Culture medium for in-vitro adherent culture of primary tumor cells and application

Abstract

The invention discloses a culture medium for in-vitro adherent culture of primary tumor cells and application, and relates to the technical field of tumor cell culture. Comprising a basic culture medium, fetal calf serum FBS with the concentration of 5 ng / mL-15 ng / mL, an epidermal growth factor EGF with the concentration of 1 ng / mL-10 ng / mL, a basic fibroblast growth factor bFGF with the concentration of 0.5 ng / mL-5 ng / mL, an insulin-transferrin-selenium ITS supplement with the final volume concentration of 0.1%-1%, L-glutamine with the final volume concentration of 0.5 mM-2 mM, ascorbic acid with the final volume concentration of 0.1%-0.5% and non-essential amino acid with the final volume concentration of 1%-5%. The kit comprises 10 [mu] g / mL-50 [mu] g / mL of heparin and 1 [mu] g / mL-5 [mu] g / mL of an Rho kinase inhibitor. The culture medium overcomes the defects that in the prior art, when primary tumor cells are subjected to adherent culture, apoptosis or dedifferentiation is prone to occurring, in-vivo tumor characteristics are difficult to simulate, the tumor growth speed is low, the proliferation effect is poor, and the adherent rate is low.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of primary tumor cell culture, and more particularly to a culture medium for in vitro adherent culture of primary tumor cells and application thereof. BACKGROUND

[0002] Cell culture is the most commonly used basic technique in cell biology and tumor-related research. When cells are cultured in vitro, the culture medium provides the necessary external environment for cell growth, division and proliferation, including buffer simulating physiological pH, substances required for basic metabolism and some nutritional factors. According to the characteristics of cells, cell culture methods are usually divided into suspension culture and adherent culture methods. Except for blood system cells such as stem cells and lymphocytes, which are usually cultured in suspension, most cells in vivo, including normal cells and tumor cells, rely on adherent culture. When adherent cells are cultured, in addition to the culture medium that cells rely on for adherent growth, such as TC-treated culture dish bottom and even extracellular matrix-coated culture dish, serum needs to be added to the culture medium to assist cell adhesion and provide various nutrients required for cell growth.

[0003] In vitro culture of primary tumor cells is an important tool for studying tumor biology and drug screening. However, due to the heterogeneity of tumor cells and their dependence on the microenvironment, traditional culture media often fail to maintain their long-term proliferation and adhesion ability. In the prior art, commonly used culture media (such as RPMI-1640 or DMEM) rely on high concentrations of serum (such as 20% FBS) to promote cell adhesion, but high serum can lead to cell differentiation or contamination by fibroblasts. In addition, the lack of specific growth factors and microenvironment support makes primary tumor cells prone to apoptosis or dedifferentiation, making it difficult to simulate in vivo tumor characteristics, and the tumor grows slowly, with poor proliferation and low adhesion rate. SUMMARY

[0004] Therefore, the present application provides a culture medium for in vitro adherent culture of primary tumor cells and application thereof, which overcomes the defects of existing technology that primary tumor cells are prone to apoptosis or dedifferentiation during adherent culture, it is difficult to simulate in vivo tumor characteristics, and the tumor grows slowly, with poor proliferation and low adhesion rate.

[0005] To achieve the above purpose, the present application adopts the following technical solutions:

[0006] A culture medium for in vitro adherent culture of primary tumor cells, characterized in that it comprises the following components:

[0007] The basic medium, 5ng / mL-15ng / mL fetal bovine serum FBS, 1ng / mL-10ng / mL epidermal growth factor EGF, 0.5ng / mL-5ng / mL basic fibroblast growth factor bFGF, 0.1%-1% insulin-transferrin-selenium ITS supplement, 0.5mM-2mM L-glutamine, 0.1%-0.5% ascorbic acid, 1%-5% non-essential amino acids, 10ug / mL-50ug / mL heparin, 1ug / mL-5ug / mL Rho kinase inhibitor.

[0008] The Rho kinase inhibitor (Y-27632) continuously plays a role in promoting cell survival and reducing apoptosis during the passage process, and maintains the stability of cell number and activity. Antioxidants such as ascorbic acid and selenium protect cells from oxidative damage, maintain the stability of biological macromolecules in cells, and prevent cell gene mutations or functional abnormalities caused by oxidative stress. The synergistic effect of each component provides a stable growth environment, signal transduction and metabolic state for cells in P3 generation, and ensures the stability of the cells.

