Culture medium, its use and microtumor culture method

Abstract

The application belongs to the field of biological medicine, and discloses a culture medium, which comprises a basic culture medium, and the basic culture medium is added with ITS-A: 1x, arginine: 15-20 mu g / ml, SPP1: 3-5 mu g / ml and S100A9: 3-5 mu g / ml. The culture medium is particularly suitable for microtumor culture of MTC model and has good cell viability. Meanwhile, the application also provides application of the culture medium and a microtumor culture method.

Description

Technical Field

[0001] This invention relates to the field of medicine, and more particularly to a culture medium and its application, and a method for culturing microtumors. Background Technology

[0002] The applicant has filed an invention patent application for a method for culturing microtumors, application number 2026105772966, entitled "Method for culturing microtumors applicable to multiple cancer types, drug screening method and drug combination".

[0003] In this project, we used Complete F Medium to culture tumor tissues: HP10069 breast cancer, EMT6 breast cancer, Hepa1-6 liver cancer, LLC lung cancer, B16 melanoma, and MC38 colon adenocarcinoma. We then used the cultured tumor tissues to verify the response to immune checkpoint blockade (ICB) and screened a number of new drug combinations.

[0004] This application is an in-depth study based on the previous project. The purpose of this project is to investigate whether there are better culture media to improve the viability of tumor cells, so as to facilitate subsequent experiments. Summary of the Invention

[0005] The purpose of this invention is to provide a culture medium that is particularly suitable for the culture of microtumor cells in the MTC model and exhibits good cell viability.

[0006] In addition, the present invention also provides an application of the culture medium and a method for culturing microtumors.

[0007] The specific solution of the present invention is as follows:

[0008] A culture medium comprising a basal culture medium, wherein the basal culture medium is added to:

[0009] ITS-A: 1x;

[0010] Arginine: 18~22μg / ml;

[0011] SPP1: 3~5μg / ml;

[0012] S100A9: 3~5μg / ml.

[0013] It should be noted that the 1x ITS-A mentioned in this invention refers to adding 100x (100-fold concentrated) ITS-A raw material to the basal culture medium to make the ITS-A raw material diluted 100 times; specifically in this case, the basal culture medium is 500ml, so the amount of 1x ITS-A added is 5ml.

[0014] In the above-mentioned culture medium, the basal culture medium contains:

[0015] ITS-A: 1x;

[0016] Arginine: 20 μg / ml;

[0017] SPP1: 4 μg / ml;

[0018] S100A9: 4μg / ml.

[0019] In the above-mentioned culture medium, the basal culture medium consists of F-12 culture medium and DMEM culture medium; the volume ratio of F-12 culture medium to DMEM culture medium is 100~150:350~400.

[0020] In the above-mentioned culture media, the volume ratio of F-12 culture medium to DMEM culture medium is 125:375;

[0021] The basal culture medium also contains:

[0022] Hydrocortisone, Epidermal Growth Factor (EGF), Insulin, Cholera toxin, Y-27632, Cefoperazone Sodium, Sulbactam Sodium.

[0023] In the above-mentioned culture medium, the hydrocortisone and epidermal growth factor EGF are provided in the form of a mixture (1,000x), and the amount is 0.5 ml added per 500 ml of basal culture medium;

[0024] The dosage of insulin (5 mg / ml) is 0.5 ml per 500 ml of basal culture medium;

[0025] The dosage of Cholera toxin (0.1 mg / mL) is 43 μL per 500 mL of basal culture medium;

[0026] The dosage of Y-27632 (10mM in sterile water) is 0.5ml per 500ml of basal culture medium;

[0027] The dosage of cefoperazone sodium and sulbactam sodium (20 μg / mL cefoperazone sodium + 10 μg / mL sulbactam sodium) is 5 ml per 500 ml of basal culture medium.

[0028] In addition, the present invention also discloses the application of culturing microtumors using any of the culture media described above.

[0029] In the above applications, the tumor type of the microtumor is one or more of breast cancer, liver cancer, lung cancer, melanoma, colon adenocarcinoma, and nasopharyngeal carcinoma.

[0030] Furthermore, this invention also discloses a microtumor culture method applicable to various cancer types, comprising the following steps:

[0031] Step 1: Cut the tumor tissue into tissue blocks with a diameter of 100~500μm;

[0032] Step 2: Place the tissue block in the gel and allow the gel to solidify;

[0033] Step 3: Contact the cured gel with the culture medium, while keeping the gel in contact with the atmosphere;

[0034] After a period of cultivation, viable culture tissues are obtained;

[0035] The culture medium is any of the culture media described above.

[0036] The cultivation method of this invention is identical to that of the prior patent application, except for the use of the culture medium.

[0037] The beneficial effects of this application are:

[0038] Through repeated screening, this invention has yielded a culture medium suitable for the MTC model with good cell viability, providing a better experimental basis for subsequent drug screening based on immune checkpoint blockade (ICB). Attached Figure Description

[0039] Figure 1 This is a flowchart of Example 1. Detailed Implementation

[0040] The present invention will now be clearly and completely described in conjunction with embodiments thereof. It should be noted that, unless specific conditions are specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0041] Before describing the method of the present invention, the raw materials used in the present invention will be described; see Table 1 for details.

