CN113403278A

CN113403278A - Culture medium and culture method of gastric cancer primary cells

Info

Publication number: CN113403278A
Application number: CN202010180617.1A
Authority: CN
Inventors: 刘青松; 黄涛; 陈程; 王文超; 任涛; 王黎
Original assignee: Precedo Pharmaceuticals Co Ltd
Current assignee: Precedo Pharmaceuticals Co Ltd
Priority date: 2020-03-16
Filing date: 2020-03-16
Publication date: 2021-09-17
Anticipated expiration: 2040-03-16
Also published as: WO2021184408A1; CN113403278B

Abstract

The invention provides a culture medium and a culture method for realizing rapid amplification of gastric cancer primary cells in vitro. The gastric cancer primary cell culture medium of the present invention comprises a primary culture medium, Rho protease inhibitor, antibiotic, insulin, non-essential amino acids, hydrocortisone, cholera toxin, glutamine, fetal calf serum, and at least one additive selected from the group consisting of B27 additive and N2 additive. By using the gastric cancer primary cell culture medium and adopting a condition reprogramming culture technology, the effective and rapid amplification of gastric cancer primary cells can be realized, so that the cells obtained by amplification keep the pathological characteristics of patients, the culture success rate and the cell amplification rate of the gastric cancer primary cells are improved, and a research basis can be provided for the personalized treatment of the patients.

Description

Culture medium and culture method of gastric cancer primary cells

Technical Field

The invention relates to the technical field of biology, in particular to a culture medium and a culture method for rapid amplification of gastric cancer primary cells, and application of the culture medium and the culture method in curative effect evaluation and screening of medicines.

Background

Gastric cancer is the most common malignant digestive tract tumor, and patients with early gastric cancer have no obvious symptoms, so that most patients with gastric cancer have developed to the middle and late stage when diagnosed. Gastric cancer cells are less sensitive to chemotherapeutic drugs, so that the chemotherapeutic effect is poor, resulting in low five-year survival rate. In recent years, with the development of science and technology, the diagnosis and treatment technology of gastric cancer is continuously improved, especially the progress of comprehensive treatment modes such as operation, endoscopic technology, radiotherapy and chemotherapy, targeted therapy and the like, the treatment effect of gastric cancer is improved, but the prognosis of comprehensive treatment for gastric cancer patients in late stage and with metastatic focus is still not ideal. Epidemiological investigation and analysis show that the number of gastric cancer cases and the number of death cases in China respectively account for 42.6 percent and 45.0 percent of the gastric cancer cases and the gastric cancer deaths worldwide, and the gastric cancer cases and the gastric cancer deaths are located at the 5 th morbidity and the 6 th mortality in 183 countries worldwide. Stomach cancer is one of the main diseases seriously harming the health of Chinese residents, and the reduction of the morbidity and mortality of stomach cancer in China becomes a major public health problem which must be directly solved.

The existing in vitro cultured gastric cancer cell lines are mainly obtained by culturing normal cells for a long period of time to be spontaneously immortalized or transfecting oncogenes that promote the immortalization of normal cells. Cell lines established by traditional methods remain the main mainstay of cell, molecular and cancer biology research. However, these methods change the genetic background of the cells, and long-term cultured cell lines are also prone to cause genomic instability, which may result in artificial changes in the phenotype of tumor cell lines and in vivo tumor cells. The complex heterogeneity of primary tumors is often absent in these cell lines, limiting the utility of these cell lines for predicting tumor cell responses, affecting the accuracy of scientific research and drug development for gastric cancer. In addition, in the process of culturing cancer cells from cells obtained from gastric cancer tissues, the conventional culture method is difficult to obtain the cancer cells, the culture process is easy to be interfered by fibroblasts, the formed clone can not be passaged, and the like, so that the application of the human gastric cancer primary cells is limited.

Xuefeng Liu et al used irradiated mouse fibroblasts and Rho-associated kinase inhibitor (Y-27632) to expand epithelial-derived cells in 2017, which has the ability to achieve unlimited growth of epithelial-derived cells without genetic manipulation (Xuefeng Liu et al, Conditional reprogramming and Long-term expansion of normal and tumor cells from human biosciences. Nat. Protoc.2017,12,439). However, the method established by Xuefeng Liu et al has a long culture period and cannot realize rapid cell expansion, thereby limiting the application of the technology.

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 a starting medium, a Rho protease inhibitor, an antibiotic, insulin, a non-essential amino acid, hydrocortisone, cholera toxin, glutamine, Fetal Bovine Serum (FBS), and an additive selected from at least one of a B27 additive and an N2 additive.

The initial culture medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640.

Among them, at least one additive selected from the group consisting of a B27 additive and an N2 additive is preferably a B27 additive, or a B27 additive and an N2 additive.

(1) The Rho protein kinase inhibitor is selected from one or more of Y27632, hydroxyfasudil and GSK429286A, and when the Rho protein kinase inhibitor is selected from Y27632, the concentration range is 2.5-40 mu M, preferably 10-20 mu M; when the fasudil is selected from hydroxyl fasudil, the concentration range is 2-32 mu M, and preferably 4-16 mu M; when the GSK429286A is selected, the concentration range is 2-32 μ M, preferably 4-16 μ M;

(2) the antibiotics are selected from one or more of streptomycin/penicillin, amphotericin B and Primocin, when the antibiotics are selected from streptomycin/penicillin, the concentration range of the streptomycin is 25-400 mu g/mL, preferably 50-200 mu g/mL, more preferably 200 mu g/m, the concentration range of the penicillin is 25-400U/mL, preferably 50-200U/mL, more preferably 200U/mL; when the amphotericin B is selected, the concentration range is 0.25-4 mug/mL, preferably 0.5-2 mug/mL; when the Primocin is selected, the concentration range is 25-400 mu g/mL, preferably 50-200 mu g/mL;

