Culture medium and culture method for primary cells of intestinal cancer

Abstract

A culture medium and a culture method for in vitro expansion of primary intestinal cancer cells are disclosed. The culture medium includes an initial culture medium, a Rho protease inhibitor, an antibiotic, gastrin, A8301, a non-essential amino acid, cholera toxin, insulin-like growth factor-1, nicotinamide, insulin, fetal bovine serum, and an additive selected from at least one of B27 additive and N2 additive. By using this culture medium, an effective and rapid expansion of primary intestinal cancer cells can be achieved. The expanded cells maintain the pathological characteristics of the patient, and the success rate of the culture of primary intestinal cancer cells and the rate of cell expansion are improved, providing a research platform for personalized treatment of patients.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, in particular to culture media and methods for rapidly expanding primary cells of intestinal cancer, and their use in drug efficacy evaluation and screening. [Background technology]

[0002] Colon cancer, a disease caused by the interaction of genetic and environmental factors, is one of the most common gastrointestinal tumors worldwide. Survey data (Non-Patent Document 1) show that colon cancer is one of the most common tumors, with both overall incidence and mortality rates on the rise, making it a malignant tumor that poses a serious threat to life and health. Surgery combined with postoperative chemotherapy is currently the main treatment for colon cancer. While improvements in surgical techniques have improved the survival rate of colon cancer patients in recent years, tumor metastasis and recurrence still pose a poor prognosis. In terms of precision colon cancer treatment, the emergence of targeted drugs has brought hope to patients with advanced colon cancer. The future lies in how to rationally select targeted drugs and develop personalized treatment plans. Continuous innovation in drug sensitivity testing technology provides strong technical support for predicting the efficacy of targeted drugs, chemotherapeutic agents, and targeted drug combinations, laying a solid foundation for the realization of personalized treatment for colon cancer patients.

[0003] Existing in vitro cultured intestinal cancer cell lines are primarily derived by spontaneously immortalizing normal cells through long-term culture or by transfecting oncogenes that promote the immortalization of normal cells. Cell lines established by traditional methods remain central to cell biology, molecular biology, and cancer biology research. However, these methods alter the genetic background of cells, and long-term cultured cell lines are prone to genomic instability, which can lead to artificial changes in tumor cell lines and tumor cell phenotypes in vivo. These cell lines typically lack the complex heterogeneity of primary tumors, limiting their application in predicting tumor cell responses and affecting the accuracy of scientific research and drug development in intestinal cancer. In addition, the process of culturing cells obtained from intestinal cancer tissue to form cancer cells is difficult using traditional culture methods, and there are issues such as interference from fibroblasts during the culture process, making it impossible to subculture the clones formed, limiting the application of human primary intestinal cancer cells.

[0004] In 2017, Xuefeng Liu et al. used irradiated mouse fibroblasts and a Rho-associated kinase inhibitor (Y-27632) to expand epithelial-derived cells. This system has the ability to achieve unlimited growth of epithelial-derived cells without genetic manipulation (Non-Patent Document 2). However, the method established by Xuefeng Liu et al. requires a long culture period and is unable to achieve rapid cell expansion, limiting the application of this technology. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Shu Zheng et al., Prevention of Colorectal Cancer, Chinese Journal of Oncology, 2004, vol. 13, NO. 1, pp1-2 [Non-patent document 2] Xuefeng Liu et al., Conditional reprogramming and long-term expansion of normal and tumor cells from human biospecimens. Nat. Protoc. 2017, 12, 439 Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding primary intestinal cancer cells in vitro.

[0007] One aspect of the present invention provides a culture medium for primary intestinal cancer cells, comprising an initial culture medium, a Rho protease inhibitor, an antibiotic, gastrin, A8301, a non-essential amino acid, cholera toxin, insulin-like growth factor-1, nicotinamide, insulin, fetal bovine serum, and an additive selected from at least one of B27 additive and N2 additive, wherein the initial culture medium is selected from the group consisting of DMEM / F12, DMEM, F12, or RPMI-1640.

[0008] Preferred embodiments of the present invention include at least one of the following aspects: (1) The Rho protease inhibitor is one or more selected from the group consisting of Y27632, fasudil, and H-1152, preferably Y27632, and the concentration is preferably 2.5 μM to 40 μM, more preferably 2.5 μM to 5 μM, and most preferably 5 μM; (2) The antibiotic is one or more selected from the group consisting of streptomycin / penicillin, amphotericin B, and Primocin, and in the case of streptomycin / penicillin, streptomycin has a concentration of 25 μg / mL to 400 μg / mL, preferably within a range of 50 μg / mL to 200 μg / mL, and more preferably 200 μg / mL; penicillin has a concentration of 25 U / mL to 400 U / mL, preferably within a range of 50 U / mL to 200 U / mL, and more preferably 200 U / mL; amphotericin B has a concentration of 0.25 μg / mL to 4 μg / mL, and preferably within a range of 0.5 μg / mL to 2 μg / mL; and Primocin has a concentration of 25 μg / mL to 400 μg / mL, and preferably within a range of 50 μg / mL to 200 μg / mL; (3) The concentration range of gastrin is preferably 1.25 nM to 20 nM, more preferably 2.5 nM to 10 nM, and most preferably 5 nM; (4) The concentration range of A8301 is preferably 25 nM to 200 nM, more preferably 200 nM; (5) The non-essential amino acids are one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and the total concentration range of the non-essential amino acids is 25 μM to 400 μM, more preferably 50 μM; (6) The concentration range of cholera toxin is preferably 1.25 ng / mL to 20 ng / mL, more preferably 2.5 ng / mL to 5 ng / mL, and most preferably 5 ng / mL; (7) The concentration range of insulin-like growth factor-1 is preferably 5 ng / mL to 405 ng / mL, more preferably 45 ng / mL; (8) The concentration range of nicotinamide is preferably 2 mM to 8 mM, more preferably 4 mM to 8 mM, and most preferably 4 mM; (9) The concentration range of insulin is preferably 0.5 μg / mL to 4 μg / mL, more preferably 1 μg / mL to 2 μg / mL, and most preferably 2 μg / mL; (10) The volume ratio of fetal bovine serum to the culture medium is preferably 2.5% (v / v) to 5% (v / v), more preferably 5% (v / v); (11) The volume ratio of the B27 additive or the N2 additive to the culture medium is preferably 1:25 to 1:200, more preferably 1:25 to 1:50, and most preferably 1:25.