[0042] Table 1 Raw Material List

[0043] gel R&D Systems Cultrex 3-D Culture Matrix™ Rat Collagen I 5 mg collagen / mL F-12 culture medium Gibco F-12 Nutrient Mixture 10x DMEM medium Gibco Dulbecco's Modified Eagle Medium (DMEM), High Glucose, GlutaMAX™Supplement 500 mL Hydrocortisone MCE Hydrocortisone 500mg / mL Epidermal growth factor (EGF) MCE EGF, Human 0.125 ng / mL insulin MCE Insulin (human) 5 mg / mL Cholera toxin MCE Cholera toxin 500ug / mL Y-27632 MCE Y-27632 25mg / mL cefoperazone Shupu Shen Cefoperazone sodium and sulbactam sodium for injection 1.5g ITS-A Gibco Insulin-Transferrin-Selenium (ITS-A), 100x Concentrate 100x SPP1 R&D Systems Recombinant Mouse Osteopontin (SPP1), CF 50 µg / bottle S100A9 R&D Systems Recombinant Mouse S100A9 Protein, CF 50 µg / bottle MitoQ Merck (Sigma-Aldrich) MitoQ Bromide 5 mg / bottle L-WRN Millipore / Sigma-Aldrich 3dGRO® L-WRN Conditioned Media Supplement 100mL B27 Gibco B-27 Serum-Free Additive (50x, Classic Standard) 50x Era MedChemExpress Estradiol 1 g / bottle HEPES Gibco HEPES buffer (1M liquid) 100 mL / bottle NAM Sigma-Aldrich Nicotinamide 250 g / bottle NAC Sigma-Aldrich N-Acetyl-L-cysteine 100 g / bottle SB202190 Sigma-Aldrich SB 202190 5 mg / bottle A83-01 Sigma-Aldrich A 83-01 5 mg / bottle

[0044] Table 2 Formulation of basal culture medium

[0045] F-12 culture medium 125mL A mixture of 0.5 mg hydrocortisone and 2.5 μg epidermal growth factor (EGF) 0.5mL insulin 0.5mL Cholera toxin 43uL Y-27632 0.5mL 20 μg / mL cefoperazone sodium + 10 μg / mL sulbactam sodium 0.5mL

[0046] Example

[0047] Establishment of a microtumor culture (MTC) model for high-throughput drug screening

[0048] refer to Figure 1 The microtumor model of the present invention includes the following structure:

[0049] Culture medium.

[0050] Gel: Cultrex 3-D Culture Matrix™ Rat Collagen I.

[0051] Tumor tissue block (4T1 breast cancer tumor-bearing tissue): pre-cut to a predetermined size: 200 μm thick and 5 mm in diameter;

[0052] Add tumor tissue blocks to the gel and allow the gel to solidify;

[0053] Culture medium and gel are cultured in a 24-well plate with an embedded culture insert; Figure 1 The culture format shown is a single-well culture; each gel contains one piece of tumor tissue.

[0054] The lower part of the gel is in contact with the culture medium, while the entire upper part of the gel is in contact with the atmosphere. The culture time is 4 days, and the culture medium is changed every 2 days. The viability of the tumor tissue blocks is evaluated as time progresses.

[0055] Cell viability was determined using the MTT assay. After culture, the Transwell chambers were placed in MTT solution (final concentration 0.5 mg / mL) and incubated at 37°C in a 5% CO2 incubator for 4 hours in the dark. The gel blocks were then removed, and the tissue sections were placed in suitable culture dishes with an appropriate amount of DMSO added and shaken thoroughly to dissolve the formazan crystals. Finally, the supernatant was aspirated, and the absorbance (OD value) at 490 nm was measured using a microplate reader to reflect the cell viability of the tissue blocks.

[0056] The screening process of this invention is shown in Tables 3 to 7 below;

[0057] It should be noted that: in the following tables of this invention, only the substances that need to be added are indicated. The basic culture medium is shown in Table 2.

[0058] Table 3 Screening Process Parameter Table

[0059] 1 0 0 0 0 0 0 0 0 100 2 2 0 0 0 0 0 0 0 96 3 0 30 0 0 0 0 0 0 98 4 0 0 2 0 0 0 0 0 110 5 0 0 0 100 0 0 0 0 120 6 0 0 0 0 4 0 0 0 122 7 0 0 0 0 0 1 0 0 120 8 0 0 0 0 0 0 1 0 123 9 0 0 0 0 0 0 0 20 133

[0060] Table 4 Screening Process Parameter Table

[0061] 10 0 0 0 0 0 100 11 15 10 1.25 0 0 135 12 0 0 0 0.5 0.5 128

[0062] Table 5 Screening Process Parameter Table

[0063] 13 0 0 0 0 0 0 0 0 0 100 14 2 1 0 0 0 0 0 0 0 110 15 2 0 20 0 0 0 0 0 0 112 16 2 0 0 100 0 0 0 0 0 115 17 2 0 0 0 0.5 0 0 0 0 110 18 2 0 0 0 0 0.5 0 0 0 93 19 2 0 0 0 0 0 / 0 0 95 20 2 0 0 0 0 0 0 4 0 107 21 2 0 0 0 0 0 0 0 1 103 It should be noted that in Table 5, NAC+HEPES+Nicotinamide refers to the individual dosages of the three: HEPES (15 mmol / L), Nicotinamide (10 mmol / L), and NAC (1.25 mmol / L).