(3) the concentration range of the insulin is 5-20 mug/mL, preferably 10-20 mug/mL;

(4) the nonessential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the total concentration range of the nonessential amino acid is 50-800 mu M, preferably 100-400 mu M;

(5) the concentration range of the hydrocortisone is 0.1-1.6 mu g/mL, preferably 0.2-0.8 mu g/mL;

(6) the concentration range of cholera toxin is 0.1-0.4 nM, preferably 0.1-0.2 nM

(7) The concentration range of the glutamine is 1-8 mM, preferably 1-4 mM;

(8) the volume ratio of Fetal Bovine Serum (FBS) to the culture medium is 1: 5-1: 40;

(9) the concentration range volume ratio of the B27 additive is 1: 25-1: 400, preferably 1: 25-1: 200;

(10) the volume ratio of the N2 additive to the culture medium is 1: 25-1: 400, preferably 1: 25-1: 100.

The invention also provides a culture method of the gastric cancer primary cells. In the method for culturing the gastric cancer primary cells of the present invention, the gastric cancer primary cell culture medium of the present invention is used to culture the gastric cancer primary cells.

In the method for culturing the gastric cancer primary cells, the cell density is 2-3 multiplied by 10⁴Per cm²Feeder cells are added. The feeder cells are irradiated NIH-3T3 cells, the irradiation source is X rays or gamma rays, and the irradiation dose is 20-50 Gy.

The method for culturing the gastric cancer primary cells further comprises the following steps:

1. isolation of gastric cancer primary cells

1.1 washing a tissue sample, such as an endoscopic specimen, with a tissue washing solution, adding a tissue digestive juice, and digesting in a constant temperature shaking table (ZQLY-180N, Chu apparatus), for example, with 8-14 ml, preferably 12 ml of tissue digestive juice; the digestion temperature range is 4-37 ℃, and preferably 37 ℃; the digestion rotating speed range is 200 rpm-350 rpm, preferably 300 rpm;

1.2, taking out and observing after digestion, stopping digestion if no obvious tissue block is found, or continuing digestion until the digestion is sufficient, wherein the digestion time range is 4-8 hours, and preferably 6 hours;

1.3 taking out after digestion is finished, centrifuging, removing supernatant, adding an initial culture medium containing serum to perform basic suspension to terminate digestion, wherein the centrifugal rotating speed range is 1200-1600 rpm, and 1500rpm is preferred; the centrifugation time is 2-6 minutes, preferably 5 minutes, and the serum-containing initial medium may be, for example, DMEM/F12 medium containing 10% fetal bovine serum.

2. Culturing Using the gastric cancer Primary cell culture Medium of the present invention

Resuspending and counting the gastric cancer primary cells obtained in the step 1 by using the gastric cancer primary cell culture medium of the invention according to the cell density of 5-10 multiplied by 10⁴Per cm²Planting the seeds into a culture dish, and simultaneously, carrying out cell density 2-3 multiplied by 10⁴Per cm²Adding the trophoblasts, culturing for 5-7 days, and then, according to the cell density of 0.5-1 multiplied by 10⁴Per cm²And supplementing and adding the feeder cells until the culture dish is full of more than 85% of cells, and performing digestion passage.

Wherein the formula of the tissue cleansing fluid in the step 1 is as follows: the DMEM/F12 basal medium contains 100-200 mu g/mL Primocin and 2% penicillin/streptomycin solution; the preparation method of the tissue digestive juice in the step 1 comprises the following steps: dissolving 1-2 mg/mL collagenase II, 1-2 mg/mL collagenase IV, 50-100U/mL deoxyribonucleic acid I, 0.5-1 mg/mL hyaluronidase, 1-3 mM calcium chloride and 1-2% bovine serum albumin in HBSS and RPMI-1640 at a volume ratio of 1: 1; the trophoblasts in the step 2 can be irradiated NIH-3T3 cells, the irradiation source is X rays or gamma rays, preferably gamma rays, and the irradiation dose is 20-50 Gy, preferably 30 Gy.

The invention also provides a drug screening method for gastric cancer diseases, which comprises the following steps:

(1) the method for culturing the gastric cancer primary cells is used for culturing the gastric cancer primary cells for drug screening;

(2) selecting a medicine to be detected and diluting according to a required concentration gradient;

(3) adding the diluted medicine to the cells cultured in (1);

(4) cell viability assays were performed.

Effects of the invention

(1) The success rate of the stomach cancer primary cell culture is improved and reaches more than 85 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 cells are not interfered by mesenchymal cells such as fibroblasts and fat cells;

(4) high amplification efficiency, as long as 10⁴The cell number of the grade can be successfully amplified to 10 within about two weeks⁶The gastric cancer primary cells with the magnitude order can be continuously passed;

(5) the culture cost is controllable: the culture medium does not need to add factors such as expensive Wnt agonist, R-spondin family protein, BMP inhibitor, FGF10 and the like, and is a simplification and improvement of the existing gastric cancer primary cell culture medium;

(6) the gastric cancer primary cells obtained by the culture of the technology have large quantity and high homogenization degree, and are suitable for screening new candidate compounds at high flux and providing high-flux drug in-vitro sensitivity function tests for patients.

Drawings

FIG. 1 is a graph showing the effect of different combinations of factors added to a gastric cancer primary cell culture medium on gastric cancer primary cell proliferation.

