A method for constructing primary cell lines of IDH-mutant gliomas

Abstract

This invention discloses a method for constructing a primary cell line for IDH-mutant glioma. This invention is the first to propose a method for constructing a primary cell line for IDH-mutant glioma. The primary cells cultured using this method retain the expression of the IDH1 R132H mutant protein even after several weeks of culture and continuously produce 2-HG at levels higher than in vivo, demonstrating high fidelity. This provides a highly reliable model for drug screening and mechanistic studies of IDH-mutant glioma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for constructing a primary cell line of IDH mutant glioma. Background Technology

[0002] Gliomas, especially glioblastomas (GBM), are highly heterogeneous and treatment-resistant. Traditional glioma cell lines (such as U87 and LN229) lose key genetic and phenotypic characteristics of the primary tumor after long-term passage in serum-containing media, making them unsuitable for translational research. Therefore, establishing primary glioma cells directly from patient surgical specimens has become the current mainstream paradigm.

[0003] IDH-mutant gliomas mainly include IDH-mutant astrocytomas and IDH-mutant oligodendrogliomas, which mostly occur in young adults aged 20-40, with a patient survival of approximately 5-15 years. Their core molecular characteristic is mutation in the IDH1 (most commonly R132H mutation) or IDH2 gene, leading to the accumulation of the metabolically abnormal product 2-hydroxyglutarate (2-HG), triggering G-CIMP methylation phenotype, metabolic reprogramming, and alterations in the immune microenvironment. Due to their low proliferative activity and unique metabolic characteristics, IDH-mutant gliomas have long lacked in vitro culture models, severely hindering the exploration of mechanisms of treatment resistance and the development of new drugs. Summary of the Invention

[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method for constructing a primary cell line of IDH mutant glioma.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the present invention provides a culture medium for culturing primary IDH mutant glioma cells.

[0006] Furthermore, the culture medium includes a basal culture medium and additives.

[0007] Furthermore, the additives include B27 Supplement, N2 Supplement, penicillin-streptomycin, EGF, bFGF, sodium pyruvate, DM-αKG, NAC, GSH, BDNF, GDNF, Laminin, and Fibronectin.

[0008] Furthermore, the basal culture medium is selected from Neurobasal-A medium.

[0009] In this invention, IDH refers to isocitrate dehydrogenase. An IDH mutation refers to the presence of an altered or "mutated" IDH gene in tumor cells of astrocytomas or oligodendromas, preventing the tumor cells from producing the IDH protein. IDH mutations are present in low-grade gliomas, grade II gliomas, melanomas, angioimmunoblastic T-cell lymphomas, myeloproliferative neoplasms, myelodysplastic syndromes (MDS), and acute myelocytic leukemia (AML). All central nervous system tumors are graded from I to IV based on the similarity of the tumor cells' appearance and behavior to cells commonly found in the central nervous system; the grading system used by most pathologists is known as the WHO grading system.

[0010] In some embodiments, the basal culture medium includes, but is not limited to, Neurobasal-A medium, Dulbecco modified Eagle medium (DMEM), basal medium (MEM), and Knockout medium. DMEM(KO DMEM), Glasgow basal medium (G) MEM), Eagle basal medium (BME), DMEM / Ham's F12, Advanced DMEM / F12 (Advanced DMEM / Ham's F12), Iscov modified Dulbecco medium, Ham's F 10. Ham'sF 12, 199 medium, RPMI 1640 medium. In a specific embodiment of the present invention, the basal medium is selected from Neurobasal-A medium.

[0011] In some embodiments, the culture medium may further contain amino acids, including L... alanine, L Arginine, L Asparagine, L Aspartic acid, L Cysteine, L Cystine, L Glutamic acid, L glycine, L Histidine, L Isoleucine, L Leucine, L Lysine, L Methionine, L Phenylalanine, L Proline, L Serine, L Threonine, L Tryptophan, L Tyrosine, L Valine and its combinations.

[0012] In some embodiments, the culture medium may also contain vitamins, including but not limited to thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), and DL-C. α Tocopherol (vitamin E), biotin (vitamin H), and menadione (vitamin K).

[0013] In some embodiments, the culture medium may also contain inorganic salts, including but not limited to salts of calcium, copper, iron, magnesium, potassium, sodium, and zinc. Salts are typically used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specifically, salts include, but are not limited to, CaCl2, CuSO4·5H2O, Fe(NO3)·9H2O, FeSO4·7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4·H2O, and ZnSO4·7H2O.