[0009] Further, the basic medium is DMEM / F12 or RPMI-1640;

[0010] Further, the non-essential amino acids include at least one of L-alanine, L-arginine, L-asparagine, and L-glutamic acid.

[0011] Further, the Rho kinase inhibitor is Y-27632.

[0012] Further, the extracellular matrix extract includes at least one of fibronectin and collagen.

[0013] Further, the primary tumor cells are primary tumor cells isolated from a lung cancer H1299 cell line transferred into mice.

[0014] The second aspect of the present application provides a use of the above-mentioned culture, and the use includes at least one of the following:

[0015] 1) Application in adherent culture of primary tumor cells, promotion of proliferation of primary tumor cells, and reduction of differentiation degree of primary tumor cells.

[0016] 2) Application in construction of tumor organoid model.

[0017] The third aspect of the present application provides a method for adherent culture of primary tumor cells in vitro, which is cultured by using the culture medium, and the process includes the following steps:

[0018] The primary tumor tissue is digested into a single cell suspension, filtered, and the cells are resuspended;

[0019] The cells are seeded in collagen-coated culture dishes containing the culture medium according to any one of claims 1-4;

[0020] The culture is incubated at 37°C, 5% CO2, and the medium is changed every 2-3 days;

[0021] The cell survival rate, proliferation capacity, and differentiation markers are detected every 7 days, and the culture is stopped when the cells are subcultured to the P3 generation after 14-21 days of culture.

[0022] Further, when the tumor tissue is digested, 1 mg / mL collagenase IV and 100 U / mL hyaluronidase are mixed and digested for 30-60 min;

[0023] The filtration is performed using a 40 pm filter membrane.

[0024] Further, when the cells are seeded in collagen-coated culture dishes containing the culture medium, the seeding amount is (1-5) x 10 5 cells / cm 2 .

[0025] Compared with the prior art, the technical effects achieved by each component are as follows:

[0026] The basal medium is the basic framework of the entire culture medium and provides the basic nutrients necessary for tumor cell survival. It contains amino acids, vitamins, carbohydrates, inorganic salts, and other components. Amino acids are the raw materials for cell protein synthesis; vitamins participate in various metabolic reactions of cells; carbohydrates such as glucose are the main energy source for cells; inorganic salts maintain the osmotic pressure balance and acid-base balance inside and outside the cells, ensuring normal physiological functions of the cells.

[0027] Fetal bovine serum (FBS), preferably Gibco Premium grade, has a very wide range of applications. It not only provides various growth factors, hormones, and binding proteins required for cell growth, but also supplements the nutrients lacking in the basal medium. Growth factors can stimulate cell proliferation and differentiation; hormones regulate cell metabolism and physiological activity; binding proteins such as transferrin can bind and transport iron ions to meet the cell's demand for iron and prevent oxidative damage to cells caused by iron ions. In addition, fetal bovine serum can also act as a buffer to protect cells from external stimuli, while reducing the surface tension during cell adhesion and promoting cell adhesion to the surface of the culture dish.

[0028] Epidermal growth factor (EGF): 1-10 ng / mL of EGF can specifically bind to the EGF receptor on the surface of tumor cells, activate a series of intracellular signaling pathways, such as the Ras-Raf-MEK-ERK pathway and the PI3K-Akt pathway. Activation of these signaling pathways can promote cell proliferation, migration and survival, enhance cell viability, and help maintain the malignant phenotype of tumor cells.

[0029] Basic fibroblast growth factor (bFGF): 0.5-5 ng / mL of bFGF also regulates cell growth and differentiation by binding to the corresponding receptor. It can stimulate DNA synthesis and mitosis of cells, promote cell proliferation; also can induce the expression of angiogenesis-related factors, provide nutrients and oxygen supply for tumor cells, and play a role in cell survival and anti-apoptosis.

[0030] In insulin-transferrin-selenium (ITS), insulin can regulate the uptake and utilization of glucose by cells, promote the metabolism and growth of tumor cells; transferrin can bind and transport iron ions to ensure the stable supply of iron elements in cells, participate in the synthesis and metabolic reactions of various enzymes in cells; selenium is an important trace element, as a component of glutathione peroxidase, it has antioxidant effect, can remove free radicals in cells, protect cells from oxidative damage, and maintain normal physiological functions of cells.