FIG. 2 is a graph showing the effect of factor concentration in gastric cancer primary cell culture medium on gastric cancer primary cell proliferation.

Fig. 3 is a photograph for observing, with a microscope, gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention.

Fig. 4 shows results of giemsa rapieri staining and identification of gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention.

Fig. 5 shows the results of immunofluorescent staining of gastric cancer primary cells cultured with the gastric cancer primary cell culture medium of the present invention.

Fig. 6 is a cell growth curve of gastric cancer primary cells cultured using the gastric cancer primary cell culture medium of the present invention.

Fig. 7 is a comparison result of culturing gastric cancer primary cells using the gastric cancer primary cell culture medium of the present invention and a conventional culture medium.

Fig. 8 shows the results of culturing gastric cancer cells using the gastric cancer primary cell culture medium of the present invention and using different generation numbers of cells for drug screening.

Detailed Description

For a better understanding of the present invention, the present invention will be further described with reference to the following examples and the accompanying drawings, which are illustrative of the present invention and are not to be construed as limiting thereof.

Example 1 Effect of factors added to gastric cancer Primary cell Medium on proliferation of gastric cancer Primary cells

(1) Preparation of gastric cancer primary cell culture medium

First, a basal medium is prepared. The basic culture medium comprises the following components: DMEM/F12 medium (available from Corning Inc.) + 10. mu. M Y27632 (available from MCE Inc.) + 100. mu.g/mL Primocin (available from Invivog Inc.).

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 of gastric cancer primary cells

1 sample selection

Gastric carcinoma solid tumor tissue samples (intraoperative/endoscopic) were obtained from patients by professional medical personnel of a professional medical institution, who all signed an informed consent. 2 soybean grains in intraoperative samples and endoscopic sample rice grains; the tissue preservation solution (manufacturer: Miltenyi Biotec) is commercially available for storage and transportation.

2 Material preparation

Preparing: NIH 3T3 cells (purchased from ATCC) irradiated with gamma-ray at a dose of 30 Gy; 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 min; taking out basal medium from 4 deg.C refrigerator 30min in advance, and taking out tissue digestive juice from-20 deg.C refrigerator 30min in advance, wherein:

tissue digestive juice: DMEM/F12 medium, collagenase II (2mg/mL), collagenase IV (2mg/mL), DNase (50U/mL), hyaluronidase (0.75mg/mL), calcium chloride (3.3mM), BSA (10 mg/mL).

Collagenase ii mentioned above was purchased from Sigma; collagenase iv was purchased from Sigma; dnase was purchased from Sigma; hyaluronidase was purchased from Sigma; calcium chloride was purchased from bio-engineering (shanghai) gmbh; BSA was purchased from Biofrox.

3 separation of samples

3.1 ultra clean bench tissue is removed from the culture dish, the blood-borne tissue is removed, rinsed 2 times with basal medium, the tissue is transferred to another culture dish and mechanically separated with a sterile scalpel, bringing the tissue mass to 1 x 1mm³Size;

3.2 sucking the cut intraoperative or endoscopic tissues into a 15mL centrifuge tube, adding 5mL of basic culture medium, uniformly mixing, and centrifuging at 1500rpm for 4 min;

3.3 abandoning the supernatant, adding a basic culture medium and digestive enzymes according to the proportion of 1:1 (note: the adding amount of the digestive enzymes is about 10mL for 1g of tumor tissues), marking the name and the number of the sample, sealing the sample by a sealing film, digesting the sample in a shaking table at the temperature of 37 ℃ and 300rpm, observing whether the digestion is finished every 1h, and judging whether particles can not be seen by naked eyes;

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 at 25 ℃ for 4 min;

3.5 abandoning the supernatant, observing whether blood cells exist, if blood cells exist, adding 10mL of blood cell lysate (purchased from Sigma company), mixing uniformly, cracking at 4 ℃ for 20min, reversing and mixing uniformly once, and centrifuging at 25 ℃ and 1500rpm for 4 min;

3.6 discard the supernatant, add 2mL of basal medium to resuspend the cells for use.

4 cell counting and processing

4.1 Observation under a lens: a small amount of the resuspended cells are transferred and spread in a culture dish, and the density and the shape of the cancer cells are observed under a microscope;

4.2 viable cell count: 10. mu.L of the resuspended cell suspension was taken, added to 80. mu.L of LGM-1 medium and 10. mu.L of trypan blue stain (manufacturer: Biochemical engineering (Shanghai) Co., Ltd.) and mixed thoroughly, 10. mu.L of the mixture was taken and added to a hemocytometer (manufacturer: Shanghai Biochemical reagent Instrument Co., Ltd., specification: 79mm 39mm 13mm), the total number of cells in four large cells under the microscope and the cell concentration (cell/mL) of 4 large cells/4 10 large cells⁴10 (dilution factor). Dead cells can be stained blue and live cells are not stained. The number of 200 cells was counted, and viable large cells (cell size) were calculated>10 μm) percent ═ 100% viable cell count/total cell count.

