CN115975940A - Culture medium and culture method of gastric cancer primary cells - Google Patents
Culture medium and culture method of gastric cancer primary cells Download PDFInfo
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- CN115975940A CN115975940A CN202210202266.9A CN202210202266A CN115975940A CN 115975940 A CN115975940 A CN 115975940A CN 202210202266 A CN202210202266 A CN 202210202266A CN 115975940 A CN115975940 A CN 115975940A
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Abstract
The present invention provides a culture medium for culturing primary gastric cancer cells, comprising at least one additive selected from the group consisting of an MST1/2 kinase inhibitor, a ROCK kinase inhibitor, a B27 additive, and an N2 additive, a basic fibroblast growth factor, CHIR99021, an epidermal growth factor, an ITS cell culture additive, SB202190, dexamethasone, N-acetyl-L-cysteine, gastrin, a8301, oncostatin M, and cholera toxin. The invention also relates to a culture method using the primary cell culture medium. The culture method uses the culture medium to culture gastric cancer primary cells on a culture vessel coated with extracellular matrix glue, so that the primary cells are rapidly proliferated.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium for culturing gastric cancer primary cells and a method for culturing the gastric cancer primary cells by using the culture medium.
Background
Gastric cancer (gastric carcinoma) is a malignant tumor originated from gastric mucosal epithelium, the incidence rate of various malignant tumors in China is the first, the good-onset age is over 50 years, and the incidence rate of male and female is 2:1. gastric cancer tends to be younger due to changes in dietary structure, increased working pressure, infection with helicobacter pylori, and the like. Gastric cancer can occur in any part of the stomach, more than half of which occur in antrum, and the greater curvature, lesser curvature, anterior and posterior walls of the stomach can be affected. Most of gastric cancers belong to adenocarcinoma, have no obvious symptoms in the early stage, or have nonspecific symptoms such as epigastric discomfort, eructation and the like, are often similar to the symptoms of chronic stomach diseases such as gastritis, gastric ulcer and the like, and are easy to ignore, so the early diagnosis rate of the gastric cancers in China is still low at present. At present, the diagnosis rate of the national early gastric cancer is still lower than 20 percent, and the 5-year survival rate of gastric cancer patients is only 27.4 percent.
In recent years, with the rise and development of molecular biology, tumor drug therapy shows a diversified trend, wherein molecular targeted drugs become hot spots for research in clinical treatment of gastric cancer due to the advantages of strong pertinence, high safety and the like. But clinically, for many treatment schemes, it is important to select a scheme suitable for the patient. Although there is gene detection as an index, some patients have no gene mutation, or some patients have multiple targeted drugs against the mutation even if some mutation, and there is a certain clinical difficulty in determining the treatment regimen. In addition to gene sequencing, in vitro primary cell culture of gastric cancer patient samples has become an important means for future in vitro prediction of therapeutic effects and guidance of clinical medication. However, the rapid in vitro acquisition of gastric cancer primary cells is a technical problem to be solved urgently.
At present, two main techniques for culturing primary cells are developed relatively mature. One is the use of irradiated feeder cells and the ROCK kinase inhibitor Y27632 to promote the growth of primary epithelial cells, a cell-conditioned reprogramming technique (Liu et al, am.j.pathol.,180, 599-607, 2012. Another technique is the in vitro 3D culture of adult stem cells to obtain organoid techniques similar to tissues and organs (Hans Clevers et al, cell,11, 172 (1-2): 373-386, 2018).
However, both of these techniques have certain limitations. The cell reprogramming technology is a technology for co-culturing autologous primary epithelial cells of a patient and murine feeder cells. When primary cells of a patient are subjected to drug sensitivity test, the existence of the murine cells can interfere with the drug sensitivity detection result of autologous primary cells of the patient; however, if the murine feeder cells are removed, the autologous primary cells of the patient are separated from the reprogramming environment, and the proliferation rate and intracellular signal pathways of the cells are obviously changed (Liu et al, am.J.Pathol.,183 (6): 1862-1870, 2013 Liu et al, cell Death Dis.,9 (7): 750, 2018), so that the response result of the autologous primary cells of the patient to the drugs is greatly influenced. The organoid technology is a technology for embedding autologous primary epithelial cells of a patient in an extracellular matrix for in vitro three-dimensional culture, and feeder cells are not needed in the technology, so that the problem of interference of mouse-derived feeder cells does not exist. However, the organoid technology requires the addition of several specific growth factors (such as Wnt proteins and R-spondin family proteins) in the culture medium, which is expensive and not suitable for clinical large-scale application. In addition, cells need to be embedded in extracellular matrix gel in the whole culture process of the organoid, the plating steps of cell inoculation, passage and drug sensitivity test are complicated and time-consuming compared with the 2D culture operation, the size of the organoid formed by the technology is not easy to control, and the situation that the inside of the organoid is necrotized due to the fact that the part of the organoid grows too large is easy to occur. Therefore, organoid techniques are less operable and adaptable than 2D culture techniques, require specialized technical personnel, and are not suitable for large-scale widespread use in clinical in vitro drug sensitivity testing (Nick Barker, nat. Cell biol.,18 (3): 246-54, 2016).