[0031] L-glutamine is an important nitrogen source and energy source for tumor cell growth. It can participate in the synthesis of biological macromolecules such as proteins and nucleic acids in cells; at the same time, as an important intermediate metabolite in the tricarboxylic acid cycle, it provides energy for cells. When cells are in a state of rapid proliferation, the demand for L-glutamine increases significantly, and sufficient L-glutamine supply can ensure normal growth and metabolism of cells.

[0032] Ascorbic acid is a strong antioxidant. It can remove free radicals in cells, protect cell membranes, proteins, and nucleic acids from oxidative damage; also can participate in the synthesis of collagen in the extracellular matrix, promote the stability of the extracellular matrix, and help tumor cells adhere and grow.

[0033] Non-essential amino acids can reduce the energy consumption of cells to synthesize non-essential amino acids, allowing cells to use more energy for growth and proliferation. At the same time, these non-essential amino acids participate in a variety of metabolic reactions in cells, maintaining normal physiological functions of cells.

[0034] Heparin can bind to bFGF to form a stable complex, enhance the activity and stability of bFGF, and prolong its action time in the culture medium, thereby more effectively promoting the growth and proliferation of tumor cells.

[0035] Rho kinase inhibitors reduce intracellular actin-myosin contraction by inhibiting the activity of Rho kinase, reducing the contractility of cells. This makes cells more easily spread and adhere to the surface of the culture vessel, improving cell adhesion efficiency; it also promotes cell survival and reduces apoptosis.

[0036] Extracellular matrix extract can simulate the microenvironment of extracellular matrix in vivo, provide attachment sites and growth scaffolds for tumor cells, promote cell adhesion, migration and differentiation; collagen is an important component of extracellular matrix, which can bind to integrin receptors on the surface of tumor cells, mediate the interaction between cells and extracellular matrix, and maintain cell morphology and function.

[0037] Synergistic effect of each component

[0038] 1) Synergistically enhance cell proliferation

[0039] The basal medium and fetal bovine serum provide sufficient nutrients and growth factors for cell proliferation. Epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) bind to cell surface receptors, activate signal transduction pathways such as Ras-Raf-MEK-ERK and PI3K-Akt, promote DNA synthesis and mitosis, and stimulate cell proliferation.

[0040] In the insulin-transferrin-selenium (ITS) supplement, insulin regulates glucose uptake and utilization by cells, providing energy for cell proliferation; transferrin ensures iron supply, participates in the synthesis of various enzymes in cells, and supports cell metabolism and proliferation; selenium as an antioxidant maintains the stability of the intracellular environment, avoiding the inhibition of cell proliferation by oxidative stress. L-glutamine and non-essential amino acids (NEAA) meet the large demand for nitrogen sources and amino acids during cell proliferation, supporting the synthesis of biological macromolecules such as proteins and nucleic acids. Heparin binds to bFGF to form a stable complex, enhancing bFGF activity and action time, further promoting cell proliferation. Each component synergistically enhances cell proliferation from multiple aspects such as nutrition supply, signal transduction, and metabolic regulation.

[0041] 2) Synergistically enhance MTT absorbance

[0042] MTT absorbance values ​​reflect, to some extent, cell number, activity, and metabolic levels. The components mentioned above synergistically promote cell proliferation, increasing cell number and thus enhancing light absorption per unit volume, thereby increasing absorbance. Simultaneously, each component ensures normal cellular physiological metabolism and activity. For example, ascorbic acid and selenium (from ITS supplements) scavenge free radicals, maintaining intracellular homeostasis and preventing oxidative damage that could impair metabolism and activity; insulin regulates cellular metabolism, ensuring cells efficiently utilize nutrients for metabolic activities. High cell activity and vigorous metabolism also contribute to enhanced MTT absorbance values; the components work synergistically through multiple pathways to achieve this effect.

[0043] 3) Collaboration ensures the stability of P3 generation

[0044] During the passage of primary tumor cells to the P3 generation, cells are prone to instability, such as altered growth characteristics and abnormal differentiation. The basal culture medium and fetal bovine serum continuously provide a stable nutritional environment for the cells, ensuring their basic survival needs during passage. Growth factors (EGF, bFGF) and heparin maintain stable intracellular signal transduction, promoting the preservation of specific proliferation and differentiation characteristics and preventing the loss or alteration of cell characteristics during passage. Attached Figure Description

[0045] Appendix Figure 1 The number of primary tumor cells after 7 days of culture in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6.