[0009] The present invention also provides a method for culturing primary intestinal cancer cells, in which the primary intestinal cancer cells are cultured using the culture medium for primary intestinal cancer cells of the present invention.

[0010] The method for culturing primary intestinal cancer cells of the present invention comprises the following steps.

[0011] 1. Isolation of Intestinal Cancer Primary Cells (1) After washing with a washing medium, the tissue sample or endoscopic sample is added with a washing medium and a tissue digestion solution (the amount of tissue digestion solution added is approximately 5 mL to 10 mL per 1 g of tumor tissue) in a ratio of 1:3, placed in a thermostatic shaker, and digested at a digestion temperature in the range of 4°C to 37°C, preferably 37°C, and a digestion rotation speed in the range of 200 rpm to 350 rpm, preferably 300 rpm. (2) Digestion can be completed when no obvious tissue mass remains, and the digestion time is in the range of 3 to 6 hours, preferably 4 hours. (3) After centrifugation at a speed of 1200 to 1600 rpm, preferably 1500 rpm, for 2 to 6 minutes, preferably 4 minutes, the supernatant is discarded and the resulting material is resuspended in DMEM / F12 medium containing 10% serum.

[0012] 2. Cultivation using the culture medium for primary colon cancer cells of the present invention The primary intestinal cancer cells obtained in step 1 above were resuspended in the culture medium for primary intestinal cancer cells of the present invention, counted, and plated in a culture dish at a density of 1 × 10 4 cells / cm 2 ~10×10 4 cells / cm 2At the same time, trophoblast cells were seeded at a cell density of 2 × 10 4 cells / cm 2 ~3×10 4 cells / cm 2 The cells are grown until they cover more than 90% of the culture dish, after which the cells are digested for passage.

[0013] Specifically, the washing medium described in step 1 is DMEM / F12 medium containing 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / mL). The tissue digestion solution described in step 1 is 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), and BSA (10 mg / mL). The trophoblast cells described in step 2 are, for example, irradiated NIH-3T3 cells, with the radiation source being X-rays or gamma rays, preferably gamma rays, and the radiation dose being 20 Gy to 50 Gy, preferably 30 Gy.

[0014] The present invention also provides a method for screening drugs for intestinal cancer, comprising the steps of: (1) culturing primary intestinal cancer cells for drug screening using the method for culturing primary intestinal cancer cells of the present invention; (2) selecting a test drug and diluting the drug based on the required concentration gradient; (3) adding a drug at various concentration gradients to the cells cultured in step (1); (4) detecting cell viability; The present invention provides a method comprising:

[0015] The technical solution of the present invention can bring about the following technical effects: (1) The success rate of primary intestinal cancer cell culture can be improved, reaching over 80%. (2) Primary intestinal cancer cells cultured in vitro can maintain the pathological characteristics of patients. (3) Cultured primary colon cancer cells are not interfered with by mesenchymal cells such as fibroblasts and adipocytes. (4) Primary colon cancer cells can be efficiently expanded, and 10 5 Starting with cell counts at levels of 10 within approximately one week 6 Large-scale cell expansion was successfully achieved, and the expanded primary colon cancer cells have the ability to be serially passaged. (5) The culture medium does not require expensive factors such as Wnt agonists, R-spondin family proteins, BMP inhibitors, and FGF10, making it possible to control culture costs. (6) This technology enables the cultivation and provision of large quantities of primary intestinal cancer cells with high uniformity, making it suitable for high-throughput screening of new candidate compounds and for high-throughput in vitro drug sensitivity functional testing in patients. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing the effect of different combinations of factors added to the culture medium for primary intestinal cancer cells on the proliferation of primary intestinal cancer cells. [Figure 2-1] 10(A) to 10(D) are graphs showing the effects of factors added at different concentrations to the culture medium for primary intestinal cancer cells of the present invention on the culture of primary intestinal cancer cells. [Figure 2-2] (E) to (J) are graphs showing the effects of different concentrations of factors added to the culture medium for primary intestinal cancer cells of the present invention on the culture of primary intestinal cancer cells. [Figure 3] (A) to (H) are micrographs of primary intestinal cancer cells obtained by culturing using the culture medium for primary intestinal cancer cells of the present invention. [Figure 4] (A) to (D) show the immunohistochemical results of cells from the original intestinal cancer tissue. [Figure 5] 1(A) to 1(D) are diagrams showing the results of immunohistochemistry of primary intestinal cancer cells cultured using the culture medium CA-1 of the present invention. [Figure 6]FIG. 1 shows the results of immunofluorescence staining of primary intestinal cancer cells cultured using the culture medium for primary intestinal cancer cells of the present invention. [Figure 7] FIG. 1 shows the cell growth curve of primary intestinal cancer cells cultured using the culture medium for primary intestinal cancer cells of the present invention. [Figure 8] 1A and 1B are diagrams showing the comparative results of culturing primary intestinal cancer cells using the culture medium CA-1 for primary intestinal cancer cells of the present invention and the conventional culture medium LXF. [Figure 9] (A) and (B) show the results of drug screening using intestinal cancer cells of different generations cultured using the culture medium for primary intestinal cancer cells of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] For a better understanding of the present invention, the following further description is provided in conjunction with embodiments and drawings. The following examples are presented for illustrative purposes only, and are not intended to limit the present invention. [Example]

[0018] Example 1. Effect of various factors added to the culture medium for primary intestinal cancer cells on the proliferation of primary intestinal cancer cells (1) Preparation of culture medium for primary colon cancer cells First, a basal medium was prepared, which consisted of DMEM / F12 medium (purchased from Corning) + 5 μM Y27632 (purchased from MCE) + 5% (v / v) fetal bovine serum (purchased from Excell Bio) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v); the commercially available concentration is 50 mg / ml).