In view of the limitations of the above technologies, there is a clinical need to develop a primary gastric cancer cell culture technology, which has a short culture period, controllable cost, and convenient operation, and is not interfered by exogenous cells. When the technology is applied to the construction of a primary gastric cancer tumor cell model, the cultured gastric cancer tumor cells can represent the biological characteristics of a gastric cancer patient. By evaluating the sensitivity of the antitumor drug on cell models derived from different cancer patients in vitro, the response rate of the antitumor drug in clinic is improved, and the pain of the patients and the waste of medical resources caused by inappropriate drugs are reduced.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium and a culture method for rapidly amplifying gastric cancer primary cells in vitro.
One aspect of the present invention is to provide a culture medium of gastric cancer primary cells, the culture medium comprising an MST1/2 kinase inhibitor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; at least one additive of B27 additive and N2 additive; basic fibroblast growth factor; CHIR99021; an epidermal growth factor; ITS cell culture additives; SB202190; dexamethasone; N-acetyl-L-cysteine; a gastrin; a8301; oncostatin M; and cholera toxin. Wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,
wherein,
R 1 selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted aryl (e.g., phenyl, naphthyl, and the like), arylC 1-C6 alkyl (e.g., benzyl, and the like), and heteroaryl (e.g., thienyl, and the like);
R 2 and R 3 Each independently selected from C1-C6 alkyl, preferably C1-C3 alkyl, more preferably methyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylolA group, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl (the heterocyclyl is selected from, for example, piperidinyl, tetrahydropyranyl, and the like);
R 6 selected from the group consisting of halogen (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).
In a preferred embodiment, the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
wherein,
R 1 selected from C1-C6 alkyl, optionally substituted with 1-2 independent R 6 Substituted phenyl, optionally substituted with 1-2 independent R 6 Substituted thienyl, and optionally substituted with 1-2 independent R 6 Substituted benzyl, R 1 More preferably optionally substituted with 1-2 independent R 6 Substituted phenyl;
R 5 selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, R 5 More preferably hydrogen;
R 6 each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl, R 6 More preferably fluorine, methyl or trifluoromethyl.
Preferably, the MST1/2 inhibitor is at least one selected from the following compounds or a pharmaceutically acceptable salt, or solvate thereof.
Most preferably, the MST1/2 kinase inhibitor of the invention is compound 1.
In an embodiment of the invention, the content of each component in the medium of the invention satisfies any one or more or all of the following:
(1) The concentration of the MST1/2 kinase inhibitor is 1.25-20 MuM, and more preferably 2.5-10 MuM;
(2) The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;
(3) The concentration of the basic fibroblast factor is 2.5-40 ng/mL, and more preferably 5-20 ng/mL;
(4) The volume ratio of the ITS cell culture additive to the culture medium is 1;
(5) The concentration of the ROCK kinase inhibitor is 2.5-40 mu M, and more preferably 5-20 mu M;
(6) The concentration of the dexamethasone is 25-400 nM, and more preferably 50-200 nM;
(7) The concentration of the CHIR99021 is 1.25-20 mu M, and more preferably 2.5-10 mu M;
(8) The concentration of the epidermal growth factor is 2.5-40 ng/mL, and more preferably 2.5-20 ng/mL;
(9) The concentration of the cholera toxin is 1.25-20 ng/mL, and more preferably 2.5-10 ng/mL;
(10) The concentration of the gastrin is 1.25-20 nM, more preferably 5-20 nM;
(11) The concentration of the SB202190 is 50-800 nM, more preferably 50-200 nM;
(12) The concentration of the N-acetyl-L-cysteine is 0.25 to 4mM, and more preferably 1 to 4mM;
(13) The concentration of the oncostatin M is 2.5-40 ng/mL, and more preferably 2.5-10 ng/mL;
(14) The concentration of the A8301 is 1.25 to 20nM, and more preferably 2.5 to 20nM.
In an embodiment of the invention, said medium further comprises a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B, and Primocin.
In a preferred embodiment, the concentration of streptomycin ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from streptomycin/penicillin, the concentration of penicillin ranges from 25 to 400U/mL, the concentration ranges from 0.25 to 4. Mu.g/mL when the antibiotic is selected from amphotericin B, and the concentration ranges from 25 to 400. Mu.g/mL when the antibiotic is selected from Primocin.
The invention also provides a culture method of the gastric cancer primary cells. In the method for culturing gastric cancer primary cells of the present invention, gastric cancer primary cells are cultured using the gastric cancer primary cell culture medium of the present invention.
The method for culturing the gastric cancer primary cells comprises the following steps.
(1) The primary cell culture medium of the invention is prepared according to the formula.
(2) The culture vessels were coated with extracellular matrix gel diluent.