[0064] Table 6 Screening Process Parameter Table

[0065] 22 0 0 0 0 0 0 0 100 23 1 100 0 0 0 0 0 125 24 1 0 4 0 0 0 0 126 25 1 0 0 1 0 0 0 130 26 1 0 0 0 / 0 0 127 27 1 0 0 0 0 0.5 0 108 28 1 0 0 0 0 0 / 101

[0066] It should be noted that in Table 6, SB202190+A83-01 refers to the dosage of SB202190 (0.5 μmol / L) and A83-01 (0.5 μmol / L); NAC+HEPES+MAC refers to the dosage of HEPES (15 mmol / L), Nicotinamid (10 mmol / L), and NAC (1.25 mmol / L).

[0067] Table 7 Screening Process Parameter Table

[0068] 29 0 0 0 0 100 30 1 20 0 0 146 31 1 20 4 0 156 32 1 20 0 1 155 33 1 20 4 1 175 34 0 20 4 1 160

[0069] The above experiments show that adding ITS-A, arginine, SPP1, and S100A9 to the basal culture medium can produce a relatively ideal cell viability culture effect.

[0070] Based on the test results of samples 30# to 33#, SPP1 and S100A9 showed obvious synergy in the presence of ITS-A and arginine.

[0071] To further clarify under what conditions SPP1 and S100A9 exhibit synergy, we further verified the experimental effects when ITS-A and arginine were present individually, and the results are shown in Table 8 below.

[0072] Table 8 Experimental Results

[0073] 45 0 0 0 0 100 46 1 0 4 0 124 47 1 0 0 1 128 48 1 0 4 1 154 49 0 20 4 0 126 50 0 20 0 1 132 51 0 20 4 1 162

[0074] As can be seen from the data in Table 8, SPP1 and S100A9 can only exhibit the best synergistic effect in the presence of ITS-A and arginine.

[0075] In summary, this invention has developed a culture medium with good cell viability that is particularly suitable for the MTC model, providing a better experimental basis for subsequent drug screening based on immune checkpoint blockade (ICB).

Claims

1. A culture medium, characterized in that, Includes a basal culture medium, wherein the basal culture medium contains: ITS-A: 1x; Arginine: 15~20μg / ml; SPP1: 3~5μg / ml; S100A9: 3~5μg / ml.

2. The culture medium according to claim 1, characterized in that, The basal culture medium contains: ITS-A: 1x; Arginine: 20 μg / ml; SPP1: 4 μg / ml; S100A9: 4μg / ml.

3. The culture medium according to claim 1, characterized in that, The basal culture medium consists of F-12 medium and DMEM medium; the volume ratio of F-12 medium to DMEM medium is 100~150:350~400.

4. The culture medium according to claim 3, characterized in that, The volume ratio of the F-12 medium to the DMEM medium is 125:375; The basal culture medium also contains: Hydrocortisone, Epidermal Growth Factor (EGF), Insulin, Cholera toxin, Y-27632, Cefoperazone, Sulbactam Sodium.

5. The culture medium according to claim 4, characterized in that, The hydrocortisone and epidermal growth factor (EGF) are provided in the form of a mixture, with a dosage of 0.5 ml per 500 ml of basal culture medium. The insulin dosage is 0.5 ml per 500 ml of basal culture medium; The dosage of Cholera toxin is 43 μL per 500 ml of basal culture medium; The dosage of Y-27632 is 0.5 ml per 500 ml of basal culture medium; The dosage of cefoperazone and sulbactam sodium is 0.5 ml per 500 ml of basal culture medium.

6. Application of culturing microtumors using the culture medium as described in any one of claims 1 to 5.

7. The application according to claim 6, characterized in that, The microtumor is one or more of the following types: breast cancer, liver cancer, lung cancer, melanoma, colon adenocarcinoma, and nasopharyngeal carcinoma.

8. A microtumor culture method applicable to various cancer types, characterized in that, Includes the following steps: Step 1: Cut the tumor tissue into tissue blocks with a diameter of 100~500μm; Step 2: Place the tissue block in the gel and allow the gel to solidify; Step 3: Contact the cured gel with the culture medium, while keeping the gel in contact with the atmosphere; After a period of cultivation, viable culture tissues are obtained; The culture medium is the culture medium as described in any one of claims 1 to 5.

Images