[0019] Different types of additives (see Table 1) were added to the basal medium to prepare culture media for primary intestinal cancer cells containing different components.

[0020] (2) Isolation and processing of primary colon cancer cells 1. Sample Selection Tissue samples (intraoperative / endoscopic) of solid colon cancer tumors were collected from patients by specialized medical personnel at specialized medical institutions, and all patients signed informed consent. The size of the intraoperative sample was 0.25 cm. 3 The size of the endoscope sample is 0.025 cm 3 A commercially available tissue preservation solution (manufacturer: Miltenyi Biotec) was used for storage and transportation.

[0021] 2. Material Preparation After surface sterilization, 15 mL sterilized centrifuge tubes, pipettors, 10 mL pipettes, and sterilized pipette tips were placed on an ultraclean work bench and irradiated with UV light for 30 minutes. The washing medium was removed from a 4°C refrigerator 30 minutes before the procedure, and the tissue digestion solution was removed from a -20°C refrigerator 30 minutes before the procedure.

[0022] Washing medium: DMEM / F12 medium containing 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available concentration is 50 mg / ml).

[0023] Tissue digestion solution: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), BSA (10 mg / mL).

[0024] The above-mentioned collagenase II, collagenase IV, DNase, and hyaluronidase were all purchased from Sigma Corporation, calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd., and BSA was purchased from Biofroxx Corporation.

[0025] 3. Sample Isolation 3.1. The tissue sample was transferred from the ultraclean work bench to a culture dish, and blood-contaminated tissue was removed. The tissue sample was washed twice with washing medium, then transferred to another culture dish and cut into 1 × 1 × 1 mm pieces with a sterile scalpel.3 The tissue was mechanically cut into tissue blocks of about 1 / 4 inch in size.

[0026] 3.2. The cut intraoperative or endoscopic tissue was aspirated into a 15 mL centrifuge tube, to which 5 mL of washing medium was added, mixed thoroughly, and then centrifuged at 1500 rpm for 4 minutes.

[0027] 3.3. The supernatant was discarded, and a 1:3 mixture of washing medium and tissue digestion solution (approximately 10 mL of tissue digestion solution was added to 1 g of tumor tissue). The samples were labeled and numbered, sealed with sealing film, and then digested in a shaker (Zhichu Instrument ZQLY-180N) at 37°C and 300 rpm. Completion of digestion was determined by the presence or absence of visible particles, observed every 30 minutes. The digestion time was 4 hours.

[0028] 3.4. After digestion was complete, the undigested tissue mass was filtered through a 100 μM filter mesh. The tissue mass on the filter mesh was washed into a centrifuge tube with wash medium to reduce cell loss. The resulting mass was centrifuged at 25°C, 1500 rpm, for 4 minutes.

[0029] 3.5. The supernatant was discarded and the resulting material was observed to determine the presence of blood cells. If blood cells were present, 8 mL of blood cell lysis solution (Sigma) was added to the resulting material, mixed thoroughly, and lysed at 4°C for 20 minutes, with one inversion during this process. The resulting material was centrifuged at 25°C, 1500 rpm, and 4 minutes.

[0030] 3.6. The supernatant was discarded, and 2 mL of DMEM / F12 medium (Excell Bio, FND500) containing 10% serum was added, and the cells were resuspended and stored.

[0031] 4. Cell Counting and Processing 4.1 Microscopic observation: A small amount of resuspended cells was plated on a culture dish, and the density and morphology of cancer cells were observed under a microscope (CNOPTEC, BDS400).

[0032] 4.2 Counting viable cells: 12 μL of the resuspended cell suspension was thoroughly mixed with 12 μL of trypan blue staining solution (Sangon Biotech (Shanghai) Co., Ltd.), and 20 μL of the mixture was added to a cell counting plate (Countstar, specification: 50 cells / box). Using a cell counter (Countstar, IC1000), the percentage of viable large cells (cells larger than 10 μm) was calculated as follows: number of viable cells / total number of cells × 100%.

[0033] (3) Primary cell culture of intestinal cancer Culture media containing various components shown in Table 1 were added to a 48-well plate at a volume of 1 mL / well. Primary intestinal cancer cells isolated from two intestinal cancer tissues (numbers OE0042 and OE(E)003) according to step (2) above were cultured in a 48-well culture plate at a density of 3 × 10 4 Cells were seeded at a cell density of 100 cells / well and cultured at 37°C under 5% CO2. After 7–10 days of culture, when the cells reached 85% growth, the culture medium was discarded and the cells were washed once with 100 μL / well of 0.05% trypsin (purchased from Gibco). After removing the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37°C and 5% CO2 for 10 minutes. Cells were observed under a microscope (CNOPTEC, BDS400), indicating complete digestion. Digestion was stopped by adding 300 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500). 20 μL of the resulting medium was added to a cell counting plate (Countstar, specification: 50 cells / box) and the total number of cells was counted using a cell counter (Countstar, IC1000). Specifically, a basal medium without any additives was used as an experimental control, and the experimental results are shown in Table 1.