(3) Culture of gastric cancer primary cells

The different components of the medium in Table 1 were added to 48-well plates in 500. mu.l/well volumes. The primary cells of gastric cancer separated from three gastric cancer tissues (sample 1, sample 2 and sample 3) according to the step (2) are 1 × 10⁴Cell density of each well was seeded in 48-well culture plates at 2X 10 cell density⁴Per cm²NIH-3T3 cells irradiated with gamma rays (irradiation dose 30Gy) were added. At 37 deg.C and 5% CO₂The culture was performed under the condition of concentration. The medium was changed every 4 days after the start of the culture. After 10 days of culture, cell counts were performed. 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

Serial number	Media additive species	Suppliers of goods	Final concentration	Grading of degree of cell proliferation promotion
					1	N2	Gibco	1:50	+
2	Epidermal growth factor	R&D	20ng/ml	○
					3	Hepatocyte growth factor	Peprotech	20ng/ml	○
4	Basic fibroblast growth factor	R&D	20ng/ml	○
					5	R-spondin1	R&D	250ng/ml	○
6	R-spondin3	R&D	250ng/ml	○
					7	Prostaglandin E2	Tocris	1μM	○
8	Insulin	Peprotech	20μg/mL	+
					9	Human leukemia inhibitory factor	Peprotech	100ng/ml	○
10	Triiodothyronine	MCE	5nM	－
					11	B27	Gibco	1:50	+
12	A8301	MCE	500nM	○
					13	SB202190	MCE	500nM	○
14	Nicotinamide	Sigma	10mM	○
					15	N-acetylcysteine	Sigma	1.25mM	－
16	Glutamine	GIBCO	4mM	+
					17	Cholera toxin	Sigma	0.1nM	+
18	Hydrocortisone	Sigma	0.8μg/mL	+
					19	Non-essential amino acids	Corning	400μM	+
20	Noggin	R&D	100ng/ml	○
					21	FBS	Excell		10％	+

Wherein "+" indicates that the medium added with the additive has the effect of promoting proliferation of three 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 showed proliferation-inhibiting effect on at least two cases of gastric cancer primary cells isolated from gastric cancer tissues; ". 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 tissues.

Example 2 Effect of combinations of different additional factors in gastric cancer Primary cell Medium on gastric cancer Primary cell proliferation

Gastric cancer primary cell culture media with different additive factor combinations are prepared according to the components in the table 2, and the proliferation promoting effect of the different additive factor combinations on gastric cancer primary cells is examined.

TABLE 2 preparation of the media with different compositions (final concentration)

Culture medium	Components
		Basic culture medium (BM)	DMEM/F12+10μM Y27632+100μg/mL Primocin
No.1	BM + 20. mu.g/mL insulin
		No.2	No.1+ 0.8. mu.g/mL hydrocortisone
No.3	No.2+ 400. mu.M non-essential amino acid
		No.4	No.3+4mM Glutamine
No.5	No.4+0.1nM cholera toxin
		No.6	No.5+10％FBS
No.7	No.6+1: 50N 2 additive
		GM-1	No.6+1: 50B 27 additive

The FBS mentioned above was purchased from Excell corporation.

Gastric cancer primary cells were obtained from gastric cancer tissue (sample 4) by the method of step 3 of step 2 of example 1, the obtained cell suspension was divided into 9 parts on average, centrifuged at 1500rpm for 4 minutes, resuspended in 200. mu.l of BM, Nos. 1 to 7 and GM-1 medium after centrifugation, and then resuspended at a viable cell density of 1X 10⁴Per cm²Seeded in 48-well plates (1 ten thousand cells per well) and subsequently at a cell density of 2X 10⁴Per cm²NIH-3T3 cells irradiated by gamma rays (irradiation dose is 30Gy) are added, and finally, the pore volumes in 48-pore plates are respectively filled to 500 microliter by using corresponding culture media, and the mixture is fully and uniformly mixed. Sterilizing the surface, and placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) for culture. And (5) carrying out passage until the cells in the 48-well plate grow to more than 85%.

On day 7 of incubation, 48 well plates were removed, rinsed for 1 minute with 200. mu.l 0.05% trypsin (from Gibco), aspirated, and 250. mu.l 0.05% trypsin (from Gibco) was added to each well, placed at 37 ℃ and 5% CO₂The reaction was carried out in an incubator for 10 minutes until the completion of the digestion of the cells was observed under a microscope (EVOS M500, Invitrogen), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, 1 ml of a basal medium was added for resuspension, and the cells were counted using a flow cytometer (JIMBIO FIL, Jiangsu microbial science Ltd.) to obtain fine cellsTotal number of cells. The results obtained from gastric cancer primary cells isolated from endoscopic tissue sample 50 are shown in fig. 1.

From the results shown in FIG. 1, it is clear that the proliferation of gastric cancer primary cells can be promoted to a different extent in the cases where the above-mentioned culture media Nos. 1 to 7 and GM-1 are used, compared with the basal culture medium. When the gastric cancer primary cell culture medium (namely GM-1 culture medium) containing Y27632, Primocin, insulin, hydrocortisone, non-essential amino acid, glutamine, cholera toxin, FBS and B27 additive is used for culturing the gastric cancer primary cell, the proliferation effect is obviously improved.

EXAMPLE 3 Effect of different concentrations of added factors on gastric cancer Primary cell proliferation

Gastric cancer primary cells were isolated and obtained from endoscopic tissues (sample 5, sample 6, sample 7, sample 8 and sample 9) according to the method of 3 of step (2) in example 1, and the obtained gastric cancer primary cells were cultured using GM-1 medium in example 2 at a viable cell density of 1X 10⁴Per cm²Seeded in 12-well plates (4.5 ten thousand cells per well) at a cell density of 2X 10⁴Per cm²Adding NIH-3T3 cells irradiated by gamma ray (irradiation dose 30Gy), and mixing. Sterilizing the surface, and placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) for culture. After the cells had expanded to 85%, the 12-well plate was removed, rinsed with 200. mu.l of 0.05% trypsin (from Gibco) for 1 minute, aspirated, and 500. mu.l of 0.05% trypsin (from Gibco) was added to each well, and the mixture was incubated at 37 ℃ and 5% CO₂The reaction was carried out in an incubator for 10 minutes until the cells were completely digested under a microscope (EVOS M500, Invitrogen), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded, 1 ml of a basal medium was added for resuspension, and the cells were counted using a flow cytometer (JIMBIO FIL, Jiangsu microbial science, Ltd.) to obtain the total number of cells. The cells obtained were used in the following culture experiments.