Specifically, the extracellular matrix glue is a low growth factor type extracellular matrix glue, and for example, commercially available Matrigel (available from BD biosciences) or BME (available from Trevigen) can be used. More specifically, the extracellular matrix gel was diluted with serum-free medium, which may be DMEM/F12 (from Corning). The dilution ratio of the extracellular matrix glue is 1. The coating method comprises the steps of adding the diluted extracellular matrix glue into a culture vessel, enabling the diluted extracellular matrix glue to completely cover the bottom of the culture vessel, standing and coating for more than 30 minutes, preferably standing and coating at 37 ℃, and preferably coating for 30-60 minutes. And (4) after the coating is finished, sucking and discarding the redundant extracellular matrix glue diluent, and using a culture vessel for later use.
(3) And (3) separating a sample from the gastric cancer solid tumor tissue to obtain gastric cancer primary cells.
Primary gastric cancer cells can be derived, for example, from gastric cancer surgical samples and biopsy endoscopic samples. Gastric cancer tissue samples are derived, for example, from surgically resected cancer tissue samples from a patient having a prescribed and consented gastric cancer tumor, and endoscopic samples are collected from the intragastric lesion via endoscopic guidance. The collection of the tissue sample is performed within half an hour after surgical resection or biopsy of the patient. More specifically, under sterile conditions, a tissue sample is excised from a non-necrotic area having a volume of 5mm 3 Above, it was transported on ice to the laboratory.
In the biological safety cabinet, the tissue sample is transferred to a cell culture dish, and blood cells on the surface of the tissue sample are washed away. Transferring the tissue sample after being moistened into another new culture dish, and using sterile surgical knife blade and surgical forceps to divide the tissue sample into the tissue samples with the volume of less than 3mm 3 The tissue fragment of (a).
Transferring the fragment of the tissue sample into a centrifuge tube, and centrifuging for 3-5 minutes at 1000-3000 r/min by using a desktop centrifuge (3-18K of Sigma company); discarding the supernatant, adding a basal medium (such as DMEM/F12 medium containing 100 mug/mL Primocin) and a tissue digestive juice (wherein the preparation method of the tissue digestive juice is that 1-2 mg/mL collagenase II, 1-2 mg/mL collagenase IV, 50-100U/mL deoxyribonuclease, 0.5-1 mg/mL hyaluronidase, 1-5 mM calcium chloride and 5-10 mg/mL bovine serum albumin are dissolved in 1640 medium) according to the proportion of 1:3, marking the sample number, sealing a sealing film, digesting by a rotary shaking table (known instrument ZQLY-180N) at 37 ℃ and 200-300, and observing whether the digestion is finished every half an hour or 1 hour; if no obvious tissue block is found, the digestion can be stopped, otherwise, the digestion is continued until the digestion is full, and the digestion time range is 4 to 8 hours. After digestion is complete, the undigested tissue mass is filtered off with a cell strainer (cell mesh size, for example, 70 to 100 μm), the tissue mass on the strainer is rinsed with basal medium, the remaining cells are rinsed into a centrifuge tube and centrifuged for 3 to 5 minutes at 1000 to 3000 rpm with a desk centrifuge. Discarding the supernatant, observing whether the residual cell mass contains blood cells, adding a blood cell lysate (purchased from Sigma company) if the residual cell mass contains the blood cells, uniformly mixing, performing lysis at 4 ℃ for 10-20 minutes, shaking for 5 minutes, uniformly mixing once, taking out after the lysis is finished, and centrifuging for 3-5 minutes at 1000-3000 r/min.
(4) Inoculating the primary gastric cancer cells separated in the step (3) into a coated culture vessel, and culturing by adopting the primary cell culture medium in the step (1).
More specifically, in one well of a multi-well plate, 2X 10 4 ～8×10 4 Per cm 2 (e.g., 4X 10) 4 Per cm 2 ) Inoculating the primary gastric cancer cell culture medium at a density of, e.g., 37 ℃ and 5% CO 2 Culturing in a cell culture box for 8-16 days, changing into a fresh primary cell culture medium every 4 days, and performing digestion passage when the primary gastric cancer cells grow to a cell density of about 80-90% of the bottom area of the porous plate.
The inoculation step does not need feeder cells, and compared with a cell condition reprogramming technology, the operation steps of culturing and irradiating the feeder cells are omitted. Compared with organoid technology, the step does not need to uniformly mix primary cells and matrigel on ice to form gel drops, and adds culture medium after the gel drops are solidified, and a culture vessel coated in advance can be directly used for primary cell inoculation. In addition, only a small amount of diluted extracellular matrix glue is needed for coating the culture vessel, compared with organoid technology, the use amount of the extracellular matrix glue with high price is saved, and the operation steps are simplified.