[0034] [Table 1]

[0035] Here, "+" indicates that, compared with the basal medium, the medium supplemented with the additive(s) has the effect of promoting the proliferation of primary intestinal cancer cells isolated from intestinal cancer tissue in at least two cases; "-" indicates that the medium supplemented with the additive(s) has the effect of inhibiting the proliferation of primary intestinal cancer cells isolated from intestinal cancer tissue in at least one case; and "◯" indicates that the medium supplemented with the additive(s) has no significant effect on the proliferation of primary intestinal cancer cells isolated from intestinal cancer tissue in at least two cases.

[0036] According to the above results, factors including non-essential amino acids, cholera toxin, insulin-like growth factor-1, B27 or N2, nicotinamide, insulin, prostaglandin E2, A8301 and gastrin were selected for further culture experiments.

[0037] Example 2. The effect of different combinations of factors added to the culture medium of primary intestinal cancer cells on the proliferation of primary intestinal cancer cells Culture media for primary intestinal cancer cells were prepared using different combinations of supplementary factors according to the ingredients shown in Table 2, and the growth-promoting effects of different combinations of supplementary factors on primary intestinal cancer cells were investigated.

[0038] [Table 2]

[0039] Following the method described in step (2)-3 of Example 1, primary intestinal cancer cells were obtained from intestinal cancer tissues (numbers OE0042 and OE(E)006). The resulting cell suspension was divided into 11 equal parts, centrifuged at 1500 rpm for 4 minutes, and then resuspended in 200 μL of BM and each of the media No. 1 to No. 10. The cells were plated in a 48-well plate at 2 × 10 4 cells / cm 2 After seeding at a viable cell density of 20,000 cells per well, γ-irradiated NIH-3T3 cells (30 Gy) (purchased from ATCC, resuspended in basal medium (BM)) were added to the wells at a density of 2 × 10 4 cells / cm 2The cells were added to the plate at a cell density of 1000 x g. Finally, the corresponding culture medium was added to each well of the 48-well plate up to a volume of 1 mL, and the resulting mixture was mixed thoroughly. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation.

[0040] After the cells had grown to cover more than 85% of the 48-well plate's surface area, the medium was discarded. The cells were washed once with 100 μL of 0.05% trypsin (purchased from Gibco). After removing the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was placed in a 37°C, 5% CO2 incubator for 10 minutes, and the cells were observed under a microscope (CNOPTEC, BDS400), indicating complete digestion. The digestion was stopped by adding 300 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500). 20 μL of the resulting medium was added to a cell counting plate (Countstar, specification: 50 cells / box) and the total number of cells was counted using a cell counter (Countstar, IC1000). Results obtained from primary intestinal cancer cells isolated from intraoperative and endoscopic tissue samples OE0042 and OE(E)006 are shown in Figure 1.

[0041] As can be seen from the results shown in Figure 1, compared with the basal medium, the above-mentioned media No. 1 to No. 10 all promoted the proliferation of primary intestinal cancer cells to different levels. The best proliferation effect was achieved when the culture medium containing Y27632, fetal bovine serum, gastrin, A8301, non-essential amino acids, cholera toxin, insulin-like growth factor-1, B27, nicotinamide, and insulin (i.e., culture medium No. 2, hereinafter referred to as "CA-1 medium") was used to culture primary intestinal cancer cells.

[0042] Example 3. Effect of different concentrations of factors added to CA-1 medium on the proliferation of primary colon cancer cells Following the method described in step (2)-3 of Example 1, primary intestinal cancer cells were obtained from endoscopic tissue samples (numbers OE(E)042, OE(E)050, and OE(E)060) and cultured using the CA-1 medium described in Example 2. The obtained primary intestinal cancer cells were plated in a 6-well plate at 1 × 10 4 cells / cm 2 NIH-3T3 cells were seeded at a viable cell density of 100,000 cells per well and then irradiated with γ-rays (30 Gy). 4 cells / cm 2 The cells were added to the plate at a cell density of 1000 kJ / well and mixed thoroughly. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C with 5% CO2. The cells were cultured and expanded in CA-1 medium until they reached a confluence of 85% or more. 500 μL of 0.05% trypsin (purchased from Gibco) was added and washed for 1 minute. After removing the trypsin, 500 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37°C with 5% CO2 for 2 to 10 minutes until the cells were completely digested. Digestion was stopped by adding 500 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500). The resulting mixture was centrifuged at 1500 rpm for 4 minutes, and the supernatant was discarded. The cell pellet was resuspended in DMEM / F12, and 20 μL of the suspension was added to a cell counting plate (manufacturer: Countstar, specifications: 50 cells / box). The total number of cells was counted using a cell counter (Countstar, IC1000). The resulting cells were used in the following culture experiments.

[0043] Next, the following 10 formulations of media were prepared for the experiment: Formulation 1: CA-1 medium without gastrin; Formulation 2: CA-1 medium without A8301; Formulation 3: CA-1 medium without non-essential amino acids; Formulation 4: Cholera toxin-free CA-1 medium, Formulation 5: CA-1 medium without insulin-like growth factor-1; Formulation 6: CA-1 medium without B27; Formulation 7: CA-1 medium without nicotinamide; Formulation 8: CA-1 medium without insulin; Formulation 9: CA-1 medium without Y-27632; Formulation 10: CA-1 medium without fetal bovine serum.

[0044] 1×10 4 20 μl of cell suspension containing cells was added to each well, and the cell suspension was diluted with 1 mL of each of the media of Formulations 1 to 10 described above.

[0045] When using the medium of formulation 1, 1 mL / well of prepared gastrin was added to a 48-well plate seeded with primary cells at final gastrin concentrations of 1.25 nM, 2.5 nM, 5 nM, 10 nM, and 20 nM, respectively, and the medium of formulation 1 was used as a blank control (BC).

[0046] When using formulation 2 medium, 1 mL / well of prepared A8301 was added to a 48-well plate seeded with primary cells at final A8301 concentrations of 25 nM, 50 nM, 100 nM, 200 nM, and 400 nM, respectively, and formulation 2 medium was used as a blank control (BC).