[0014] In some embodiments, the culture medium may also contain sugars that can serve as a carbon energy source. Sugars include, but are not limited to, glucose, galactose, mannose, and fructose. Among these, glucose is preferred, and D-glucose (dextrose) is more preferred.

[0015] In some embodiments, the culture medium may also contain trace elements. These trace elements include barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0016] Furthermore, in the culture medium: The final concentration of B27 supplement is 0.5×-1.5×, N2 The final concentrations of the supplements were 0.5×-1.5×, penicillin-streptomycin 50U / mL-150U / mL, EGF 10ng / mL-30ng / mL, bFGF 10ng / mL-30ng / mL, sodium pyruvate 0.5mM-1.5mM, DM-αKG 50µM-150µM, NAC 0.5mM-1.5mM, GSH 1µM-3µM, BDNF 5ng / mL-15ng / mL, GDNF 5ng / mL-15ng / mL, laminin 0.5µg / mL-1.5µg / mL, and Fibronectin 1µg / mL-3µg / mL.

[0017] Furthermore, in the culture medium: The final concentrations of B27 Supplement, N2 Supplement, penicillin-streptomycin, EGF, bFGF, sodium pyruvate, DM-αKG, NAC, GSH, BDNF, GDNF, laminin, and Fibronectin were 1 µg / mL and 2 µg / mL, respectively.

[0018] Furthermore, the IDH-mutant glioma includes gliomas carrying IDH1 gene mutations.

[0019] Furthermore, the IDH-mutant gliomas include IDH-mutant astrocytomas and IDH-mutant oligodendrogliomas.

[0020] Furthermore, the IDH1 gene mutation includes the R132H site mutation.

[0021] A second aspect of the present invention provides a method for culturing primary cells of IDH-mutant glioma.

[0022] Furthermore, the method includes culturing IDH-mutant glioma tissue samples using the culture medium described in the first aspect of the present invention.

[0023] In some embodiments, the IDH-mutant glioma tissue sample is isolated from a patient with IDH-mutant astrocytoma or IDH-mutant oligodendroglioma.

[0024] Furthermore, the method includes: 1) Use Matrigel substrate to coat the culture containers; 2) Cut the IDH mutant glioma tissue sample into 0.5 mm³ tissue blocks and transfer them to the culture container coated in step 1) above for static culture; 3) After static culture is completed, add the culture medium described in the first aspect of the present invention to the culture container and culture.

[0025] In some embodiments, common culture container forms include, but are not limited to: multi-well cell culture plates (such as 6-well, 12-well, 24-well, 48-well, or 96-well plates), cell culture dishes (such as 35 mm, 60 mm, or 100 mm diameter culture dishes), cell culture flasks (such as T25, T75, or T175 culture flasks), chamber slides, or microplates. In this invention, the culture container is preferably a multi-well cell culture plate coated with Matrigel matrix gel.

[0026] Furthermore, the Matrigel matrix gel was incubated at 37°C for 1 hour.

[0027] Furthermore, the Matrigel matrix gel was diluted with pre-cooled PBS at a ratio of 1:30.

[0028] Furthermore, the pre-cooling temperature is 0~4℃.

[0029] Furthermore, the static incubation time is 15 minutes, and the static incubation temperature is 37°C.

[0030] Furthermore, the culturing conditions in step 3) are as follows: culturing in a constant temperature incubator at 37°C and 5% CO2.

[0031] Furthermore, the culture medium described in the first aspect of the present invention is preheated.

[0032] Furthermore, the preheating temperature is 37°C; Furthermore, the method also includes a fluid replacement step.

[0033] Furthermore, the fluid replacement is performed by changing half the fluid every 3 days.

[0034] In this invention, half-volume medium replacement refers to the procedure of removing approximately half the volume of old culture medium from the culture container and replenishing it with an equal volume of freshly prepared culture medium during cell culture. Specifically, this typically involves aspirating and discarding approximately 50% (v / v) of the old culture supernatant from the wells or dishes, then slowly adding fresh culture medium containing the same concentration of growth factors and additives along the sidewall of the culture container until the liquid level returns to its initial volume. This procedure aims to remove metabolic waste and apoptotic cell debris while maintaining a dynamic balance of cytokines and nutrients in the culture system, avoiding drastic fluctuations in the cell microenvironment and damage to adherent cells caused by physical shear forces during a full medium replacement.

[0035] Furthermore, the IDH-mutant glioma includes gliomas carrying IDH1 gene mutations.