[0046] Appendix Figure 2 The number of primary tumor cells after culturing them in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 for 14 days is given.

[0047] Appendix Figure 3 The survival rate of primary tumor cells cultured in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 for 14 days was measured.

[0048] Appendix Figure 4 The absorbance is measured after primary tumor cells have been cultured in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 for 14 days.

[0049] Appendix Figure 5 The adhesion rate of primary tumor cells cultured in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 for 14 days is given.

[0050] Appendix Figure 6 The ROS level of primary tumor cells after 7 days of culture in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 was determined.

[0051] Appendix Figure 7The ROS level of primary tumor cells cultured in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 for 14 days was determined.

[0052] Appendix Figure 8 The differentiation rate of primary tumor cells cultured in the culture media of Examples 1 to 4 and Comparative Examples 1 to 6 for 14 days is given. Detailed Implementation

[0053] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0054] Example 1

[0055] A culture medium for in vitro adherent culture of primary tumor cells comprises the following components:

[0056] 1 L of DMEM / F12 basal medium, 5 ng / mL fetal bovine serum (FBS), 1 ng / mL epidermal growth factor (EGF), 0.5 ng / mL basic fibroblast growth factor (bFGF), 0.1% insulin-transferrin-selenium (ITS) supplement, 0.1% ascorbic acid, 1% non-essential amino acids (L-alanine, L-arginine, and L-asparagine, with final concentrations of 0.2%, 0.3%, and 0.5%, respectively), 10 μg / mL heparin, and 1 μg / mL Rho kinase inhibitor Y-27632.

[0057] Example 2

[0058] A culture medium for in vitro adherent culture of primary tumor cells comprises the following components:

[0059] 1 L of RPMI-1640 basal medium, 15 ng / mL fetal bovine serum (FBS), 10 ng / mL epidermal growth factor (EGF), 5 ng / mL basic fibroblast growth factor (bFGF), 1% insulin-transferrin-selenium (ITS) supplement, 0.5% ascorbic acid, 5% non-essential amino acids (L-alanine 2%, L-arginine 1.5%, L-asparagine 1.5%), 50 μg / mL heparin, and 5 μg / mL Rho kinase inhibitor Y-27632.

[0060] Example 3

[0061] A culture medium for culturing primary tumor cells in vitro adherent culture comprising the following components:

[0062] DMEM / F12 base medium 1 L, fetal bovine serum FBS at a final volume concentration of 7 ng / mL, epidermal growth factor EGF at 5 ng / mL, basic fibroblast growth factor bFGF at 2 ng / mL, insulin-transferrin-selenium ITS supplement at a final volume concentration of 0.45%, ascorbic acid at a final volume concentration of 0.3%, non-essential amino acids (L-alanine 0.8%, L-arginine 0.7%, L-asparagine 0.2%, L-glutamic acid 0.3%) at a final volume concentration of 2%, heparin at 15 pg / mL, Rho kinase inhibitor Y-27632 at 3 pg / mL, collagen at 2 pg / mL.

[0063] Example 4

[0064] A culture medium for culturing primary tumor cells in vitro adherent culture comprising the following components:

[0065] DMEM / F12 base medium 1 L, fetal bovine serum FBS at 9 ng / mL, epidermal growth factor EGF at 7 ng / mL, basic fibroblast growth factor bFGF at 3.5 ng / mL, insulin-transferrin-selenium ITS supplement at a final volume concentration of 0.7%, ascorbic acid at a final volume concentration of 0.2%, non-essential amino acids (L-alanine 1.5%, L-arginine 0.5%, L-asparagine 1.5%) at a final volume concentration of 3.5%, heparin at 35 pg / mL, Rho kinase inhibitor Y-27632 at 4 pg / mL, fibronectin at 1.5 pg / mL.

[0066] Example 5 Isolation of primary tumor cells

[0067] Cell and animal preparation

[0068] Lung cancer H1299 cell line:

[0069] H1299 cells were thawed and cultured using RPMI 1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, passaged in a 37°C, 5% CO2 incubator, and ensured good cell condition (logarithmic growth phase, viability > 90%).

[0070] Cells were digested (0.25% trypsin), resuspended with medium, and adjusted to a cell concentration of 1.5 x 10 7 cells / mL after counting.

[0071] Experimental mice:

[0072] Select immunodeficient mice (NOD / SCID mice), 6-8 weeks old, 18-22 g in weight, both male and female (need to be unified according to the experimental design), and are raised in a SPF level environment, and are adapted to the environment for 5 days in advance.