[0047] When using the medium of formulation 3, 1 mL / well of the prepared non-essential amino acids was added to a 48-well plate seeded with primary cells at final concentrations of 25 μM, 50 μM, 100 μM, 200 μM, and 400 μM, respectively, and the medium of formulation 3 was used as a blank control (BC).

[0048] When using the medium of formulation 4, 1 mL / well of prepared cholera toxin was added to the 48-well plate seeded with primary cells at final concentrations of cholera toxin of 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml, 10 ng / ml, and 20 ng / ml, respectively, and the medium of formulation 4 was used as a blank control (BC).

[0049] When using the medium of formulation 5, 1 mL / well of prepared insulin-like growth factor-1 was added to a 48-well plate seeded with primary cells to achieve final insulin-like growth factor-1 concentrations of 5 ng / ml, 15 ng / ml, 45 ng / ml, 135 ng / ml, and 405 ng / ml, respectively, and the medium of formulation 5 was used as a blank control (BC).

[0050] When using the medium of formulation 6, 1 mL / well of prepared B27 was added to a 48-well plate seeded with primary cells at final concentrations of 12.5x, 25x, 50x, 100x, and 200x, respectively, and the medium of formulation 6 was used as a blank control (BC).

[0051] When using Formulation 7 medium, 1 mL / well of prepared nicotinamide was added to a 48-well plate seeded with primary cells to final concentrations of nicotinamide of 1 mM, 2 mM, 4 mM, 8 mM, and 16 mM, respectively, and Formulation 7 medium was used as a blank control (BC).

[0052] When using Formulation 8 medium, 1 mL / well of prepared insulin was added to a 48-well plate seeded with primary cells to final insulin concentrations of 0.25 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, and 4 μg / ml, and Formulation 8 medium was used as a blank control (BC).

[0053] When using formulation 9 medium, 1 mL / well of prepared Y-27632 was added to a 48-well plate seeded with primary cells at final concentrations of 2.5 μM, 5 μM, 10 μM, 20 μM, and 40 μM, respectively, and formulation 9 medium was used as a blank control (BC).

[0054] When using Formulation 10 medium, 1 mL / well of prepared fetal bovine serum was added to a 48-well plate seeded with primary cells at the following fetal bovine serum addition rates: 2.5% (v / v), 5% (v / v), 10% (v / v), 20% (v / v), and 40% (v / v), and Formulation 10 medium was used as a blank control (BC).

[0055] After the cells had expanded to approximately 85% of the 48-well plate, they were digested and counted. The fold expansion was calculated based on the cell count in the blank control (BC). The results are shown in Figures 2A-2J. In Figures 2A-2J, the ratios represent the ratio of the cell count obtained by one subculture in each medium to the cell count obtained by one subculture in the corresponding blank control. A ratio greater than 1 indicates that the conditioned medium containing different concentrations of factors or small molecular compounds has a better growth-promoting effect than the blank control culture medium, while a ratio less than 1 indicates that the conditioned medium containing different concentrations of factors or small molecular compounds has a weaker growth-promoting effect than the blank control culture medium.

[0056] According to the results shown in Figures 2A to 2J, the amount of gastrin is preferably 1.25 nM to 20 nM, more preferably 2.5 nM to 10 nM, and even more preferably 5 nM; the amount of A8301 is preferably 25 nM to 200 nM, and more preferably 200 nM; the amount of non-essential amino acids is preferably 25 μM to 400 μM, and more preferably 50 μM; the amount of cholera toxin is preferably 1.25 ng / ml to 20 ng / ml, more preferably 2.5 ng / ml to 5 ng / ml, and even more preferably 5 ng / ml; the amount of insulin-like growth factor-1 is preferably 5 ng / ml to 405 ng / ml, and more preferably 45 ng / ml; and the volume of B27 The concentration is preferably 25-fold to 200-fold, more preferably 25-fold to 50-fold, and even more preferably 25-fold; the amount of nicotinamide in the culture medium is preferably 2 mM to 8 mM, more preferably 4 mM to 8 mM, and even more preferably 4 mM; the amount of insulin is preferably 0.5 μg / ml to 4 μg / ml, more preferably 1 μg / ml to 2 μg / ml, and even more preferably 2 μg / ml; the amount of Y-27632 is preferably 2.5 μM to 40 μM, more preferably 2.5 μM to 5 μM, and even more preferably 5 μM; and the volume content of fetal bovine serum is preferably 2.5% (v / v) to 5% (v / v), and more preferably 5% (v / v).

[0057] Example 4. Cultivation and identification of primary cells of intestinal cancer (1) Primary cell culture of intestinal cancer Following the method described in step (2)-3 of Example 1, primary intestinal cancer cells were obtained from endoscopic tissue samples (numbers OE(E)019, OE(E)028, OE(E)021, OE(E)023, OE(E)026, OE(E)025, OE(E)024, and OE(E)004) and cultured using the CA-1 medium described in Example 2. The obtained primary intestinal cancer cells were plated in a 6-well plate at 1 × 10 4 cells / cm 2 NIH-3T3 cells were seeded at a viable cell density of 100,000 cells per well and then irradiated with γ-rays (30 Gy). 4 cells / cm 2 After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation.

[0058] Cultured primary intestinal cancer cells were observed under a microscope (EVOS M500, Invitrogen). Figures 3A to 3H are photographs taken under a 10x objective lens. The cells were densely arranged under the microscope and had a slightly irregular shape.