[0036] Furthermore, the IDH-mutant gliomas include IDH-mutant astrocytomas and IDH-mutant oligodendrogliomas.

[0037] Furthermore, the IDH1 gene mutation includes the R132H site mutation.

[0038] A third aspect of the present invention provides a primary cell line of IDH-mutant glioma.

[0039] Furthermore, the primary cells are constructed using the method described in the second aspect of this invention.

[0040] Furthermore, the primary cells can retain the IDH mutant genotype and maintain good proliferation status even after long-term culture.

[0041] The inventors of this invention have for the first time successfully cultured primary cells of IDH-mutant glioma using patient-derived IDH1 R132H mutant astrocytoma and IDH1 R132H mutant oligodendroglioma tissue samples. These primary cells can retain the IDH mutant genotype and maintain a good proliferative state after long-term culture, and can serve as a cell model for studying the pathogenesis and pathogenic factors of IDH-mutant glioma, screening therapeutic drugs, or predicting efficacy.

[0042] The fourth aspect of this invention provides the application of the culture medium described in the first aspect of this invention in the construction of primary IDH mutant glioma cells.

[0043] The fifth aspect of this invention provides the application of the primary cells described in the third aspect of this invention in screening drugs for the treatment of IDH-mutant gliomas, constructing drug screening models for IDH-mutant gliomas, predicting the efficacy of radiotherapy, predicting the efficacy of concurrent chemoradiotherapy, and studying the pathogenesis or pathogenic factors of IDH-mutant gliomas.

[0044] In this invention, treatment and its grammatical variations refer to clinical interventions that attempt to alter the natural course of disease in the treated individual, and may be performed for prevention or in the course of clinicopathology. Desired therapeutic effects include, but are not limited to, preventing disease onset or recurrence, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing disease progression, improving or alleviating the disease state, and mitigating or improving prognosis.

[0045] The present invention also provides a method for predicting the treatment sensitivity of IDH-mutant gliomas, the method comprising contacting the primary cells described in the third aspect of the present invention with a test drug.

[0046] Furthermore, the tested drugs include targeted drugs and chemotherapy drugs.

[0047] Furthermore, the method also includes contacting the primary cells described in the third aspect of the invention with radiation.

[0048] Further, the method includes measuring the primary cells in contact with the test drug, and determining the sensitivity of the test drug based on changes in the morphology and number of the primary cells. If the number of primary cells decreases or the growth of primary cells is inhibited, it indicates that the primary cells are sensitive at that test drug concentration.

[0049] In this invention, contacting the primary cells described in the third aspect of the invention with the test drug means placing the primary cells and the test drug in a contactable state. For example, contacting the primary cells with the test drug in the third aspect of the invention can be achieved by adding the test drug to a solution containing the primary cells. The test drug of this invention can be a low-molecular-weight compound, protein (e.g., antibody), DNA, RNA, low-molecular-weight interfering RNA, or antisense oligonucleotide. The test drug can be, for example, an agent for treating diseases or cancers other than IDH-mutant gliomas. The test drug can be, for example, a mixture of one or more substances. Preferably, the test drug is a single substance.

[0050] Advantages and beneficial effects of the present invention: This invention proposes for the first time a method for constructing primary cell lines of IDH-mutant gliomas. The cells cultured in this invention can still retain the expression of the IDH1 R132H mutant protein after several weeks of culture and continuously produce 2-HG at levels higher than in vivo, with high fidelity. This provides a highly reliable model for drug screening and mechanism research of IDH-mutant gliomas. Attached Figure Description

[0051] Figure 1 The immunohistochemical results of IDH1 R132H mutant astrocytoma are shown; among them, Figure 1 A, Figure 1 B Figure 1 C shows the immunohistochemical results of the experimental group culture medium, the culture medium with the oxidant replaced, and the control culture medium, respectively; Figure 1 D shows the quantitative detection results of IDH1 R132H mutation in different culture media; Figure 2 The OD values ​​of cells cultured in the experimental group and the culture medium with the replaced oxidant are shown. 450 result; Figure 3 The experimental and control culture media showed the OD values ​​of IDH1 R132H mutant astrocytomas cultured in both media. 450 result; Figure 4 The experimental and control culture media showed the OD values ​​of IDH1 R132H mutant oligodendrogliomas. 450 result. Detailed Implementation

[0052] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations.

