 48-well culture plate with 500 μl/well to completely cover the bottom of the wells of the culture plate. The resultant was let stand for 1 hour in a 37° C. incubator. After 1 hour, the extracellular matrix dilution was removed to provide a Matrigel-coated plate.
[0095] 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.
[0096] Next, different kinds and concentrations of additive factors (Table 3) were added to the basic medium (BM), so as to prepare culture mediums for ESCC 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 BM + 50, 25, 12.5, 7.5 ng/ml EGF growth factor (EGF) Biological BM + hepatocyte Sino BM + 20, 10, 5, 2.5 ng/ml HGF growth factor (HGF) Biological BM + insulin-like Sino BM + 20, 10, 5, 2.5 ng/ml IGF-1 growth factor 1 Biological (IGF-1) BM + fibroblast Sino BM + 20, 10, 5, 2.5 ng/ml FGF growth factor (FGF) Biological BM + N2 additive Gibco BM + 1/50, 1/100, 1/200, 1/400 diluting ratio of N2 BM + B27 additive Gibco BM + 1/25, 1/50, 1/100, 1/200 diluting ratio of B27 BM + insulin- Insulin, BM + 1/25, 1/50, 1/100, 1/200 transferrin-selenium transferrin, diluting ratio of insulin- complex (ITS) sodium transferrin-selenium complex selenite all stock solution (the stock purchased solution contains 500 μg/ml from Sigma insulin, 250 μg/ml transferrin, and 250 ng/ml sodium selenite in DMEM/F-12) BM + Y27632 MCE BM + 20, 10, 5, 2.5 μM Y27632 BM + Compound 1 Preparation BM + 6, 3, 1.5, 0.75 μM Example Compound 1 BM + CHIR99021 MCE BM + 20, 10, 5, 2.5 μM CHIR99021 BM + DMH-1 MCE BM + 6, 3, 1.5, 0.75 μM DMH-1 BM + A83-01 MCE BM + 1000, 500, 250, 125 nM A83-01 BM + SB202190 MCE BM + 1000, 500, 250, 125 nM SB202190 BM + Noggin Sino BM + 200, 100, 50, 25 ng/ml Biological Noggin
[0097] The culture mediums with different components were added at a volume of 500 μl/well to 48-well plates which were coated with extracellular matrix gel (Matrigel). ESCC tumor cells (No. 0F0064) isolated from ESCC tissue according to the same method described in Example 1 were inoculated in a Matrigel-coated 48-well culture plate at a cell density of 2×10.sup.4 cells/cm.sup.2. 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 ESCC tumor cells (No. 0F0064) were cultured under different medium formulations. The culture mediums were renewed every 4 days after the start of the culture. After 12 days of culture, cell counts were performed. As an experiment control, the basis medium (BM) without any additive was used. The results were shown in
[0098] (2) Effects of Combination of Y27632 and Compound 1
[0099] Extracellular matrix gel (Matrigel®) (purchased from BD Biosciences) was diluted at 1:100 in serum-free DMEM/F12 medium to prepare an extracellular matrix dilution, which was added to a 48-well culture plate with 500 μl/well to completely cover the bottom of the wells of the culture plate. The resultant was let stand for 1 hour in a 37° C. incubator. After 1 hour, the extracellular matrix dilution was removed to provide a Matrigel-coated plate.
[0100] Different concentrations of Y27632 and Compound 1 (Table 4) were added to the basic medium BM, so as to prepare culture mediums for ESCC epithelial cells containing different additive components.