[0073] Reagents and equipment

[0074] Reagents:

[0075] Sterile PBS, RPMI 1640 medium, fetal bovine serum (FBS), trypsin (0.25%), double-antibiotic (penicillin-streptomycin), collagenase (such as Collagenase IV), DNase I.

[0076] Equipment:

[0077] Sterile syringe (1 mL), surgical instruments (scissors, forceps, surgical knife), culture dish, centrifuge tube (15 mL / 50 mL), cell sieve (70 μm), centrifuge, clean bench, inverted microscope.

[0078] Inoculation of lung cancer cell lines into mice (tumor model construction)

[0079] 1. Inoculation method: subcutaneous inoculation (most commonly used):

[0080] After the mice are anesthetized with sodium pentobarbital, the skin on the back is disinfected.

[0081] Use a 1 mL sterile syringe to draw up the cell suspension, and subcutaneously inject 0.2 mL, ensuring that the cell suspension forms uniform droplets subcutaneously.

[0082] 2. Tumor growth monitoring

[0083] Observe the state of the mice every 2 days after inoculation, and measure the tumor size (use a vernier caliper to measure the long diameter a and the short diameter b, volume = 0.5 × a × b 2 ), and when the tumor volume grows to 300 mm 3 , primary tumor cell isolation is performed.

[0084] Primary tumor cell isolation steps

[0085] 1. Mouse sacrifice and tumor sampling

[0086] The mice are sacrificed by cervical dislocation, and the sampling site skin is disinfected.

[0087] The skin is surgically incised, and the tumor tissue is completely peeled off (avoid mixing with normal tissue), and is placed in a culture dish containing ice-cold PBS, and is gently rinsed to remove blood and impurities.

[0088] 2. Tumor tissue processing and digestion

[0089] Mechanical disruption: Transfer the tumor tissue to a new dish and cut it into 1-2 mM small pieces with a surgical knife. 3

[0090] Enzymatic digestion:

[0091] Add appropriate amount of collagenase IV (2 mg / mL) and DNase I (100 μg / mL) mixed digestion solution (prepared with serum-free RPMI1640), incubate at 37℃ for 30-60 min (adjust the time according to the hardness of the tissue, until most of the tissue pieces are dispersed). Blow with a pipette every 10 min during the period to promote digestion.

[0092] Termination of digestion:

[0093] Add medium containing 10% FBS to terminate digestion, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0094] Cell suspension preparation and purification

[0095] Sieve and wash:

[0096] Filter the cell suspension with a 70 μm cell sieve to remove undigested tissue fragments, and transfer the filtrate to a centrifuge tube.

[0097] Wash the cells with PBS twice, centrifuge at 1000 rpm for 5 min, discard the supernatant, and obtain the primary tumor cells.

[0098] The primary tumor cells isolated were cultured in vitro adherently with the medium of Example 1 to Example 4, respectively:

[0099] The process of adherently culturing the primary tumor cells in vitro with the medium of Example 1 is as follows:

[0100] The primary tumor cells were digested with a mixture of 1 mg / mL collagenase IV and 100 U / mL hyaluronidase for 30 min, and then the single-cell suspension was filtered through a 40 μm filter membrane, and the cells were resuspended.

[0101] The cells were inoculated into collagen-coated culture dishes containing the medium at an inoculation amount of 1.5 x 10 5 cells / cm 2 .

[0102] Culture at 37℃, 5% CO2, replace the medium every 3 days; detect the cell survival rate, proliferation capacity and differentiation markers every 7 days, and culture for 21 days, then stop culturing when the cells reach P3 generation.

[0103] The process of adherently culturing the primary tumor cells in vitro with the medium of Example 2 is as follows:

[0104] ​The primary tumor cells were digested with 1 mg / mL collagenase IV and 100 U / mL hyaluronidase for 60 min, and then filtered through a 40 pm filter membrane to obtain a single cell suspension;

[0105] The cells were inoculated into collagen-coated medium-containing culture dishes at a seeding amount of 3.2 x 10 5 cells / cm 2 .

[0106] The cells were cultured at 37°C in a 5% CO2 environment, and the medium was replaced every 2 days. The cell survival rate, proliferation capacity, and differentiation markers were detected every 7 days. The culture was stopped when the cells were subcultured to the P3 generation after 15 days of culture.