Next, the following 9 media formulations were prepared for the experiments:

formula 1: GM-1 medium components in example 2 do not contain non-essential amino acids;

and (2) formula: the GM-1 medium component of example 2 is glutamine-free;

and (3) formula: GM-1 medium of example 2 contains no B27 in its components;

and (4) formula: the GM-1 medium component of example 2 contained no insulin;

and (5) formula: GM-1 medium component of example 2 contained no cholera toxin;

and (6) formula: the GM-1 medium component of example 2 is free of hydrocortisone;

and (3) formula 7: GM-1 medium component of example 2 contains no Y27632;

and (4) formula 8: GM-1 medium component in example 2 contained no FBS;

formula 9: GM-1 medium in example 2.

The digested cell suspensions were diluted with the GM-1 medium of formulations 1-9 and example 2, respectively, and seeded into 24-well plates at 500. mu.l volumes per 2 ten thousand cells.

When the culture medium of formula 1 is used, 5 concentration gradients of non-essential amino acids are prepared according to final concentrations of 800. mu.M, 400. mu.M, 200. mu.M, 100. mu.M and 50. mu.M, and 500. mu.L of the prepared non-essential amino acids are added to a 24-well plate inoculated with primary cells; and control wells (BC) were set using medium of formula 1.

When the medium of formulation 2 was used, 5 glutamine concentration gradients were prepared at final concentrations of 8mM, 4mM, 2mM, 1mM, and 0.5mM, and 500. mu.l of prepared glutamine per well was added to 24-well plates inoculated with primary cells; and control wells (BC) were set using medium of formula 2.

When the culture medium of formula 3 is used, 5 concentration gradients of B27 additives are prepared according to final concentrations of 1:400, 1:200, 1:100, 1:50 and 1:25 respectively, and 500 microliters of the prepared B27 additives are added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 3.

When the culture medium of formula 4 is used, 5 concentration gradients of insulin are prepared according to final concentrations of 40. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL and 2.5. mu.g/mL, and 500 microliters of prepared insulin per well are added to a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 4.

When the culture medium of formula 5 is used, 5 concentration gradients of cholera toxin are prepared according to final concentrations of 0.4nM, 0.2nM, 0.1nM, 0.05nM and 0.025nM respectively, and 500 microliters of prepared cholera toxin per well are added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 5.

When the culture medium of formula 6 is used, 5 concentration gradients of hydrocortisone are prepared according to final concentrations of 1.6. mu.g/mL, 0.8. mu.g/mL, 0.4. mu.g/mL, 0.2. mu.g/mL and 0.1. mu.g/mL respectively, and 500. mu.L of the prepared hydrocortisone per well is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 6.

When the culture medium of formula 7 is used, 5 concentration gradients of Y27632 are prepared according to final concentrations of 40. mu.M, 20. mu.M, 10. mu.M, 5. mu.M and 2.5. mu.M, and 500. mu.L of prepared Y27632 per well is added to a 24-well plate inoculated with primary cells; and control wells (BC) were set using medium of formula 7.

When the culture medium of formula 8 is used, 5 concentration gradients of FBS are prepared according to final concentrations of 1:40, 1:20, 1:10, 1:5 and 1:2.5 respectively, and 500 microliters of the prepared FBS is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 8.

When the culture medium of formula 9 is used, 5 concentration gradients of N2 additive are prepared according to final concentrations of 1:400, 1:200, 1:100, 1:50 and 1:25 respectively, and 500 microliters of the prepared N2 additive is added into a 24-well plate inoculated with primary cells respectively; and control wells (BC) were set using medium of formula 9.

Simultaneously, 1 ten thousand irradiated NIH-3T3 cells were added to each well as feeder cells.

When the cells were expanded to about 85% of the 24 wells and digested, the ratio was calculated with reference to the number of cells in the control well (BC), and the results are shown in FIGS. 2 (A) to (I), respectively. In FIG. 2, 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 different concentration factors is better than that of the culture medium in the control hole, and the ratio is less than 1, which indicates that the proliferation promoting effect of the prepared culture medium containing different concentration factors is weaker than that of the culture medium in the control hole.

As shown in FIG. 2, the concentration range of the non-essential amino acid is 50-800. mu.M, and the proliferation effect is most obvious when the non-essential amino acid is added into cells with the concentration of 400. mu.M; the concentration range of the glutamine is 1-8 mM, and the effect of cell proliferation is most obvious when the glutamine is added into cells with the concentration of 4 mM; the concentration range of B27 is 1: 25-1: 400, and the cell proliferation effect is most obvious when the concentration is 1: 50; the concentration range of the insulin is 5-20 mug/mL, and the proliferation effect of the added cells is most obvious when the concentration is 10 mug/mL; the concentration range of the cholera toxin is 0.1-0.4 nM, and the cell proliferation effect is most obvious when the cholera toxin is added into the cell with the concentration of 0.1 nM; the concentration range of the hydrocortisone is 0.1-1.6 mu g/mL, and the cell proliferation effect is most obvious when the hydrocortisone is added into the cell with the concentration of 0.8 mu g/mL; the concentration range of Y27632 is 2.5-40 mu M, and the proliferation effect is most obvious when the concentration is 10 mu M and the cell is added; the concentration range of the FBS is 1: 5-1: 40, and the cell proliferation effect is most obvious when the FBS is added in a ratio of 1: 10; the concentration range of N2 is 1: 25-1: 400, and the cell proliferation effect is most obvious when the N2 is added in a ratio of 1: 50.