[0059] (2) Immunohistochemical identification of colon cancer cells subcultured from colon cancer tissue Approximately 0.25cm 3 Cancer tissue (sample number: OE(O)001) was collected intraoperatively from a patient with intestinal cancer and immersed in 1 mL of 4% paraformaldehyde for fixation. Using the method described in Example 3, sample OE(O)001 was continuously cultured up to the fifth passage using the medium CA-1 of the present invention. The 4% paraformaldehyde-fixed tissue or cells were embedded in paraffin and cut into 4 μM-thick tissue sections using a microtome. Then, conventional immunohistochemical assays (see Li et al., Nature Communication, (2018) 9: 2983 for specific procedures) were performed. The primary antibodies used were ki-67, CK20, CDX-2, and villin (all purchased from CST).

[0060] Figures 4A-4D and 5A-5D are comparative photographs of immunohistochemistry results for intestinal cancer tumor cells obtained by culturing the original tissue cells using the CA-1 medium of the present invention. Figures 4A and 5A are photographs of intestinal cancer tissue labeled with Ki-67 antibody and the cells obtained after expansion and culture, respectively. Figures 4B and 5B are photographs of intestinal cancer tissue labeled with CK20 antibody and the cells obtained after expansion and culture, respectively. Figures 4C and 5C are photographs of intestinal cancer tissue labeled with CDX-2 antibody and the cells obtained after expansion and culture, respectively. Figures 4D and 5D are photographs of intestinal cancer tissue labeled with villin antibody and the cells obtained after expansion and culture, respectively. These results confirm that when intestinal cancer tumor cells (sample number: OE(O)001) cultured using the present technology were cultured up to passage 5, the expression of intestinal cancer-related biomarkers on the cells was essentially consistent with that of the original tissue section from which the cells were derived. This suggests that the cells cultured by the technique of the present invention maintain the original pathological characteristics of the cancer tissue of patients with intestinal cancer.

[0061] (3) Immunofluorescence identification of primary intestinal cancer cells after subculture Sample OE(O)001 was cultured in CA-1 medium from Example 2 until cells reached 85% growth. 500 μL of 0.05% trypsin (purchased from Gibco) was added and washed for 1 minute. After removing the trypsin, 500 μL of 0.05% trypsin was added to each well. The plate was then placed in an incubator at 37°C and 5% CO2 for 2 to 10 minutes until the cells were completely digested. 500 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500) was added to stop the digestion. The mixture was centrifuged at 1500 rpm for 4 minutes, and the supernatant was discarded. The mixture was resuspended in 500 μL of CA-1 medium. Cultured primary intestinal cancer cells were identified by immunofluorescence staining.

[0062] Cultured primary intestinal cancer cells were seeded onto a cell slide (purchased from Thermo Fisher) and cultured in an incubator at 37°C with 5% CO2 until the cells attached to the wall, and then cultured for an additional 2 to 3 days.

[0063] When the cells had grown to cover 80% of the bottom surface, the culture medium was discarded. The slides were washed once with PBS (purchased from Shanghai Sangon), and 300 μL of 4% paraformaldehyde (biosharp, BL539A) was added. The slides were then left to stand at room temperature for 20 minutes to fix the cells. The slides were then washed three times with PBS for 5 minutes. Next, a 5% volumetric BSA (purchased from Shanghai Sangon) solution was prepared using PBS + 0.3% Triton X-100 (purchased from Shanghai Sangon). Blocking was performed in a 37°C water bath for 30 minutes. Antibodies were diluted in primary antibody diluent (Beyotime, P0023A). Specific antibodies against ki-67, CK20, CDX-2, and villin (all purchased from CST) were diluted 1:50. The blocking solution was removed, and the prepared primary antibodies were added. The slides were then incubated overnight in a 4°C refrigerator. Specifically, CK20 was expressed in nearly all intestinal adenocarcinomas, and colonic adenocarcinomas were identified as CDX-2(+) and villin(+).

[0064] The next day, the slides were removed from the 4°C refrigerator, allowed to equilibrate to room temperature, and then incubated at 37°C for 1 hour. They were then washed with PBS for 5 minutes, and this was repeated three times. Primary antibody dilution solution was prepared for secondary antibody dilution. Fluorescent secondary antibodies (purchased from Thermo Fisher, rabbit or mouse) were diluted 1:1000 with an excitation light of 488 nm. The slides were incubated in the dark at room temperature for 1 hour, and then washed with PBS for 5 minutes, and this was repeated three times.

[0065] DAPI (purchased from Sigma) was diluted 1:1000 with PBS, stained for 5 minutes at room temperature in the dark, washed with PBS for 5 minutes, and repeated three times. Imaging was performed under a microscope (EVOS M500, Invitrogen), photographed, and recorded.

[0066] Figure 6 shows the immunofluorescence staining and identification of primary intestinal cancer cells cultured in vitro from sample OE(O)001. The photograph was taken under a 20x objective lens using fluorescence. As shown in Figure 6, Ki-67, CK20, CDX-2, and villin were expressed, suggesting that the sample was colorectal adenocarcinoma cells. The diagnostic results for primary cells cultured using the CA-1 medium of the present invention were consistent with those for intestinal cancer tissue. Cells cultured using the technology of the present invention maintain the original pathological characteristics of patients with intestinal cancer.

[0067] Example 5. Statistics of initial culture period and cell number of primary colon cancer cells, and calculation of population doubling (PD) values Primary intestinal cancer cells were obtained from four intestinal cancer tissue samples (numbers OE0042, OE(E)003, OE(E)006, and OE(O)001) according to the method described in step (2)-3 of Example 1. The obtained primary intestinal cancer cells were cultured using the CA-1 medium described in Example 2, and the cells were cultured in a T25 flask at a density of 2 × 10 4 cells / cm 2 The cells were seeded at a viable cell density of 1000 and cultured. After expanding to 95%, the cells were digested and counted. At the same time, the number of days of culture until digestion was recorded as one culture period. Under these experimental conditions, the cells were continuously cultured, and the resulting cells were expanded to different passages. Each passage was counted after digestion, and the corresponding culture period was recorded. The PD value was calculated according to the formula: population doubling (PD) = 3.32 × log10 (total number of cells after digestion / initial number of seeded cells). For this formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

[0068] Figure 7 shows the growth curves of four primary cells under the culture conditions of the intestinal cancer primary cells of the present invention, plotted using Graphpad Prism software. The horizontal axis represents the number of days in cell culture, and the vertical axis represents the cumulative cell growth fold, which represents the fold increase in cells during the culture period. The higher the value, the greater the fold increase in cells within a certain period, i.e., the more cells have increased. The slope represents the rate of cell growth. Figure 7 confirms that when the intestinal cancer primary cells are continuously cultured and expanded for at least 80 days using the CA-1 medium of the present invention, the cell growth rate remains substantially unchanged, and the ability to continuously expand remains intact.