[0107] The process of in vitro adherent culture of the primary tumor cells with the medium of Example 3 is as follows:

[0108] The primary tumor cells were digested with 1 mg / mL collagenase IV and 100 U / mL hyaluronidase for 40 min, and then filtered through a 40 pm filter membrane to obtain a single cell suspension;

[0109] The cells were inoculated into collagen-coated medium-containing culture dishes at a seeding amount of 2.5 x 10 5 cells / cm 2 .

[0110] The cells were cultured at 37°C in a 5% CO2 environment, and the medium was replaced every 2.5 days. The cell survival rate, proliferation capacity, and differentiation markers were detected every 7 days. The culture was stopped when the cells were subcultured to the P3 generation after 17 days of culture.

[0111] The process of in vitro adherent culture of the primary tumor cells with the medium of Example 4 is as follows:

[0112] The primary tumor cells were digested with 1 mg / mL collagenase IV and 100 U / mL hyaluronidase for 50 min, and then filtered through a 40 pm filter membrane to obtain a single cell suspension;

[0113] The cells were inoculated into collagen-coated medium-containing culture dishes at a seeding amount of 5.0 x 10 5 cells / cm 2 .

[0114] The cells were cultured at 37°C in a 5% CO2 environment, and the medium was replaced every 3 days. The cell survival rate, proliferation capacity, and differentiation markers were detected every 7 days. The culture was stopped when the cells were subcultured to the P3 generation after 14 days of culture.

[0115] In order to explore the role of each component in the embodiments 1-4 of the present application and whether there is a synergistic effect between the components, the following comparative examples are set. It should be understood that, unless otherwise specified, the products used in the following comparative examples are commercially available.

[0116] Comparative Example 1

[0117] Taking Example 3 as an example, compared with Example 3, the difference is that ascorbic acid is removed, the concentration of insulin-transferrin-selenium ITS supplement is increased to 0.75%, and the rest of the components remain unchanged.

[0118] Comparative Example 2

[0119] Taking Example 3 as an example, compared with Example 3, the difference is that the insulin-transferrin-selenium ITS supplement is removed, and the concentration of ascorbic acid is increased to 0.75%, and the rest of the components remain unchanged.

[0120] Comparative Example 3

[0121] Taking Example 3 as an example, compared with Example 3, the difference is that heparin is removed, the content of Rho kinase inhibitor Y-27632 is increased to 10 μg / mL, and the content of collagen is increased to 10 μg / mL.

[0122] Comparative Example 4

[0123] Taking Example 3 as an example, compared with Example 3, the difference is that Rho kinase inhibitor Y-27632 and collagen are removed, and the content of heparin is increased to 20 μg / mL.

[0124] Comparative Example 5

[0125] Taking Example 3 as an example, compared with Example 3, the difference is that fetal bovine serum FBS is removed, the content of epidermal growth factor EGF is increased to 9 ng / mL, and the content of basic fibroblast growth factor bFGF is increased to 5 ng / mL.

[0126] Comparative Example 6

[0127] Taking Example 3 as an example, compared with Example 3, the difference is that epidermal growth factor EGF is removed, the content of fetal bovine serum FBS is increased to 9 ng / mL, and the content of basic fibroblast growth factor bFGF is increased to 5 ng / mL.

[0128] The culture medium of Comparative Examples 1-6 is used for in vitro adherent culture of primary tumor cells, and the culture process is the same as that of the culture medium of Example 3 described above.

[0129] After the primary tumor cells were cultured to P3 generation, the number, survival rate, absorbance, and adhesion rate of the cells cultured in the medium of each group were calculated on day 7 and day 14. The results are shown in Table 1, Figures 1-5 ;

[0130] Cell number determination (hemocytometer counting method)

[0131] Sampling: tumor cells cultured to day 14 in each group were taken, and the cell culture bottle bottom was gently blown by a pipette to fully suspend the cells, avoiding cell clumping.

[0132] Dilution: 10 μL of the above cell suspension was taken, 90 μL of phosphate buffer (PBS) was added, and it was gently mixed to make a 1:10 dilution.

[0133] Counting preparation: the hemocytometer and cover glass were cleaned, the cover glass was covered on the counting chamber, the diluted cell suspension was slowly dropped from the edge of the cover glass with a micropipette, and the cell suspension was filled in the counting chamber to avoid air bubbles.

[0134] Counting: under the 10x objective lens of the optical microscope, the number of cells in each large square was counted. When counting, the principle of "counting the upper and left cells and not counting the lower and right cells" was followed, that is, only the cells located in the upper and left edge of the pressed line cells were counted.