Example 4 culture and characterization of gastric cancer Primary cells

Gastric cancer primary cells obtained from an endoscopic tissue sample (sample 10) according to the method of 3 of step (2) of example 1 and cultured using the GM-1 medium of example 2 were cultured to obtain gastric cancer primary cells at a viable cell density of 1X 10⁴Per cm²Seeded in 12-well plates (4.5 ten thousand cells per well) at a cell density of 2X 10⁴Per cm²Adding NIH-3T3 cells irradiated by gamma ray (irradiation dose 30Gy), and mixing. Sterilizing the surface, and placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) for culture.

On day 12, the cultured gastric cancer primary cells were observed with a microscope (EVOS M500, Invitrogen), and photographs taken under 4-fold objective lens and 10-fold objective lens in fig. 3 (a) and (B), respectively, were taken, and the cells were closely arranged under the lens and were slightly irregular in morphology.

On the 12 th day, the cultured cells were rinsed with 200. mu.l of 0.05% trypsin (from Gibco) for 1 minute, aspirated, and 500. mu.l of 0.05% trypsin (from Gibco) was added to each well, and the mixture was incubated at 37 ℃ and 5% CO₂The reaction was carried out in an incubator for 10 minutes until complete digestion of the cells was observed under a microscope (EVOS M500, Invitrogen), and after centrifugation at 1500rpm for 4 minutes, the supernatant was discarded and 500. mu.l of GM-1 medium was added for resuspension, and the cultured gastric cancer primary cells were identified.

100 microliters of the gastric cancer primary cell suspension smear is taken for carrying out Rui's-Giemsa staining identification. The dyeing method is as follows.

(1) Air drying the cell suspension smear, dripping 1 drop of Rui's-Giemsa A liquid (purchased from Besoh corporation), then dripping 3 drops of Rui's-Giemsa B liquid (purchased from Besoh corporation), mixing uniformly and staining for 3 minutes;

(2) flushing with running water, wherein the dye solution cannot be poured out firstly during flushing, and the dye solution is flushed with the running water to prevent sediment from being deposited on the specimen;

(3) dried and photographed under a microscope (Oxlinbas CX 41).

FIG. 4 shows the results of Reishi Giemsa staining identification of gastric cancer primary cells after in vitro culture. Fig. 4 (a) and (B) are photographs taken with a 4-fold objective lens and a 10-fold objective lens, respectively, and the nuclei are large and deep-stained, and correspond to the characteristics of cancer cells.

And (3) performing immunofluorescence staining on the gastric cancer primary cells cultured by the sample. The dyeing method is as follows.

(1) Climbing sheet

Cultured gastric cancer primary cells were seeded on a cell slide (purchased from Saimer fly) and placed at 37 ℃ with 5% CO₂Culturing in an incubator until the cells adhere to the wall.

(2) Fixing

PBS (from Shanghai Producer) was prepared into 4% formaldehyde (from Sigma) and stored in a refrigerator at 4 ℃ for further use.

② after the cells adhere to the wall, abandoning the culture solution, and fixing the cells on 4% formaldehyde ice for 30 minutes. PBS (from shanghai bio-workers) for 5 min x3 times.

(3) Transparent (avoid light)

Preparing a transparent liquid: PBS + 0.3% H₂O₂(from Shanghai Producer) + 0.3% Triton X-100 (from Shanghai Producer).

② transparent: discard PBS, add transparent solution, avoid light, shake table (about 100 rpm) transparent 30 minutes, PBS wash 5 minutes x3 times.

(4) Sealing of

5% BSA (from Shanghai Producer) was formulated for blocking using PBS + 0.3% Triton X-100 and blocked for 30min at 37 ℃.

(5) Primary antibody incubation

PBS + 0.3% Triton X-100 is prepared for diluting antibodies, specific antibodies Pan-keratin, MUC1, P63 and Napsina (the antibodies are all purchased from CST company) are diluted according to a ratio of 1:50, a sealing solution is discarded, a prepared primary antibody diluent is added, and the mixture is incubated overnight in a refrigerator at 4 ℃, wherein the Pan-keratin epithelial tissue is highly expressed, the MUC1 is highly expressed in gastrointestinal cancer, the P63 is highly expressed in squamous cancer, and the Napsina is highly expressed in adenocarcinoma cells.

② taking out the mixture at 4 ℃, balancing the mixture to room temperature, continuing to incubate for 1 hour at 37 ℃, washing the mixture with PBS for 5 minutes and 3 times.

(6) Second antibody incubation (avoid light)

PBS + 0.3% Triton X-100 was prepared for dilution of the secondary antibody, a fluorescent secondary antibody (purchased from Samera Feiki) with excitation light 488 and rabbit species was diluted at a ratio of 1:1000, incubated for 1h at room temperature in the dark, washed with PBS for 5 minutes X3 times.

(7) DAPI staining (avoid light)

DAPI (purchased from Sigma) was diluted in 1000PBS, stained for 5 min at ambient temperature in the dark, and washed 5 min x3 times with PBS. Images were taken under a microscope (EVOS M500, Invitrogen) and recorded by photography.

Fig. 5 shows the results of immunofluorescence staining identification of gastric cancer primary cells after in vitro culture, which are pictures photographed by fluorescence under a 10-fold objective lens. As shown in the figure, Pan-keratin, MUC1 and P63 are highly expressed, and Napsina is low expressed, which indicates that the sample is the squamous carcinoma of stomach, and the result is consistent with the result of hospital pathological diagnosis.