Optionally, after the inoculated primary gastric cancer cells are cultured for 8-16 days, when cell clones formed in a culture container are converged to reach bottom area of 80%, removing supernatant, adding 0.5-2mL0.05% pancreatin (purchased from Thermo Fisher company) for cell digestion, and incubating for 5-20 minutes at room temperature; then 1 to 4mL of a DMEM/F12 culture medium containing, for example, 5% (v/v) fetal bovine serum, 100U/mL penicillin and 100. Mu.g/mL streptomycin is used to resuspend the digested cells, centrifuged at 1000 to 3000 rpm for 3 to 5 minutes, the primary cell culture medium of the present invention is used to resuspend the digested single cells, and the resulting cell suspension is placed in an extracellular matrix gel-coated T25 cell culture flask for further expansion culture. The coating operation of the T25 cell culture bottle is the same as the step (2).
The amplified gastric cancer primary cells grow in 2D, so that the conditions of non-uniform size of organoid, internal necrosis of overgrown organoid and the like caused by organoid technical amplification are avoided.
The beneficial effects of the invention include:
(1) The success rate of the primary cell culture of the gastric cancer is improved and reaches over 90 percent;
(2) Ensuring that the in vitro primary cultured gastric cancer primary cells can maintain the pathological characteristics of patients;
(3) The cultured primary gastric cancer epithelial cells are not interfered by fibroblasts, and purified gastric cancer epithelial cells can be obtained;
(4) The components of the culture medium do not contain serum, so the culture medium is not influenced by the quality and quantity of serum of different batches;
(5) The amplification efficiency is high, the gastric cancer primary cells can be rapidly cultured, and the amplified gastric cancer primary cells can be subjected to continuous passage;
(6) In the passage step, the operation and dissociation of matrigel are not needed, and the digestion passage of the cells can be completed within 10-15 minutes;
(7) The culture cost is controllable, and expensive factors such as Wnt agonist, R-spondin family protein, noggin protein, BMP inhibitor and the like do not need to be added into the culture medium;
(8) The gastric cancer primary cells obtained by the culture of the technology have large quantity, and are suitable for screening candidate compounds at high flux and providing high-flux medicine in-vitro sensitivity function tests for patients.
Drawings
Fig. 1A to 1N are graphs showing the effect of different concentrations of factors added to the gastric cancer primary cell culture medium of the present invention on the proliferation of gastric cancer primary cells.
Detailed Description
For a better understanding of the present invention, the present invention is further described below with reference to the following examples and the accompanying drawings. The following examples are intended to illustrate the invention and are not intended to limit it.
[ preparation example of MST1/2 kinase inhibitor ]
In the present specification, an MST1/2 kinase inhibitor refers to any inhibitor that directly or indirectly down-regulates MST1/2 signaling. In general, MST1/2 kinase inhibitors, for example, bind to and reduce the activity of MST1/2 kinase. Due to the structural similarity of MST1 and MST2, MST1/2 kinase inhibitors may also be compounds that bind to and reduce the activity of MST1 or MST2, for example.
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) benzene
Sulfonamide 1
Methyl 2-amino-2- (2,6-difluorophenyl) acetate (A2): after 2-amino-2- (2,6-difluorophenyl) acetic acid (2.0 g) was added to the round bottom flask, methanol (30 ml) was added followed by thionyl chloride (1.2 ml) dropwise under ice-bath. The reaction system was reacted at 85 ℃ overnight. After the reaction was complete, the solvent was evaporated to dryness under reduced pressure to give a white solid which was used directly in the next step.
Methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (A3): to a round bottom flask was added methyl 2-amino-2- (2,6-difluorophenyl) acetate (2 g), followed by acetone (30 ml) and potassium carbonate (2.2 g), then the system was cooled to-10 ℃ with an ice salt bath, followed by the slow addition of 2,4-dichloro-5-nitropyrimidine (3.1 g) in acetone. The reaction was stirred at room temperature overnight. After the reaction, the reaction mixture was filtered, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by pressure silica gel column chromatography to obtain Compound A3.LC/MS: m + H359.0.
2-chloro-7- (2,6-difluorophenyl) -7,8-dihydropteridin-6 (5H) -one (A4): to a round bottom flask was added methyl 2- ((2-chloro-5-nitropyrimidin-4-yl) amino) -2- (2,6-difluorophenyl) acetate (2.5 g) followed by acetic acid (50 ml) and iron powder (3.9 g). The reaction was stirred at 60 ℃ for two hours. After the reaction was completed, the solvent was evaporated to dryness under reduced pressure, and the resultant was neutralized to be alkaline with saturated sodium bicarbonate. The mixture was extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A4.LC/MS: m + H297.0.
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 to a round bottom flask, cooled to-35 deg.C, iodomethane (0.9 mL) was added followed by sodium hydride (615 mg) and the reaction was stirred for an additional two hours. After the reaction, water was added to quench, ethyl acetate was used for extraction, and the organic phase was washed with water and saturated brine, respectively, and then dried over anhydrous sodium sulfate. Filtering the organic phase, and evaporating to dryness under reduced pressure to obtain a crude product. Washing the crude product with diethyl ether to obtain a compound A5.LC/MS: m + H325.0.