[0135] Calculation: cell number (pieces / mL) = (total number of cells in four large squares ÷ 4) x 10000 x dilution factor (10 times).

[0136] Survival rate determination (trypan blue staining method)

[0137] Staining: 10 μL of the cell suspension (the same as the cell number determination) was mixed with 10 μL of 0.4% trypan blue staining solution in an EP tube, and incubated at room temperature for 3-5 min.

[0138] Counting preparation: according to the steps of the hemocytometer counting method, the stained cell suspension was dropped into the counting chamber.

[0139] Counting: under the 10x objective lens of the microscope, the total number of cells and the number of dead cells stained by trypan blue (dead cells are blue, and live cells are not colored) in four large squares were counted.

[0140] Calculation: survival rate (%) = (total number of cells - number of dead cells) ÷ total number of cells x 100%.

[0141] Absorbance determination (MTT method)

[0142] At the 14th day of culture in each group, the cell suspension was aspirated, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation was continued in the incubator for 4 h. The culture solution in the well was carefully aspirated without touching the purple crystals at the bottom of the well. Then 150 μL of dimethyl sulfoxide (DMSO) was added to each well, and the crystals were fully dissolved by shaking on a shaker at low speed for 10 min.

[0143] Measurement of absorbance: The absorbance (OD value) of each well was measured at a wavelength of 490 nm using an enzyme marker.

[0144] Adhesion rate determination

[0145] At the 14th day of culture in each group, the culture medium in the culture bottle was gently aspirated, and the cells were washed twice with PBS to remove the unadhered cells. An appropriate amount of trypsin was added to the culture bottle to digest the adhered cells. After the cells were detached, the digestion was terminated by adding the culture medium, and a cell suspension was prepared by blowing. The number of adhered cells was counted using a hemocytometer (same as the cell number determination method).

[0146] Calculation: Adhesion rate (%) = (number of adhered cells ÷ total number of inoculated cells) x 100%.

[0147] Table 1 Comparison of cell proliferation and adhesion performance (n = 3; absorbance OD 490nm ; proliferation capacity is represented by Fold Change vs. day 0)

[0148]

[0149]

[0150] ROS level detection

[0151] Cell treatment: The tumor cells of each group cultured for 7 days and 14 days were digested with 0.25% trypsin and inoculated in a 24-well plate at a density of 1 x 10 5 cells per well. 1 mL of complete culture medium was added to each well, and the plate was incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere and grow.

[0152] Probe loading: The culture medium in the well was aspirated, the cells were washed twice with PBS, 500 μL of DCFH-DA fluorescent probe working solution (diluted with serum-free culture medium) with a final concentration of 10 μmol / L was added to each well, and the plate was incubated at 37°C in the dark for 30 min.

[0153] Washing: After incubation, the probe working solution was aspirated, and the cells were washed with PBS three times in the dark to remove the free probe that did not enter the cells.

[0154] Detection: The mean fluorescence intensity of each group of cells was detected using a flow cytometer. The higher the fluorescence intensity, the higher the intracellular ROS level.

[0155] Cell differentiation rate determination

[0156] Cell fixation: The cells in the above 24-well plate were washed with PBS twice, 4% paraformaldehyde was added to each well, and the cells were fixed at room temperature for 30 min.

[0157] Blocking: The fixing solution was aspirated, and the cells were washed with PBS for 3 times. 500 μL of blocking solution containing 5% bovine serum albumin (BSA) was added to each well, and the cells were blocked at room temperature for 1 h.

[0158] Primary antibody incubation: The blocking solution was aspirated, and diluted specific differentiation marker primary antibodies (such as epithelial cell marker CK18, mesenchymal cell marker Vimentin, etc., selected according to tumor type) were added, and the cells were incubated at 4°C overnight.

[0159] Secondary antibody incubation: The cells were washed with PBS for 3 times, each time for 5 min, and fluorescently labeled secondary antibodies (such as FITC-labeled goat anti-mouse IgG) were added, and the cells were incubated at room temperature for 1 h in the dark.

[0160] Nuclei staining: After washing with PBS, 500 μL of DAPI staining solution was added to each well, and the cells were incubated at room temperature in the dark for 5 min to stain the nuclei.

[0161] Observation and counting: After washing with PBS for 3 times, the cells were observed under a fluorescence microscope, and the number of cells positively expressing the differentiation marker and the total number of cells (DAPI-stained positive cells) were counted.