[0069] Example 6. Comparison of culture effects with existing culture media (1) Preparation of culture medium The culture medium described in the literature (Non-Patent Document 2) has a formulation of DMEM / F12 medium + 250 ng / ml amphotericin B (Selleck) + 10 μg / ml gentamicin (MCE) + 0.1 nM cholera toxin + 0.125 ng / ml EGF + 25 ng / ml hydrocortisone + 10 μM Y27632 + 10% FBS (hereinafter referred to as "LXF" medium).

[0070] (2) Obtaining and culturing primary colon cancer cells Primary intestinal cancer cells were obtained from intraoperative tissue samples (OE(E)003, OE0042) according to the method described in step (2)-3 of Example 1, and then cultured with and without trophoblast cells.

[0071] When using trophoblast cells, the cells were plated in a 48-well plate at 3 × 10 cells per well using the above LXF medium and the CA-1 medium of Example 2. 4 cells / cm 2 NIH-3T3 cells were seeded at a viable cell density of 30,000 cells per well and then irradiated with γ-rays (30 Gy). 4 cells / cm 2The cells were added at a cell density of 100 μL. Finally, the corresponding culture medium was added to each well of the 48-well plate up to a volume of 500 μL, and the resulting mixture was mixed thoroughly. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation. After the cells had grown to cover more than 85% of the 48-well plate area, they were subcultured.

[0072] When trophoblast cells were not present, cells were cultured in a 48-well plate at 3 × 10 cells per well using the above LXF medium and the CA-1 medium of Example 2. 4 cells / cm 2 After seeding at a viable cell density of 30,000 cells per well, 500 μL of the corresponding culture medium was added to each well of the 48-well plate and mixed thoroughly. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation. After the cells had grown to cover more than 85% of the 48-well plate area, they were subcultured.

[0073] On day 7 of culture, the 48-well plate was removed and the medium discarded. The cells were washed once with 100 μL of 0.05% trypsin (purchased from Gibco). After removing the trypsin, 200 μL of 0.05% trypsin was added to each well. The plate was placed in an incubator at 37°C and 5% CO2 for 10 minutes, and the cells were observed under a microscope (CNOPTEC, BDS400), indicating that the cells were completely digested. Then, 300 μL of DMEM / F12 medium containing 10% serum (Excell Bio, FND500) was added to stop the digestion. 20 μL of the resulting medium was added to a cell counting plate (Countstar, specification: 50 cells / box), and the total number of cells was counted using a cell counter (Countstar, IC1000). The counting results are shown in Figures 8A and 8B.

[0074] As can be seen from the results shown in Figures 8A and 8B, compared with LXF medium, CA-1 significantly promoted the proliferation of primary intestinal cancer cells, regardless of the presence or absence of trophoblast cells, and its effect was better than that of LXF medium used in conventional techniques. Furthermore, in the presence of trophoblast cells (Figure 8B), the promoting effect on the proliferation of primary intestinal cancer cells was more pronounced.

[0075] Example 7. Use of primary colon cancer cells obtained by expanding using the culture medium of the present invention for drug screening 1. Cell Culture and Plating Following the same method as described in Example 1, primary intestinal cancer cells were isolated from intestinal cancer intraoperative / endoscopic samples (OE(E)003, OE0042) and cultured in CA-1 medium. After the cells were expanded to 85% of the plate, they were digested and subcultured as passage 1. The cells were digested and counted according to the steps described in Example 1, and 5.76 × 10 cells were obtained. 4 The cells were placed in a loading slot (purchased from Corning) at a viable cell density of 3000 cells / mL and mixed thoroughly. After thorough mixing, the cells were seeded into a 384-well milky white cell culture plate (purchased from Corning) for culture at a volume of 50 μL per well and a cell number of 3000 cells / well. The plate was sealed by adding CA-1 medium from the edge of the plate, and the sample name and test time using CellTiter-Glo (purchased from Promega) were written on the plate. The surface was disinfected with 75% alcohol (purchased from LIRCON), and the resulting cells were cultured in an incubator at 37°C and 5% CO2. The first, second, third, fourth, and fifth passages of the cultured cells were selected for drug screening, respectively, to test the drug sensitivity of serial passages of primary cells cultured using the medium of the present invention.

[0076] 2. Preparation of Candidate Drugs Seven concentration gradients of two drugs (daunorubicin and dasatinib; both purchased from MCE) were prepared according to the table below, added to a 384-well plate (purchased from Thermo Fisher) at a volume of 30 μL per well, and stored for use.

[0077] [Table 3]

[0078] 3. High-throughput Drug Loading The prepared drug plate was removed and placed at room temperature.

[0079] The plate was centrifuged at 1000 rpm for 1 minute at room temperature in a centrifuge (Beckman) and then removed. A high-throughput automated loading system (JANUS, Perkin Elmer) was used for high-throughput drug loading. 0.1 μL of the corresponding concentration of candidate drug was added to each well of a 384-well plate containing cultured colon cancer cells. After drug loading, the surface of the 384-well plate was disinfected and placed in an incubator. Cell viability was measured after 72 hours.