4- ((7- (2,6-difluorophenyl) -5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl) amino) benzenesulfonamide (1): into a round bottom flask was added 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). The reaction was stirred at 120 ℃ overnight. After the reaction is finished, filtering, and washing by methanol and ether to obtain the compound 1.LC/MS: m + H461.1.
2. Preparation of other MST1/2 inhibitor compounds of the invention
Other MST1/2 inhibitor compounds of the invention were synthesized in analogy to compound 1 and their structural and mass spectral data are shown in the table below.
Example 1 Effect of factors added to gastric cancer Primary cell culture Medium on proliferation of gastric cancer Primary cells
(1) Preparation of gastric cancer primary cell culture medium
First, a basal medium containing an initial medium is prepared. The starting medium may be selected from DMEM/F12, DMEM, F12 or RPMI-1640, commonly used in the art. In this example, the basic medium formulation is: DMEM/F12 medium (from Corning) + 100. Mu.g/mL Primocin (from InvivoGen, 0.2% (v/v), commercial product concentration 50 mg/mL).
Different additives (see table 1) are added into the basic culture medium to prepare the gastric cancer primary cell culture medium containing different additive components.
(2) Isolation and processing of gastric cancer primary cells
1 sample selection
Gastric cancer solid tumor tissue samples (intraoperative) were obtained from patients by professional medical personnel at a professional medical institution, all signed with informed consent. Intraoperative sample 0.25cm 3 And the commercial tissue preservation solution (manufacturer: miltenyi Biotec) is adopted for storage and transportation.
2 Material preparation
And (3) sterilizing the surfaces of a 15mL sterile centrifuge tube, a pipette, a 10mL pipette, a sterile gun head and the like, and then placing the sterilized surfaces into an ultra-clean workbench for ultraviolet irradiation for 30 minutes. Basal medium was removed from the 4 ℃ freezer 30 minutes earlier and tissue digest was removed from the-20 ℃ freezer 30 minutes earlier.
The formula of the tissue digestive juice comprises the following components: 1640 Medium (Corning, 10-040-CVR), collagenase II (2 mg/mL), collagenase IV (2 mg/mL), DNase (50U/mL), hyaluronidase (0.75 mg/mL), calcium chloride (3.3 mM), bovine serum albumin BSA (10 mg/mL).
Collagenase ii, collagenase iv, dnase, hyaluronidase mentioned above were all purchased from Sigma company; calcium chloride was purchased from bio-engineering (shanghai) gmbh; BSA was purchased from biofloxx.
3 separation of samples
3.1 taking tissue samples in a Petri dish in a clean bench, removing blood-carrying tissue, rinsing 2 times with a basal medium, transferring the tissue to another Petri dish, mechanically separating with a sterile scalpel, dividing the tissue block into 1 x 1mm 3 Size;
3.2 sucking the cut intraoperative tissue into a 15mL centrifuge tube, adding 5mL basic culture medium, uniformly mixing, and centrifuging at 1500rpm for 4 minutes;
3.3 abandoning the supernatant, adding the basal medium and the tissue digestive juice in a proportion of 1:3 (note: the adding amount of the tissue digestive juice is 1g of tumor tissue and about 10mL of the tissue digestive juice), marking the name and the number of the sample, sealing the sample by a sealing film, digesting the sample in a shaking table (ZQLY-180N) at 300rpm at 37 ℃, observing whether the digestion is finished every 30 minutes, and judging that no visible particles exist;
3.4 after the digestion is finished, filtering out undigested tissue agglomerates by a 100-micron filter screen, flushing the tissue agglomerates on the filter screen into a centrifuge tube by using a basic culture medium to reduce cell loss, and centrifuging at 1500rpm for 4 minutes at 25 ℃;
3.5 abandoning the supernatant, observing whether blood cells exist, if the blood cells exist, adding 8mL of blood cell lysate (purchased from Sigma company), mixing uniformly, cracking at 4 ℃ for 20 minutes, reversing and mixing uniformly for once, and centrifuging at 25 ℃ at 1500rpm for 4 minutes;
3.6 discard the supernatant, add 2mL of basal medium to resuspend the cells for use.
4 cell count and treatment
4.1 Observation under a lens: a small amount of the resuspended cells are removed and spread in a culture dish, and the density and the morphology of the cancer cells are observed under a microscope (CNOPTEC, BDS 400);
4.2 viable cell count: after 12. Mu.L of the resuspended cell suspension was taken and mixed well with 12. Mu.L of trypan blue stain (manufacturer: biotechnology (Shanghai) Co., ltd.), 20. Mu.L of the mixture was taken and added to a cell counting plate (manufacturer: countstar, specification: 50 plates/cassette), and the percentage of viable large cells (cell size >10 μm) = viable cell count/total cell count 100% was calculated under a cell counter (Countstar, IC 1000).