TABLE-US-00005 TABLE 4 Preparation of culture mediums containing different components (final concentrations are shown) Medium NO. Composition NO. 1 BM + 1 μM Compound 1 + 1 μM Y27632 NO. 2 BM + 1 μM Compound 1 + 3 μM Y27632 NO. 3 BM + 1 μM Compound 1 + 10 μM Y27632 NO. 4 BM + 2 μM Compound 1 + 1 μM Y27632 NO. 5 BM + 2 μM Compound 1 + 3 μM Y27632 NO. 6 BM + 2 μM Compound 1 + 10 μM Y27632 NO. 7 BM + 3 μM Compound 1 + 1 μM Y27632 NO. 8 BM + 3 μM Compound 1 + 3 μM Y27632 NO. 9 BM + 3 μM Compound 1 + 10 μM Y27632 NO. 10 BM + 4 μM Compound 1 + 1 μM Y27632 NO. 11 BM + 4 μM Compound 1 + 3 μM Y27632 NO. 12 BM + 4 μM Compound 1 + 10 μM Y27632 NO. 13 BM + 6 μM Compound 1 + 1 μM Y27632 NO. 14 BM + 6 μM Compound 1 + 3 μM Y27632 NO. 15 BM + 6 μM Compound 1 + 10 μM Y27632 NO. 16 BM + 10 μM Compound 1 + 1 μM Y27632 NO. 17 BM + 10 μM Compound 1 + 3 μM Y27632 NO. 18 BM + 10 μM Compound 1 + 10 μM Y27632
[0101] The culture mediums with different components were added at a volume of 500 μl/well to 48-well plates which were coated with extracellular matrix gel (Matrigel). ESCC tumor cells (No. 0F0063) isolated from ESCC tissue according to the method described in Example 1 were inoculated in a Matrigel-coated 48-well culture plate at a cell density of 2×10.sup.4 cells/cm.sup.2. 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 ESCC tumor cells (No. 0F0063) were cultured under different medium formulations. The culture mediums were renewed every 4 days after the start of the culture. After 12 days of culture, cell counts were performed. As an experiment control, the basis medium BM was used. The results were shown in
[0102] (3) Effects of Increasing Factors in the Culture Mediums on the Proliferation of Primary ESCC Cells Obtained by the Method of the Invention
[0103] Extracellular matrix gel (Matrigel®) (purchased from BD Biosciences) was diluted at 1:100 in serum-free DMEM/F12 medium to prepare an extracellular matrix dilution, which was added to a 48-well culture plate with 500 μl/well to completely cover the bottom of the wells of the culture plate. The resultant was let stand for 1 hour in a 37° C. incubator. After 1 hour, the extracellular matrix dilution was removed to provide a Matrigel-coated plate.
[0104] Different small molecules, additives and growth factors (Table 5) were sequentially added to the basic medium BM, respectively, so as to prepare culture mediums for ESCC epithelial cells containing different additive components.
TABLE-US-00006 TABLE 5 Preparation of culture mediums containing different components (final concentrations are shown) Medium NO. Composition NO. 1 BM + 3 μM Compound 1 NO. 2 NO. 1 + 10 μM Y27632 NO. 3 NO. 2 + 500 nM A83-01 NO. 4 NO. 3 + 500 nM SB202190 NO. 5 NO. 4 + 1:50 B27 additive NO. 6 NO. 5 + 10 ng/mL HGF NO. 7 NO. 6 + 50 ng/mL EGF NO. 8 NO. 7 + 1:50 insulin-transferrin-selenium complex stock solution (the stock solution contains 500 μg/ml insulin, 250 μg/ml transferrin, and 250 ng/ml sodium selenite in DMEM/F-12) NO. 9 NO. 8 + 100 ng/mL Noggin NO. 10 NO. 9 + 10 ng/mL FGF NO. 11 NO. 10 + 10 ng/m IGF-1 NO. 12 NO. 11 + 10 ng/mL FGF10 (purchased from Sino Biological) NO. 13 NO. 12 + 7 μg/mL bovine pituitary extract (purchased from M&C Gene Technology) NO. 14 NO. 13 + 0.4 μg/mL hydrocortisone (purchased from Sigma) NO. 15 NO. 14 + 400 μM non-essential amino acids (purchased from Gibco) NO. 16 NO. 15 + 1 mM glutamine (purchased from Gibco) NO. 17 NO. 16 + 10 mM HEPES balanced solution (purchased from Gibco) NO. 18 NO. 17 + 100 ng/mL RSPO1 (purchased from Sino Biological) NO. 19 NO. 18 + 5% fetal bovine serum (volume ratio) (purchased from ExCell Bio)