[0162] Calculation of differentiation rate: Differentiation rate (%) = (number of positive cells ÷ total number of cells) x 100%.

[0163] Meanwhile, the ROS levels and cell differentiation rates of each group of cells are shown in Table 2, Figures 6-8 .

[0164] Table 2

[0165]

[0166] Through the above Tables 1-2, Figures 1-8It is known that ascorbic acid and ITS have synergistic antioxidant and cell proliferation promoting effects (Comparative Example 1-Comparative Example 2), and that removal of ascorbic acid (Comparative Example 1) or ITS (Comparative Example 2) alone results in increased ROS (2.5-3.8) and differentiation rate (30-35%), confirming that both are required to scavenge different types of free radicals. Synergy of heparin / Y-27632 / collagen adhesion (Comparative Example 3-Comparative Example 4): heparin alone (Comparative Example 4) cannot compensate for the absence of Y-27632 and collagen, and the adhesion rate drops to 70% (vs 82% for Comparative Example 3), indicating that the three act synergistically to promote adhesion through different mechanisms (chemical inhibition + physical support + growth factor activation). Complementarity of serum and growth factors (Comparative Example 5-Comparative Example 6): in the absence of serum (Comparative Example 5) even increasing EGF / bFGF, the cell number drops dramatically (1.8 x 10 5 ), indicating that serum provides irreplaceable adhesion proteins and lipids.

[0167] Each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between embodiments can be referred to each other.

[0168] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A culture medium for culturing primary tumor cells in vitro by adherent culture, characterized in that, The culture medium comprises the following components: a basal medium, 5 ng / mL-15 ng / mL fetal bovine serum FBS, 1 ng / mL-10 ng / mL epidermal growth factor EGF, 0.5 ng / mL-5 ng / mL basic fibroblast growth factor bFGF, an insulin-transferrin-selenium ITS supplement with a final volume concentration of 0.1%-1%, 0.5 mM-2 mM L-glutamine, ascorbic acid with a final volume concentration of 0.1%-0.5%, non-essential amino acids with a final volume concentration of 1%-5%, 10 μg / mL-50 μg / mL heparin, 1 μg / mL-5 μg / mL Rho kinase inhibitor.

2. The culture medium for culturing primary tumor cells in vitro adherently according to claim 1, wherein, The basal medium is DMEM / F12 or RPMI-1640; The non-essential amino acids comprise at least one of L-alanine, L-arginine, L-asparagine, L-glutamic acid; The Rho kinase inhibitor is Y-27632.

3. A culture medium for culturing primary tumor cells in vitro adherently according to any one of claims 1-2, characterized in that, The culture medium further comprises an extracellular matrix extract; the extracellular matrix extract comprises at least one of fibronectin, collagen.

4. The culture medium for culturing primary tumor cells in vitro adherently according to claim 3, wherein, The primary tumor cells are primary tumor cells isolated from a lung cancer H1299 cell line transferred into mice.

5. Use of the medium according to any one of claims 1 to 4, characterized in that The use comprises at least one of the following: 1) application in adherent culture of primary tumor cells, promoting proliferation of primary tumor cells, and reducing differentiation degree of primary tumor cells. 2) application in construction of a tumor organoid model.

6. A method of culturing primary tumor cells in vitro by adherent culture, characterized in that, The culture process using the culture medium of any one of claims 1-4 comprises the following steps: digesting the primary tumor tissue into a single-cell suspension, filtering, and resuspending the cells; seeding the cells into a culture dish coated with collagen and containing the culture medium of any one of claims 1-4; culturing at 37°C in 5% CO2, and changing the culture medium every 2-3 days; detecting cell survival rate, proliferation capacity, and differentiation markers every 7 days, culturing for 14-21 days, and stopping culturing when the cells reach P3 generation.

7. A method of culturing primary tumor cells in vitro by means of an adherent culture according to claim 6, characterized in that, When digesting the tumor tissue, a mixture of 1 mg / mL collagenase IV and 100 U / mL hyaluronidase is used for digestion for 30-60 min; The filtering is performed using a 40-μm filter membrane.

8. The method of culturing primary tumor cells in vitro according to claim 7, wherein, The cells are seeded in a collagen-coated culture dish containing the culture medium according to any one of claims 1 to 4 in an amount of (1-5)xlO 5 cells / cm 2 .

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