[0053] Example I. Experimental Materials 1. Tissue samples: Three fresh surgical samples each of IDH1 R132H mutant astrocytoma and oligodendroglioma, all from the Department of Neurosurgery, Beijing Tiantan Hospital, Capital Medical University (IDH1 R132H mutant astrocytoma samples were numbered A1, A2, and A3; IDH1 R132H mutant oligodendroglioma samples were numbered O1, O2, and O3).

[0054] 2. Culture media and additives: Neurobasal-A medium (Gibco, catalog number 10888022), B27 supplement (Gibco, catalog number 17504044), N2 supplement (Gibco, catalog number 17502048), penicillin-streptomycin (Gibco, catalog number 15140122), recombinant human EGF (PeproTech, catalog number AF-100-15), bFGF (PeproTech, catalog number 100-18B), sodium pyruvate (Sigma-Aldrich, catalog number P2256), dimethyl α-ketoglutarate (DM-αKG, Sigma-Aldrich, catalog number 349631), N-acetylcysteine ​​amide (NACA, Sigma-Aldrich, catalog number A0737), L-reduced glutathione (GSH, Sigma-Aldrich, catalog number G4251), β-... Mercaptoethanol (BME, Sigma-Aldrich, catalog number M6250), recombinant human brain-derived neurotrophic factor (BDNF, PeproTech, catalog number xy-011Ga01), recombinant human glial cell-derived neurotrophic factor (GDNF, PeproTech, catalog number 450-10), and laminin (Laminin, Gibco, catalog number 23017015).

[0055] 3. Matrix adhesive: Matrigel (Corning, catalog number CLS354234).

[0056] II. Cultivation Methods (1) On ice, dilute Matrigel 1:30 with pre-cooled PBS, coat 24-well plates, add 200 µL to each well, and incubate at 37°C for 1 hour; (2) Under sterile conditions, the fresh tumor tissue was washed with PBS and mechanically cut into tissue blocks of about 0.5 mm³ using a scalpel blade; (3) Use tweezers to gently transfer the tissue blocks into the coated 24-well plate, placing 4-5 blocks in each well; let stand in a 37℃ incubator for 15 minutes to allow the tissue blocks to adhere slightly; (4) Slowly add 500 µL of the corresponding 37°C pre-warmed culture medium to the wells of the control group and the present invention group respectively; place the culture plate in a constant temperature incubator at 37°C and 5% CO2 for incubation; Control group culture medium (NC): Neurobasal-A medium (basal medium) + 1× B27 Supplement + 1× N2 Supplement + penicillin-streptomycin (100 U / mL) + EGF (20 ng / mL) + bFGF (20 ng / mL); Experimental group culture medium (IDH Medium): Neurobasal-A medium (basal medium) + 1× B27 Supplement + 1× N2 Supplement + penicillin-streptomycin (100 U / mL) + EGF (20 ng / mL) + bFGF (20 ng / mL) + sodium pyruvate (1 mM) + DM-αKG (100 µM) + NAC (1 mM) + GSH (2 µM) + BDNF (10 ng / mL) + GDNF (10 ng / mL) + Laminin (1 µg / mL) + Fibronectin (2 µg / mL); Oxidizing agent replacement medium (IDH Medium-BME): Based on the experimental group medium, GSH was replaced with BME (50 µM), while the other components and concentrations remained unchanged; (5) Change half of the medium every 3 days. Seed 5000 cells per well in 96-well plates from different culture media and culture them in their respective culture media. (6) On days 1, 4, 7, 11, 14, 17 and 21 of culture, the absorbance (OD value) at 450 nm was measured by CCK-8 method and the growth curve was plotted.

[0057] III. Experimental Results Immunohistochemical results of IDH-R132H mutations showed that after 10 days of culture in the optimized medium (IDH Medium) of this invention, the IDH mutation rate in tumor tissues remained high, indicating that this medium is suitable for culturing IDH-mutant cells. After 10 days of culture in the medium with a replaced oxidant (IDH Medium-BME), the IDH mutation rate in tumor tissues decreased, indicating that this medium damaged IDH-mutant cells. After 10 days of culture in the conventional medium (NC), the IDH mutation rate in tumor tissues decreased significantly, indicating that this medium is unsuitable for culturing IDH-mutant cells. Figure 1 ).

[0058] The CCK-8 results showed that both IDH-mutant astrocytomas and IDH-mutant oligodendromas cultured using the optimized medium (IDH Medium) of this invention were able to survive long-term and maintain a good proliferative state. While both IDH-mutant astrocytomas and IDH-mutant oligodendromas cultured using the medium with the replaced oxidant (IDH Medium-BME) were able to survive long-term, their proliferation was slower. Figure 2 ).