[0105] The culture mediums with different components were added at a volume of 500 μl/well to 48-well plates which were coated with extracellular matrix gel (Matrigel), and simultaneously, the BM medium was used as the experiment control. ESCC tumor cells (No. 0F0065) isolated from ESCC tissue according to the method described in Example 1 were inoculated in a Matrigel-coated 48-well culture plate at a cell density of 2×10.sup.4 cells/cm.sup.2. 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 ESCC tumor cells (No. 0F0065) were cultured under different medium formulations. After 10 days of culture, cell counts were performed. The results were shown in
[0106] (4) Effects of Different Concentrations of the Additive Factors on the Proliferation of Primary ESCC Cells Obtained in the Invention
[0107] Extracellular matrix gel (Matrigel®) (manufactured by BD Biosciences) was diluted at 1:100 in serum-free DMEM/F12 medium to prepare an extracellular matrix dilution, which was added to a 48-well culture plate with 200 μl/well to completely cover the bottom of the wells of the culture plate. The resultant was let stand for 1 hour in a 37° C. incubator. After 1 hour, the extracellular matrix dilution was removed to provide a Matrigel-coated plate.
[0108] Preparation of the culture medium for primary ESCC epithelial cells of the invention (abbreviated as FEM): to the basic medium (BM), was added epidermal growth factor EGF at the final concentration of 50 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 FEM medium), B27 additive at the diluting ratio of 1:50, Compound 1 at the final concentration of 3 μM, Y27632 at the final concentration of 10 μM, TGFβ1 inhibitor A83-01 at the final concentration of 500 nM, and P38/MAPK inhibitor SB202190 at the final concentration of 500 nM, to prepare the culture medium for primary ESCC epithelial cells.
[0109] ESCC epithelial cells originated from cancer tissue were isolated and obtained from the cancer tissue of an ESCC patient (Sample No. 0F0065) using the same method as in Example 1. Next, ESCC tumor 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 Matrigel® (purchased from BD Biosciences)-coated 12-well plate at a density of 4×10.sup.4 cells/cm.sup.2. 2 mL of the prepared culture medium for primary ESCC epithelial cells FEM 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 500 μL 0.05% trypsin (purchased from Gibco) was added 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 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.
[0110] Next, the following 8 culture mediums with different formulations were prepared for experiments:
[0111] Formulation 1: FEM medium composition without B27 additive;
[0112] Formulation 2: FEM medium composition without insulin-transferrin-selenium complex;
[0113] Formulation 3: FEM medium composition without epidermal growth factor;
[0114] Formulation 4: FEM medium composition without hepatocyte growth factor;
[0115] Formulation 5: FEM medium composition without Y27632;
[0116] Formulation 6: FEM medium composition without Compound 1;
[0117] Formulation 7: FEM medium composition without A83-01;
[0118] Formulation 8: FEM medium composition without SB202190.
[0119] The digested cell suspension was diluted with the above Formulations 1-8 respectively, and were seeded into a 48-well plate with 10,000 cells and 250 μL volume per well.
[0120] When using the medium of Formulation 1, to a 48-well plate inoculated with primary cells was added the prepared B27 additive, with 250 μL per well, such that the final concentrations of B27 additive were 1:200, 1:100, 1:50 and 1:25, respectively; the medium of Formulation 1 was used as the control well (BC).
[0121] 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 1:200, 1:100, 1:50 and 1:25, respectively (corresponding to the final concentrations of insulin/transferrin/sodium selenite of 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).
[0122] When using the medium of Formulation 3, to a 48-well plate inoculated with primary cells was added the prepared epidermal growth factor, with 250 μL per well, such that the final concentrations of epidermal growth factor were 100 ng/mL, 50 ng/mL, 25 ng/mL, and 12.5 ng/mL, respectively; the medium of Formulation 3 was used as the control well (BC).