(3) Culture of gastric cancer primary cells
Mixing extracellular matrix glue
An extracellular matrix diluent was prepared by diluting the culture medium (manufactured by BD Biotech) at a ratio of 1:100 using serum-free DMEM/F12, and 500. Mu.l/well of the extracellular matrix diluent was added to a 48-well plate so as to completely cover the bottom of the well of the plate. The mixture was allowed to stand in an incubator at 37 ℃ for 1 hour. After 1 hour, the extracellular matrix diluent was removed to give a Matrigel-coated culture plate.
The gastric cancer primary cells obtained in the above step were resuspended and counted in pre-cooled DMEM/F12. Different components of the medium (Table 1) were added in 500. Mu.l/well volumes to 48-well plates coated with extracellular matrix gel (Matrigel). Counting gastric cancer primary cells at 2 × 10 4 Per cm 2 The cell density of (A) was inoculated into a 48-well Matrigel-coated culture plate, surface-sterilized, and subjected to 37 ℃ C., 5% CO 2 Incubators (purchased from semer fly) cultured the same number of freshly isolated gastric cancer tumor cells (No. GC-001) under different medium formulation conditions. The medium was changed every 4 days after the start of the culture. After 12 days of culture, cell counting was performed, and the effects of each factor on promoting the proliferation of gastric cancer primary cells were compared. Among them, as an experimental control, a basal medium without any additive was used, and the experimental results are shown in table 1.
TABLE 1 additional ingredients in culture Medium and organoid proliferation promoting Effect
Wherein "+" indicates that the medium added with the additive has the function of promoting proliferation of at least two cases of gastric cancer primary cells separated from gastric cancer tissues compared with a basic medium; "-" indicates that the medium to which the additive was added exhibited an inhibitory effect on the proliferation of at least one example of gastric cancer primary cells isolated from gastric cancer tissue; ". Smallcircle" indicates that the medium to which the additive was added had no significant effect on the proliferation of at least two of the gastric cancer primary cells isolated from gastric cancer tissue.
Based on the above results, factors such as MST1/2 kinase inhibitor compound 1, ROCK kinase inhibitor Y27632, at least one additive selected from B27 additive and N2 additive, basic fibroblast growth factor, CHIR99021, epidermal growth factor, ITS cell culture additive, SB202190, dexamethasone, N-acetyl-L-cysteine, gastrin, a8301, oncostatin M, cholera toxin, and the like were selected for further culture experiments.
Example 2 proliferation of gastric cancer Primary cells by Medium-supplemented factors at different concentrations
Gastric cancer primary cells were obtained from intraoperative tissue samples (nos. GC-002, GC-003) according to the method of (2) of example 1, and primary cell culture was performed using the medium formulation in table 2 below.
TABLE 2 culture Medium formulation (final concentration)
When the culture medium of formula 1 is used, 200. Mu.L of the prepared compound 1 per well is added to 48-well plates inoculated with primary cells on the basis of formula 1, and the final concentrations of the compound 1 are 1.25. Mu.M, 2.5. Mu.M, 5. Mu.M, 10. Mu.M and 20. Mu.M, respectively; and control wells (BC) were set using medium of formula 1. The final concentrations of the other additives in the series of media were the same as in GC-2.2 medium. The following experiments for formulations 1 to 14 were also performed in the same manner and will not be described in detail.
When the culture medium of formula 2 is used, 200. Mu.L of prepared Y27632 per well is added to a 48-well plate inoculated with primary cells on the basis of formula 2, and the final concentrations of Y27632 are 2.5. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M and 40. Mu.M respectively; and control wells (BC) were set using medium of formula 2.
When the culture medium of formula 3 is used, 200 μ L of B27 prepared per well is added to 48-well plates inoculated with primary cells on the basis of formula 3, the final concentrations of B27 are 1; and control wells (BC) were set using medium of formula 3.
When the culture medium of the formula 4 is used, 200 mu L of prepared basic fibroblast factors are respectively added into a 48-well plate inoculated with primary cells on the basis of the formula 4, and the final concentrations of the basic fibroblast factors are respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 4.
When the culture medium of formula 5 is used, 200 μ L of prepared CHIR99021 is added to 48-well plates inoculated with primary cells on the basis of formula 5, wherein the final concentrations of CHIR99021 are 1.25 μ M, 2.5 μ M, 5 μ M, 10 μ M and 20 μ M respectively; and control wells (BC) were set using medium of formula 5.
When the culture medium of the formula 6 is used, 200 mu L of the prepared epidermal growth factor per well is respectively added into a 48-well plate inoculated with primary cells on the basis of the formula 6, and the final concentrations of the epidermal growth factor are respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formulation 6.
When the culture medium of formula 7 is used, 200 μ L of prepared ITS cell culture additive per well is added to 48-well plates inoculated with primary cells on the basis of formula 7, and the final concentrations of ITS cell culture additive are 1; and control wells (BC) were set using medium of formula 7.