[0059] Three IDH-mutant astrocytomas cultured using conventional medium (NC) failed to survive long-term, while three IDH-mutant astrocytomas cultured using the optimized medium (IDH Medium) of this invention were able to survive long-term and maintain a good proliferative state. Figure 3 ).

[0060] Three IDH-mutant oligodendroglioma strains cultured in conventional medium (NC) failed to survive long-term, while three IDH-mutant oligodendroglioma strains cultured in the optimized medium (IDH Medium) of this invention were able to survive long-term and maintain a good proliferative state. Figure 4 ).

[0061] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A culture medium for culturing primary IDH-mutant glioma cells, characterized in that, The culture medium includes a basal culture medium and additives; The additives include B27 Supplement, N2 Supplement, penicillin-streptomycin, EGF, bFGF, sodium pyruvate, DM-αKG, NAC, GSH, BDNF, GDNF, Laminin, and Fibronectin. The basal culture medium was selected from Neurobasal-A medium.

2. The culture medium according to claim 1, characterized in that, In the culture medium: The final concentration of B27 supplement is 0.5×-1.5×, N2 The final concentrations of the supplements were 0.5×-1.5×, penicillin-streptomycin 50U / mL-150U / mL, EGF 10ng / mL-30ng / mL, bFGF 10ng / mL-30ng / mL, sodium pyruvate 0.5mM-1.5mM, DM-αKG 50µM-150µM, NAC 0.5mM-1.5mM, GSH 1µM-3µM, BDNF 5ng / mL-15ng / mL, GDNF 5ng / mL-15ng / mL, laminin 0.5µg / mL-1.5µg / mL, and Fibronectin 1µg / mL-3µg / mL.

3. The culture medium according to claim 1, characterized in that, In the culture medium: The final concentrations of B27 Supplement, N2 Supplement, penicillin-streptomycin, EGF, bFGF, sodium pyruvate, DM-αKG, NAC, GSH, BDNF, GDNF, laminin, and Fibronectin were 1 µg / mL and 2 µg / mL, respectively.

4. The culture medium according to claim 1, characterized in that, The IDH-mutant gliomas include gliomas carrying IDH1 gene mutations; Preferably, the IDH-mutant glioma includes IDH-mutant astrocytoma and IDH-mutant oligodendroglioma; Preferably, the IDH1 gene mutation includes the R132H site mutation.

5. A method for culturing primary IDH-mutant glioma cells, characterized in that, The method includes culturing IDH mutant glioma tissue samples using the culture medium according to any one of claims 1-4.

6. The method according to claim 5, characterized in that, The method includes: 1) Use Matrigel substrate to coat the culture containers; 2) Cut the IDH mutant glioma tissue sample into 0.5 mm³ tissue blocks and transfer them to the culture container coated in step 1) above for static culture; 3) After static culture is completed, add the culture medium described in any one of claims 1-4 to the culture container and culture.

7. The method according to claim 6, characterized in that, The Matrigel matrix gel was incubated at 37°C for 1 hour. Preferably, the static incubation time is 15 minutes, and the static incubation temperature is 37°C. Preferably, the culturing conditions in step 3) are as follows: culturing in a constant temperature incubator at 37°C and 5% CO2. Preferably, the culture medium according to any one of claims 1-4 is preheated; Preferably, the preheating temperature is 37°C; Preferably, the method further includes a liquid replacement step; Preferably, the fluid replacement is performed by changing half the fluid every 3 days; Preferably, the IDH-mutant glioma includes gliomas carrying IDH1 gene mutations; Preferably, the IDH-mutant glioma includes IDH-mutant astrocytoma and IDH-mutant oligodendroglioma; Preferably, the IDH1 gene mutation includes the R132H site mutation.

8. A primary cell line of IDH-mutant glioma, characterized in that, The primary cells are prepared by the method according to any one of claims 5-7; Preferably, the primary cells retain the IDH mutant genotype and maintain good proliferation after long-term culture.

9. The use of the culture medium according to any one of claims 1-4 in the construction of primary IDH mutant glioma cells.

10. The application of the primary cells described in claim 8 in screening drugs for the treatment of IDH-mutant gliomas / constructing drug screening models for IDH-mutant gliomas / radiotherapy efficacy prediction models / concurrent chemoradiotherapy efficacy prediction models / research on the pathogenesis or pathogenic factors of IDH-mutant gliomas.

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