[0123] 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 20 ng/mL, 10 ng/mL, 5 ng/mL, and 2.5 ng/mL, respectively; the medium of Formulation 4 was used as the control well (BC).
[0124] 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 20 μM, 15 μM, 12.5 μM, 10 μM, 7.5 μM, 5 μM, and 2.5 μM, respectively; the medium of Formulation 5 was used as the control well (BC).
[0125] 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 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1.5 μM, and 0.75 μM, respectively; the medium of Formulation 6 was used as the control well (BC).
[0126] When using the medium of Formulation 7, to a 48-well plate inoculated with primary cells was added the prepared A83-01, with 250 μL per well, such that the final concentrations of A83-01 were 1000 nM, 500 nM, 250 nM, and 125 nM, respectively; the medium of Formulation 7 was used as the control well (BC).
[0127] When using the medium of Formulation 8, to a 48-well plate inoculated with primary cells was added the prepared SB202190, with 250 μL per well, such that the final concentrations of SB202190 were 1000 nM, 500 nM, 250 nM, and 125 nM, respectively; the medium of Formulation 8 was used as the control well (BC).
[0128] 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
[0129] According to the results of
Example 3
[0130] Culture of Human Primary ESCC Epithelial Cells
[0131] (1) Culture of Primary ESCC Tumor Cells Derived from ESCC Tissue
[0132] ESCC epithelial cells originated from cancer tissue were isolated and obtained from the cancer tissue of an ESCC patient (Sample No. 0F0062) using the same method as in Example 1. Next, ESCC tumor 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 Matrigel® (purchased from BD Biosciences)-coated 12-well plate at a density of 4×10.sup.4 cells/cm.sup.2. 2 mL of the prepared culture medium for primary ESCC epithelial cells FEM 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.
[0133]
[0134] (2) Comparison of the Culture Results of Primary ESCC Tumor Cells Derived from ESCC Tissue Under the Conditions of with and without Coating of Extracellular Matrix Gel
[0135] The primary ESCC tumor cells (No. 0F0065) derived from cancer tissues were isolated from the cancer tissues of one ESCC patient, using the same method of Example 1. Next, equal numbers (4×10.sup.4 cells/cm.sup.2) of the primary ESCC tumor cells 0F0065 were inoculated into a Matrigel® (purchased from BD Biosciences)-coated 12-well plate and a 12-well plate without any treatment, respectively. 2 mL of the prepared culture medium for primary ESCC epithelial cells FEM was added to the 12-well plates, which were then placed in a 37° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher) for culture after disinfection.
Example 4
[0136] Effect of Different Culture Mediums on Promoting Proliferation of ESCC Tissue-Derived Primary ESCC Tumor Cells
[0137] (1) Comparison of the Influences of Different Culture Mediums on Clone Formation of Primary Cells and Proliferation Effects of Primary Cells
[0138] Culture medium for primary ESCC epithelial cell FEM was prepared in the same method of Example 2, and basic medium BM was prepared as control. As another control, a culture medium for cell conditional reprogramming technology FM 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 6. Simultaneously, as a further control, a culture medium for primary esophageal carcinoma cells KM was prepared. For the preparation steps, see Karin J. Purdie et al., Cancer Cell Culture: Methods and Protocols, Second Edition, Methods in Molecular Biology, vol. 731, 151-159, 2011. The formulation of the culture medium is shown in Table 7. Moreover, as an additional control, a commercial medium EpiCult™ Plus Medium (hereinafter also referred to as “EPI medium”) was purchased from Stemcell, and the formulation of the culture medium is shown in Table 8.