Example 5 Primary culture period and cell count statistics of gastric cancer Primary cells and calculation of the dosage double (PD) value

Gastric cancer primary cells were obtained from 5 samples of gastric cancer tissue samples (samples 11 to 15) according to the method of 3 of step (2) in example 1. The obtained gastric cancer primary cells were cultured in the GM-1 medium of example 2 at a viable cell density of 1X 10⁴Per cm²The cells were seeded in a 6-well plate and cultured, digested and counted after the cells were expanded to 85%, and the number of days of culture until digestion was recorded as one culture cycle. The culture was continued under the experimental conditions, the cells obtained by amplification were subjected to different generations of amplification, digestion was performed for each generation, the corresponding culture period was counted, and PD was calculated according to the formula of amplification double (PD) 3.32 log10 (total number of cells after digestion/initial number of seeded cells). As shown in FIG. 6, the abscissa represents the number of days of cell culture, the ordinate represents the cumulative cell growth rate, and represents the cell growth rate in the culture cycle, and a larger value represents a larger number of times the cells have been grown in a given cycle, i.e., a larger number of cells have been grown, and the slope represents the cell growth rate.

As can be seen from FIG. 6, when 5 samples were cultured using the gastric cancer primary cell culture medium of the present invention, the rate of cell expansion remained substantially unchanged for at least 80 days, and the ability to continue the expansion was maintained.

As shown in Table 1, the average culture period obtained by culturing 5 samples was 14.4 days, and the average number of cells obtained by amplification was 86.87 ten thousand.

TABLE 3 tissue culture period for gastric cancer samples

Note: cumulative cell doubling of 3.32 log10 (total number of cells/number of initial seed after digestion)

Example 6 comparison of culture Effect with existing Medium

(1) Preparation of culture medium

The FM culture medium is a common culture medium in the prior art, and the formula of the FM culture medium is as follows: DMEM/F12 medium + 10% FBS + 5. mu.g/ml insulin +250ng/ml amphotericin B (from Selleck Inc.) + 10. mu.g/ml gentamycin (from MCE Inc.) +0.1nM cholera toxin +0.125ng/ml EGF +25ng/ml hydrocortisone + 10. mu. M Y27632;

(2) acquisition and culture of gastric cancer primary cells

Gastric cancer primary cells were obtained from the intraoperative tissue sample (sample 16) according to the method of 3 of step (2) of example 1, and cultured in the presence of feeder cells and in the absence of feeder cells, respectively.

When feeder cells were present, the FM medium and the GM-1 medium of example 2 were used, respectively, at a viable cell density of 1X 10⁴Per cm²Seeded in 48-well plates (1 ten thousand cells per well) and subsequently at a cell density of 2X 10⁴Per cm²NIH-3T3 cells irradiated by gamma rays (irradiation dose is 30Gy) are added, and finally, the pore volumes in 48-pore plates are respectively filled to 500 microliter by using corresponding culture media, and the mixture is fully and uniformly mixed. Sterilizing the surface, and placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) for culture. And (5) carrying out passage until the cells in the 48-well plate grow to more than 85%.

In the absence of feeder cells, the above FM medium and GM-1 medium of example 2 were used, respectively, at a viable cell density of 1X 10⁴Per cm²The cells were inoculated into 48-well plates (1 ten thousand cells per well), and the well volumes in the 48-well plates were filled up to 500. mu.l each with the corresponding medium, and mixed well. Sterilizing the surface, and placing at 37 deg.C and 5% CO₂Incubators (purchased from Saimeri fly) for culture. And (5) carrying out passage until the cells in the 48-well plate grow to more than 85%.

On day 7 of incubation, 48 well plates were removed, rinsed for 1 minute with 200 microliters of 0.05% trypsin (from Gibco), and after aspiration 250 microliters of 0.05% trypsin (from Gibco) was added to each well. Placing at 37 ℃ and 5% CO₂The reaction was carried out in an incubator for 10 minutes until the cells were completely digested under a microscope (EVOS M500, Invitrogen) and then the digestion was terminated. Centrifuging at 1500rpm for 4min, discarding supernatant, adding 1 ml of basic culture medium, resuspending, counting with flow image counter (JIMBIO FIL, Ohio Microbiol., Jiangsu),the total number of cells was obtained, and the counting results are shown in FIG. 7.

As can be seen from the results in fig. 7, GM-1 significantly promotes the amplification of gastric cancer primary cells compared to FM medium, regardless of the presence or absence of trophoblasts, and the effect is superior to that of FM medium used in the prior art, and the effect of promoting the amplification of gastric cancer primary cells is more significant in the presence of trophoblasts.

Example 7 amplification of gastric cancer Primary cells Using the culture Medium of the present invention for drug screening

1. Cell culture and plating

From the obtained gastric cancer endoscopic sample (sample 17), gastric cancer primary cells were isolated and cultured using GM-1 medium in the same manner as in example 1, and digestion passaging was performed until the cells were expanded to 85% to obtain one generation. And respectively taking the cultured cells of the 1 st generation, the 2 nd generation, the 3 rd generation, the 4 th generation and the 5 th generation for drug screening.

The number of cell digestions was counted according to the procedure of example 1, and the cells were cultured in a viable cell density of 4X 10 using GM-1 medium⁴After the cells were mixed well in the sample addition well (purchased from corning), the cells were cultured in 384-well opaque white cell culture plates (purchased from corning) in a volume of 50. mu.L per well and in a number of 2000 cells/well. GM-1 medium plates were added from the edges of the well plates and labeled with the sample name and CellTiter-Glo (from Promega) assay time. Sterilizing with 75% alcohol (purchased from Lierkang) on surface, standing at 37 deg.C and 5% CO₂Culturing in incubator, and adding medicine after 24 hr.