[0080] 4. Cell Viability Detection CellTiter-Glo luminescent reagent (purchased from Promega) was removed from a 4°C refrigerator, 10 mL of the reagent was added to the loading slot, and the 384-well test plate was removed from the incubator. 10 μL of CellTiter-Glo luminescent reagent was added to each well. After allowing to stand for 10 minutes, the assay was performed using a multifunction microplate reader (Envision, Perkin Elmer).

[0081] 5. Data Processing Calculate the cell inhibition rates of cells treated with different drugs according to the formula: cell inhibition rate (%) = 100% - chemiluminescence value of drug-loaded wells / chemiluminescence value of control wells × 100%, and calculate the 50% inhibitory concentration (IC) of the drug on the cells using graphpad prism software. 50 ) was calculated. The results are shown in Figures 9A and 9B.

[0082] When intestinal cancer cells cultured in the intestinal cancer primary cell culture medium of the present invention are used for drug screening, it can be seen from Figures 9A and 9B that the inhibitory effect of the same drug on cultured cells of different passages remains substantially the same (the inhibition curves are substantially consistent). Even cells from the same patient have different sensitivities to various drugs at the maximum blood concentration in the human body. This result can determine the effectiveness of drugs in clinical use for patients with intestinal cancer, and at the same time, it can be confirmed that the sensitivity of tumor cells of different passages obtained according to the culture method of this patent to drugs remains stable. [Industrial Applicability]

[0083] The present invention provides a culture medium and a culture method for culturing or expanding primary intestinal cancer cells in vitro, and the cultured cells can be used for evaluating or screening the efficacy of drugs, making the present invention suitable for industrial application.

[0084] Although the present invention has been described in detail in this specification, the present invention is not limited thereto, and those skilled in the art can make modifications according to the principles of the present invention. Therefore, it should be understood that any modifications made according to the principles of the present invention fall within the protection scope of the present invention.

Claims

1. an initial culture medium, a Rho protease inhibitor, an antibiotic, gastrin, A8301, a non-essential amino acid, a cholera toxin, insulin-like growth factor-1, nicotinamide, insulin, fetal bovine serum, and an additive selected from at least one of a B27 additive and an N2 additive; the initial culture medium is selected from the group consisting of DMEM / F12, DMEM, F12 or RPMI-1640; The Rho protease inhibitor is one or more selected from the group consisting of Y27632, fasudil, and H-1152, and the concentration is 2.5 μM to 40 μM; the antibiotic is one or more selected from the group consisting of streptomycin / penicillin, amphotericin B and primocin, in which in the case of streptomycin / penicillin, streptomycin has a concentration in the range of 25 μg / mL to 400 μg / mL, penicillin has a concentration in the range of 25 U / mL to 400 U / mL, amphotericin B has a concentration in the range of 0.25 μg / mL to 4 μg / mL, and primocin has a concentration in the range of 25 μg / mL to 400 μg / mL; the concentration of said gastrin is 1.25 nM to 20 nM; The concentration of A8301 is 25 nM to 200 nM; the non-essential amino acid is one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and the total concentration is 25 μM to 400 μM; the concentration of the cholera toxin is between 1.25 ng / mL and 20 ng / mL; the concentration of the insulin-like growth factor-1 is 5 ng / mL to 405 ng / mL; the concentration of said nicotinamide is 2 mM to 8 mM; the insulin has a concentration of 0.5 μg / mL to 4 μg / mL; The volume concentration of the fetal bovine serum in the culture medium is 2.5% (v / v) to 5% (v / v); A culture medium for primary cells of intestinal cancer, characterized in that the volume ratio of said B27 additive or said N2 additive to said culture medium is 1:25-1:

200.

2. The Rho protease inhibitor is one or more selected from the group consisting of Y27632, fasudil, and H-1152, and the concentration is 2.5 μM to 5 μM; the antibiotic is one or more selected from the group consisting of streptomycin / penicillin, amphotericin B and primocin, in which in the case of streptomycin / penicillin, streptomycin has a concentration in the range of 50 μg / mL to 200 μg / mL, penicillin has a concentration in the range of 50 U / mL to 200 U / mL, amphotericin B has a concentration in the range of 0.5 μg / mL to 2 μg / mL, and primocin has a concentration in the range of 50 μg / mL to 200 μg / mL; The concentration of the gastrin is 2.5 nM to 10 nM; the concentration of the cholera toxin is between 2.5 ng / mL and 5 ng / mL; the concentration of the nicotinamide is 4 mM to 8 mM; the insulin has a concentration of 1 μg / mL to 2 μg / mL; The culture medium for primary intestinal cancer cells according to claim 1, characterized in that the volume ratio of the B27 additive or the N2 additive to the culture medium is 1:25-1:

50.

3. A method for culturing primary intestinal cancer cells, comprising culturing the primary intestinal cancer cells using the culture medium for primary intestinal cancer cells according to claim 1 or 2.

4. The primary intestinal cancer cells were cultured in a culture dish at 1×10 ４ cells / cm ２ ~10x10 ４ cells / cm ２ and trophoblast cells were seeded at a cell density of 2 × 10 ４ cells / cm ２ ~3×10 ４ cells / cm ２ and using a culture medium for primary intestinal cancer cells according to claim 1 or 2 for the culture.

5. The culture method according to claim 4, characterized in that the trophoblast cells are irradiated NIH-3T3 cells, the radiation source is X-rays or γ-rays, and the radiation dose is 30 Gy to 50 Gy.

6. A method for screening drugs for intestinal cancer, comprising: (1) culturing primary intestinal cancer cells using the method for culturing primary intestinal cancer cells according to any one of claims 3 to 5; (2) selecting a test drug and diluting the drug based on the required concentration gradient; (3) adding the drug at various concentration gradients to the cells cultured in step (1); (4) detecting cell viability; and A method comprising:

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