TABLE-US-00007 TABLE 6 Composition of culture medium for cell conditional reprogramming technology FM 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-00008 TABLE 7 Composition of culture medium KM (Keratinocyte medium) Final Medium composition Supplier concentration Epidermal growth factor Sino Biological 10 ng/ml Liothyronine Triiodothyronin MCE 13 ng/ml Transferrin Sino Biological 5 μg/ml Cholera Toxin Sigma-Aldrich 8.4 ng/ml Hydrocortisone Sigma-Aldrich 0.4 μg/ml Insulin Sigma-Aldrich 5 μg/ml Fetal bovine serum Gibico 10 vol. % DMEM medium Corning 65 vol. % Ham's F12 Nutrient Mixture Gibico 25 vol. %
TABLE-US-00009 TABLE 8 Composition of commercial medium EpiCult ™ Plus Medium (EPI) Medium composition Supplier Final concentration EpiCult ™ Plus Basal Medium Stemcell 98 vol. % EpiCult ™ Plus Supplement Stemcell 2 vol. % Hydrocortisone Stemcell 480 ng/ml
[0139] By using the same method of Example 1, the primary ESCC tumor cells (No. 0F0060) derived from ESCC tissues were obtained. Next, the cells were cultured at the same density (4×10.sup.4 cells/cm.sup.2) under the following five culture conditions:
[0140] A. The technology of the invention: the primary ESCC tumor cells were inoculated into a 24-well plate coated with Matrigel® (manufactured by BD Biosciences) at a density of 4×10.sup.4 cells/cm.sup.2, cultured using 2 mL of the culture medium for primary ESCC epithelial cells FEM of the invention;
[0141] B. Cell conditional reprogramming technology: the primary ESCC tumor cells were inoculated onto γ-ray irradiated mouse fibroblast cell line J2 cells (purchased from Kerafast) at a density of 4×10.sup.4 cells/cm.sup.2, cultured in a 24-well plate using the cell conditional reprogramming medium FM (the detailed steps, see Liu et al., Am J Pathol, 183(6): 1862-1870, 2013);
[0142] C. The primary ESCC tumor cells were inoculated into a Matrigel® (manufactured by BD Biosciences)-coated 24-well plate at a density of 4×10.sup.4 cells/cm.sup.2, and cultured in the 24-well plate using 2 mL of the culture medium KM;
[0143] D. The primary ESCC tumor cells were inoculated into a Matrigel® (manufactured by BD Biosciences)-coated 24-well plate at a density of 4×10.sup.4 cells/cm.sup.2, and cultured in the 24-well plate using 2 mL of the commercial medium EPI;
[0144] E. The primary ESCC tumor cells were inoculated into a Matrigel® (manufactured by BD Biosciences)-coated 24-well plate 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.
[0145] In the above five cultures, the cells cultured under the five culture conditions wherein the mediums were renewed every 4 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).
[0146] To the primary ESCC cancer cells (No. 0F0060) 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 500 μL 0.05% trypsin (purchased from Gibco) was added 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 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 364,000. The cells cultured under the other four culture conditions were digested and counted in the same operation process as above. The total number of cells cultured by using the mediums FM, KM, EPI and BM were 315000, 91000, 98000 and 84000, respectively.
[0147] The photos of cells in
[0148]
[0149] (2) Continuous Cultivation and Growth Curve of Primary ESCC Tumor Cells in Different Culture Mediums
[0150] The culture medium for primary ESCC epithelial cell FEM and the culture mediums BM, KM, FM, and EPI as controls were obtained by using the same method as in (1) of this Example.
[0151] The ESCC tissue originated primary ESCC tumor cells (No. 0F0075) were cultured in the following five culture mediums, and then digested, passaged and counted by using the same method as in (1) of this Example.
[0152] 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.
[0153] The following is the formula for calculating the cell population doubling number of primary ESCC epithelial cells under different culture conditions:
[0154] 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.
[0155]
[0156] The photos of cells in
Example 5
[0157] Identification of Primary ESCC Tumor Cells Derived from Cancer Tissues
[0158] (1) Immunohistochemical Identification of Primary ESCC Tissues and ESCC Cells after Passage Culture
[0159] Cancer tissue (Sample No. 0F0053) about the size of a mung bean was taken from a surgical resection sample of an ESCC patient, and immersed in 1 mL of 4% paraformaldehyde. ESCC epithelial cells (Sample No. 0F0053) were obtained from the remaining cancer tissue in the same method of Example 1. Sample 0F0053 was continuously cultured to the sixth passage using the culture medium FEM of the invention according to the method of Example 3.