2. Screening drug formulations

2 drugs (cytarabine, bortezomib; both from MCE) were prepared in 7 concentration gradients according to the following table, and 30. mu.L of each drug was added to a 384-well plate (from Saimer Miller) and stored at-20 ℃ until use.

TABLE 4 preparation of cytarabine and bortezomib drug additive solution

Final concentration (μ M)	Concentration of formulation (μ M)
		10	5000
3.33	1666.7
		1.11	555.6
0.37	185.2
		0.12	61.7
0.04	20.6
		0.01	6.9

3. High flux dosing

The prepared drug plate is taken out, placed at room temperature, placed in a centrifuge (Sigma company 3-18K) after being completely melted, centrifuged at 1000rpm at room temperature for 1 minute and taken out. High-throughput dosing was performed using a high-throughput automated loading system (JANUS, Perkin Elmer). Adding 0.1 microliter of screening medicine with corresponding concentration into each well of the 384-well plate cultured with the gastric cancer cells, after the medicine is added, sterilizing the surface of the 384-well plate, transferring the 384-well plate into an incubator to continue culturing, and measuring the cell activity after 72 hours.

4. Cell viability assay

The CellTiter-Glo luminescence reagent (purchased from Promega) was taken out from a refrigerator at 4 ℃ and 10ml of the reagent was put in a sample addition tank, a 384-well plate to be tested was taken out from an incubator, 10. mu.L of the CellTiter-Glo luminescence reagent was added to each well, and the plate was left to stand for 10min and then detected using a multifunctional microplate reader (Envision, Perkin Elmer).

5. Data processing

The cell viability after the cells acted with different drugs was calculated according to the formula of cell viability (%). wells chemiluminescence/control wells chemiluminescence x 100%, and half inhibition of drug action on cells was calculated using graphpad prism software (IC 50). The results are shown in FIG. 8.

As can be seen from fig. 8, when the gastric cancer cells cultured in the gastric cancer primary cell culture medium of the present invention were subjected to drug screening, the inhibitory effects of the same drug on different generations of cultured cells were substantially consistent (the inhibitory curves were substantially consistent).

Claims

1. A gastric cancer primary cell culture medium comprising:

a starter culture medium, a Rho protease inhibitor, an antibiotic, insulin, a non-essential amino acid, hydrocortisone, a cholera toxin, glutamine, fetal calf serum, and at least one additive selected from the group consisting of a B27 additive and an N2 additive,

the initial culture medium is selected from DMEM/F12, DMEM, F12 or RPMI-1640.

2. Gastric cancer primary cell culture medium according to claim 1, characterized in that:

the Rho protein kinase inhibitor is selected from one or more of Y27632, hydroxyfasudil and GSK429286A, and when the Rho protein kinase inhibitor is selected from Y27632, the concentration range is 2.5-40 mu M, preferably 10-20 mu M; when the fasudil is selected from hydroxyl fasudil, the concentration range is 2-32 mu M, and preferably 4-16 mu M; when the GSK429286A is selected, the concentration range is 2-32 μ M, preferably 4-16 μ M;

the antibiotics are selected from one or more of streptomycin/penicillin, amphotericin B and Primocin, when the antibiotics are selected from streptomycin/penicillin, the concentration range of the streptomycin is 25-400 mu g/mL, preferably 50-200 mu g/mL, more preferably 200 mu g/m, the concentration range of the penicillin is 25-400U/mL, preferably 50-200U/mL, more preferably 200U/mL; when the amphotericin B is selected, the concentration range is 0.25-4 mug/mL, preferably 0.5-2 mug/mL; when the Primocin is selected, the concentration range is 25-400 mu g/mL, preferably 50-200 mu g/mL;

the concentration range of the insulin is 5-20 mug/mL, preferably 10-20 mug/mL;

the nonessential amino acid is one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the total concentration range of the nonessential amino acid is 50-800 mu M, preferably 100-400 mu M;

the concentration range of the hydrocortisone is 0.1-1.6 mu g/mL, preferably 0.2-0.8 mu g/mL;

the concentration range of cholera toxin is 0.1-0.4 nM, preferably 0.1-0.2 nM

The concentration range of the glutamine is 1-8 mM, preferably 1-4 mM;

the volume ratio of the fetal calf serum to the culture medium is 1: 5-1: 40;

the volume ratio of the B27 additive to the culture medium is 1: 25-1: 400, preferably 1: 25-1: 200;

the volume ratio of the N2 additive to the culture medium is 1: 25-1: 400, preferably 1: 25-1: 100.

3. A method for culturing primary gastric cancer cells is characterized by comprising the following steps:

culturing gastric cancer primary cells using the gastric cancer primary cell culture medium of claim 1 or 2.

4. The culture method according to claim 3, wherein:

in the culture, the cell density is 2-3 × 10⁴Per cm²Feeder cells are added.

5. The culture method according to claim 4, wherein:

the feeder cells are irradiated NIH-3T3 cells, the irradiation source is X rays or gamma rays, and the irradiation dose is 30-50 Gy.

6. The method for screening the drugs for gastric cancer is characterized by comprising the following steps:

(1) culturing the gastric cancer primary cells by using the method for culturing the gastric cancer primary cells according to any one of claims 3 to 5;

(3) adding the diluted medicine to the cells cultured in (1);

(4) cell viability assays were performed.