When the culture medium of formula 8 is used, 200 μ L of prepared SB202190 is added to 48-well plates inoculated with primary cells based on formula 8, respectively, with final concentrations of SB202190 of 50nM, 100nM, 200nM, 400nM, and 800nM, respectively; and control wells (BC) were set using medium of formula 8.
When the culture medium of the formula 9 is used, prepared dexamethasone is added into 48-well plates inoculated with primary cells respectively by 200 mu L per well on the basis of the formula 9, and the final concentrations of the dexamethasone are respectively 25nM, 50nM, 100nM, 200nM and 400nM; and control wells (BC) were set using medium of formula 9.
When the culture medium of the formula 10 is used, 200 mu L of prepared cholera toxin per well is respectively added into a 48-well plate inoculated with primary cells on the basis of the formula 10, and the final concentrations of the cholera toxin are respectively 1.25ng/mL, 2.5ng/mL, 5ng/mL, 10ng/mL and 20ng/mL; and control wells (BC) were set using medium of formula 10.
When the medium of formulation 11 was used, 200. Mu.L of N-acetyl-L-cysteine prepared in a final concentration of 0.25mM, 0.5mM, 1mM, 2mM, 4mM, respectively, was added to each well of 48-well plates inoculated with primary cells based on formulation 11; and control wells (BC) were set using medium of formula 11.
When the culture medium of formula 12 is used, 200 μ L of each well of the prepared gastrin was added to 48-well plates inoculated with primary cells based on formula 12, respectively, at final concentrations of gastrin of 1.25nM, 2.5nM, 5nM, 10nM, and 20nM, respectively; and control wells (BC) were set using medium of formula 12.
When the culture medium of the formula 13 is used, 200 mu L of the prepared antitumor agent M is added into a 48-well plate inoculated with primary cells on the basis of the formula 13, and the final concentration of the antitumor agent M is respectively 2.5ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 40ng/mL; and control wells (BC) were set using medium of formula 13.
When the culture medium of formula 14 is used, 200 μ L of the prepared A8301 is added to each well of formula 14 in 48-well plates inoculated with primary cells, and the final concentrations of the A8301 are 1.25nM, 2.5nM, 5nM, 10nM and 20nM, respectively; and control wells (BC) were set using medium of formula 14.
When the cells were expanded to about 85% of the 48 wells and digested, the number of cells in the control well (BC) was counted, and the proliferation fold was calculated with reference to the number of cells in the control well (BC), and the data collected from 2 samples are shown in FIGS. 1A to 1N. In FIGS. 1A to 1N, the ratio is the ratio of the number of cells obtained by one-pass culture using each medium to the number of cells obtained by one-pass culture using the corresponding control well. The ratio is more than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is better than that of a control Kong Peiyang medium; if the ratio is less than 1, the proliferation promoting effect of the prepared culture medium containing factors or small molecular compounds with different concentrations is weaker than that of the culture medium of the control hole.
According to the results of FIGS. 1A to 1N, the content of the MST1/2 kinase inhibitor compound 1 is preferably 1.25 to 20. Mu.M, more preferably 2.5 to 10. Mu.M; the concentration of Y27632 is preferably 2.5 to 40. Mu.M, more preferably 5 to 20. Mu.M; the volume concentration of B27 is preferably 1; the concentration of the basic fibroblast is preferably 2.5-40 ng/mL, and more preferably 5-20 ng/mL; the concentration of CHIR99021 is preferably 1.25 to 20. Mu.M, more preferably 2.5 to 10. Mu.M; the concentration of the epidermal growth factor is preferably 2.5-40 ng/mL, and more preferably 2.5-20 ng/mL; the volume concentration of ITS cell culture additive to the culture medium is preferably 1; the concentration of SB202190 is preferably 50 to 800nM, more preferably 50 to 200nM; the concentration of dexamethasone is preferably 25-400 nM, more preferably 50-200 nM; the concentration of cholera toxin is preferably 1.25-20 ng/mL, more preferably 2.5-10 ng/mL; the concentration of N-acetyl-L-cysteine is preferably 0.25 to 4mM, more preferably 1 to 4mM; the concentration of gastrin is preferably 1.25 to 20nM, more preferably 5 to 20nM; the concentration of the oncostatin M is preferably 2.5-40 ng/mL, and more preferably 2.5-10 ng/mL; the concentration of A8301 is preferably 1.25 to 20nM, more preferably 2.5 to 20nM.
Industrial applicability
The invention provides a culture medium and a culture method for culturing gastric cancer primary cells, which can be used for evaluating and screening the curative effect of medicaments by the cultured gastric cancer primary cells. Thus, the present invention is suitable for industrial applications.
Although the present invention has been described in detail herein, the present invention is not limited thereto, and modifications can be made by those skilled in the art based on the principle of the present invention, and thus, it is to be understood that various modifications made in accordance with the principle of the present invention are within the scope of the present invention.