[0160] Immunohistochemistry assay was used to detect the expression of important ESCC-related biomarkers in the original tissue of Sample 0F0053 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 (CK) (purchased from CST), p63 antibody (purchased from CST), p53 antibody (purchased from CST), and Ki67 antibody (purchased from R&D).
[0161]
[0162] (2) Copy Number Variation Analysis of ESCC Tissues and ESCC Cells Digested and Cultured from Cancer Tissues
[0163] The ESCC tumor cells (No. 0F0025) were continuously cultured using the culture medium FEM of the invention according to the method of Example 3. For the ESCC tumor cells that were cultured and passaged in vitro for the first, second and third times (P1, P2 and P3, respectively) and the cancer tissues directly obtained from ESCC patients by surgery, the genomic DNA of the cells was extracted using the DNeasy blood & tissue Kit (manufactured by QIAGEN). Normal esophageal tissues from the same patient were collected, and the genomic DNA was extracted by the same method, which was used as background control. Next, whole exome sequencing was performed on the genomic DNA of cells and tissue samples (see Hans Clevers et al., Cell, 11; 172 (1-2): 373-386, 2018 for specific operation steps), and the sequencing results were compared with the reference genome. CNVkit software was used to analyze the comparison results. CBS segmentation (circular binary segmentation algorithm) was used to analyze the gene copy number of the whole genome, features of regions with similar position and copy number changes were extracted by rolling median software, and the results were plotted according to the chromosome order. It is confirmed from
Example 6
[0164] Effects of Removing Single Factor or Combination of Factors from Culture Medium on Continuous Expansion of Primary ESCC Cells
[0165] The culture medium for primary ESCC epithelial cell FEM was prepared in the same method of Example 2. As a control, a basic medium (hereinafter also referred to as “BM”) was prepared using the same method of Example 2. In addition, six other different culture mediums were prepared according to Table 9.
TABLE-US-00010 TABLE 9 Culture mediums with different compositions (final concentrations are shown) Culture medium Composition FEM without DMEM/F12 + 100 μg/mL Primocin + 1:50 B27 Compound 1 additive + 50 ng/ml EGF + 10 ng/ml HGF + 1/50 diluting ratio ITS complex + 10 μM Y27632 + 500 nM A83-01 + 500 nM SB202190 FEM without DMEM/F12 + 100 μg/mL Primocin + 1:50 B27 Y27632 additive + 50 ng/ml EGF + 10 ng/ml HGF + 1/50 diluting ratio ITS complex + 3 μM Compound 1 + 500 nM A83-01 + 500 nM SB202190 FEM without DMEM/F12 + 100 μg/mL Primocin + 1:50 B27 Compound 1 additive + 50 ng/ml EGF + 10 ng/ml HGF + 1/50 and Y27632 diluting ratio ITS complex + 500 nM A83-01 + 500 nM SB202190 FEM without DMEM/F12 + 100 μg/mL Primocin + 1:50 B27 A83-01 additive + 50 ng/ml EGF + 10 ng/ml HGF + 1/50 diluting ratio ITS complex + 3 μM Compound 1 + 10 μM Y27632 + 500 nM SB202190 FEM without DMEM/F12 + 100 μg/mL Primocin + 1:50 B27 SB202190 additive + 50 ng/ml EGF + 10 ng/ml HGF + 1/50 diluting ratio ITS complex + 3 μM Compound 1 + 10 μM Y27632 + 500 nM A83-01 FEM without DMEM/F12 + 100 μg/mL Primocin + 1:50 B27 A83-01 and additive + 50 ng/ml EGF + 10 ng/ml HGF + 1/50 SB202190 diluting ratio ITS complex + 3 μM Compound 1 + 10 μM Y27632
[0166] ESCC tissue-originated primary ESCC tumor cells (No. 0F0061) were obtained by using the same method as in Example 1. Then, the primary ESCC tumor cells were inoculated in a Matrigel® (manufactured by BD Biosciences)-coated 24-well plate at a density of 4×10.sup.4 cells/cm.sup.2. 1 mL of the culture medium for primary ESCC epithelial cells (FEM) of the invention was used, which was then placed in a 37° C., 5% CO.sub.2 incubator (purchased from Thermo Fisher) for culture.
[0167] 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 400 μL 0.05% trypsin (purchased from Gibco) was added 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 800 μL 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. The cells were incubated into another 24-well culture plate coated with extracellular matrix gel at a density of 2×10.sup.4 cells/cm.sup.2 for further culture. The cells were inoculated into the 24-well culture plate coated with extracellular matrix gel at the same density using other seven different mediums for culture.
[0168] 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 2×10.sup.4 cells/cm.sup.2 again and cultured continuously using the respective culture mediums of different compositions.
[0169] The following is the formula for calculating the cell population doubling number of primary ESCC epithelial cells under different culture conditions:
[0170] 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.
[0171]
[0172] The photos of cells in
[0173] As can be seen from the results of
Example 7
[0174] Xenograft Tumorigenesis Experiments of Cancer Tissue-Derived Primary ESCC Tumor Cells in Mice
[0175] ESCC tumor cells (No. 0F0056) were isolated and obtained from the cancer tissues of one pathologically diagnosed ESCC patient by using the same method as in Example 1. 0F0065 was cultured using the culture medium FEM of the invention according to the method of Example 3, and when the number of ESCC tumor cells reached 1×10.sup.7, the ESCC tumor cells were digested and collected by using the same method of Example 4. The culture medium for ESCC tumor cells of the invention FEM 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 ESCC tumor cells, and the resultant was injected into the ESCC 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 ESCC tumor cells were observed and photographed every three days.
[0176] Tumor formation can be observed in both of the two tumor cell inoculation sites of the mice on day 15 after tumor cell inoculation. From day 15 to day 30, the tumor proliferation in mice was obvious. This indicates that the cancer tissue-derived ESCC tumor cells cultured by the culture method of the invention have tumorigenicity in mice.
Example 8
[0177] Drug Sensitivity Functional Test of Cancer Tissue-Derived EACC Tumor Cells
[0178] Taking a surgical resection sample from an ESCC patient as an example, it is verified that the ESCC tumor cells cultured from the patient-derived ESCC tumor samples can be used to test the sensitivity of the tumor cells of the patient to different drugs.
[0179] 1. Plating of primary ESCC tumor cells: Cell suspensions of isolated ESCC tumor cells (No. 0F0060 and No. 0F0062) obtained according to the method of Example 1, were inoculated in a Matrigel® (purchased from BD Biosciences)-coated 12-well plate at a density of 4×10.sup.4 cells/cm.sup.2. 2 mL of the prepared culture medium for primary ESCC epithelial cells FEM 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 500 μL 0.05% trypsin (purchased from Gibco) was added 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 FEM 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.
[0180] 2. Drug Gradient Experiments:
[0181] (1) The drug storage plates were prepared by gradient dilution method: 10 μL of the drug stock solutions to be tested (the concentration of the drug stock solution was determined based on 2 times of the maximum blood concentration Cmax of the drug in the human body) were respectively taken, and added to 0.5 mL EP tubes containing 20 μL of DMSO; 10 μL of solutions from the above EP tubes were pipetted 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), Mitomycin (purchased from MCE), Hydroxycamptothecin (purchased from MCE), and Erlotinib (purchased from MCE).
[0182] (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 ESCC tumor 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.
[0183] (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.
[0184] (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.
[0185] (5) The drug sensitivity testing results were shown in
[0186]
INDUSTRIAL APPLICABILITY
[0187] The invention provides a culture medium and a culture method for culturing or expanding primary ESCC epithelial cells in vitro. The cultured cells can be used in the efficacy evaluation and screening of drug. Therefore, the invention is applicable in the industry.
[0188] 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.