Claims (10)
1. A culture medium for gastric cancer primary cells, characterized by comprising an MST1/2 kinase inhibitor; a ROCK kinase inhibitor selected from at least one of Y27632, fasudil, and H-1152; at least one additive of B27 additive and N2 additive; basic fibroblast growth factor; CHIR99021; an epidermal growth factor; ITS cell culture additives; SB202190; dexamethasone; N-acetyl-L-cysteine; a gastrin; a8301; oncostatin M; and a cholera toxin,
wherein the MST1/2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt, or solvate thereof,
wherein,
R 1 selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted aryl, arylC 1-C6 alkyl and heteroaryl;
R 2 and R 3 Each independently selected from C1-C6 alkyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, and C3-C6 heterocycloC 1-C6 alkyl;
R 6 selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
2. The culture medium of claim 1, wherein
R 1 Selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and optionally substituted with 1-2 independent R 6 Substituted phenyl, naphthyl, benzyl and thienyl;
R 2 and R 3 Each independently selected from C1-C3 alkyl;
R 4 and R 5 Each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC 1-C6 alkyl, C1-C6 alkoxyC 1-C6 alkyl, piperidinylC 1-C6 alkyl, and tetrahydropyranyl C1-C6 alkyl;
R 6 selected from the group consisting of halogen, C1-C6 alkyl, C1-C6 alkoxy, and C1-C6 haloalkyl.
3. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt, or solvate thereof,
wherein,
R 1 selected from C1-C6 alkyl, optionally substituted by 1-2 independent R 6 Substituted phenyl, optionally substituted with 1-2 independent R 6 Substituted thienyl, and optionally substituted with 1-2 independent R 6 A substituted benzyl group;
R 5 selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl;
R 6 each independently selected from halogen, C1-C6 alkyl, and C1-C6 haloalkyl.
4. The culture medium of claim 3, wherein
R 1 Is optionally substituted by 1-2 independent R 6 Substituted phenyl;
R 5 is hydrogen;
R 6 preferably fluorine, methyl or trifluoromethyl。
5. The culture medium of claim 1, wherein the MST1/2 kinase inhibitor is selected from at least one of the following compounds or a pharmaceutically acceptable salt thereof:
6. a culture medium according to any one of claims 1 to 5, wherein the content of each component in the culture medium satisfies any one or more or all of the following:
the concentration of the MST1/2 kinase inhibitor is 1.25-20 mu M;
the volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;
the concentration of the basic fibroblast is 2.5-40 ng/mL;
the volume ratio of the ITS cell culture additive to the culture medium is 1;
the concentration of the ROCK kinase inhibitor is 2.5-40 mu M;
the concentration of the dexamethasone is 25-400 nM;
the concentration of the CHIR99021 is 1.25-20 mu M;
the concentration of the epidermal cell growth factor is 2.5-40 ng/mL;
the concentration of the cholera toxin is 1.25-20 ng/mL;
the concentration of the gastrin is 1.25-20 nM;
the concentration of the SB202190 is 50-800 nM;
the concentration of the N-acetyl-L-cysteine is 0.25 to 4mM;
the concentration of the tumor suppressor M is 2.5-40 ng/mL;
the concentration of the A8301 is 1.25-20 nM.
7. A culture medium according to any one of claims 1 to 5, wherein the content of each component in the culture medium satisfies any one or more or all of the following:
the concentration of the MST1/2 kinase inhibitor is 2.5-10 mu M;
the volume ratio of the B27 or N2 cell culture additive to the culture medium is 1;
the concentration of the basic fibroblast factor is 5-20 ng/mL;
the volume ratio of the ITS cell culture additive to the culture medium is 1;
the concentration of the ROCK kinase inhibitor is 5-20 mu M;
the concentration of the dexamethasone is 50-200 nM;
the concentration of the CHIR99021 is 2.5-10 mu M;
the concentration of the epidermal growth factor is 2.5-20 ng/mL;
the concentration of the cholera toxin is 2.5-10 ng/mL;
the concentration of the gastrin is 5-20 nM;
the concentration of the SB202190 is 50-200 nM;
the concentration of the N-acetyl-L-cysteine is 1 to 4mM;
the concentration of the oncostatin M is 2.5-10 ng/mL;
the concentration of the A8301 is 2.5-20 nM.
8. The culture medium according to any one of claims 1 to 5, further comprising:
a starting medium selected from DMEM/F12, DMEM, F12 or RPMI-1640; and
an antibiotic selected from one or more of streptomycin/penicillin, amphotericin B and Primocin.
9. The culture medium according to any one of claims 1 to 5, wherein said medium is free of Wnt agonists, R-spondin family proteins, noggin proteins, BMP inhibitors.
10. A method for culturing primary gastric cancer cells is characterized by comprising the following steps:
(1) Preparing a culture medium according to any one of claims 1 to 9;
(2) Coating a culture vessel with an extracellular matrix gel diluent selected from at least one of Matrigel and BME;
(3) Inoculating primary gastric cancer epithelial cells separated from gastric cancer tissues in a culture vessel coated with extracellular matrix glue, and culturing by using the culture medium in the step (1).
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