Establishment method and culture medium formula of ventricular canal membrane tumor organoid

Abstract

The invention discloses a construction method and a culture medium formula of a ventricular canal membrane tumor organ. By utilizing the culture medium formula, the organoid can be effectively promoted to form an ideal form; compared with other culture media, the culture medium has the advantages that the organoid cultured by the culture medium has a more regular circular or elliptical structure, the edge is clear and regular, and various organs exist independently without mutual adhesion. According to the culture medium, the morphological stability and consistency of the organoid are remarkably improved, and a reliable basis is provided for research and drug screening of the ventricular tubuloma.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically, it relates to a method for constructing ependymoma organoids and a culture medium formulation. Background Technology

[0002] Ependymomas are primary central nervous system tumors originating from the ventricular system of the brain or the central canal of the spinal cord. They are usually caused by abnormal proliferation or malignant transformation of ependymal cells and have a high recurrence rate and risk of disability. According to the World Health Organization Classification of Tumors of the Central Nervous System (5th Edition), ependymomas are classified into grades I, II, and III, with grade III being anaplastic ependymomas. Grade III anaplastic ependymomas are highly malignant, exhibit more complex biological behavior, are more difficult to treat, and have significantly increased clinical risks. Based on age of onset, anaplastic ependymomas can be divided into adult and pediatric types. Pediatric anaplastic ependymomas mainly occur in the ventricular system and usually respond well to treatment; while adult anaplastic ependymomas mostly occur in the spinal cord, and treatment mainly involves surgery combined with adjuvant radiotherapy, but the response to chemotherapy is relatively limited. Compared with pediatric anaplastic ependymomas, adult anaplastic ependymomas have a poorer prognosis and a rapid disease progression. Due to the clinical characteristics and treatment challenges of adult anaplastic ependymomas, there is an urgent need to develop an in vitro model suitable for adult anaplastic ependymomas in order to conduct in-depth research on their pathogenesis and optimize clinical treatment plans, thereby improving treatment outcomes and prognosis.

[0003] To deepen the understanding of the pathogenesis of anaplastic ependymoma and optimize treatment strategies, organoid models offer a novel technological platform for related research. As a three-dimensional culture system, organoids can simulate the microenvironment of tissues or organs in vitro, exhibiting high biological similarity and functional reconstruction capabilities. They have become important tools for studying tumor biological characteristics, screening anti-tumor drugs, and developing personalized treatments. Particularly in studying tumor signaling pathways, the dynamic changes in the tumor microenvironment, and their drug response mechanisms, organoid models provide an experimental basis highly consistent with in vivo tumors. Compared to traditional two-dimensional cell culture, three-dimensional organoids can more realistically reflect key characteristics of tumor cells under physiological conditions, such as proliferation, differentiation, migration, and drug resistance. Previous studies have reported the successful in vitro construction of organoids for pediatric ependymoma using specific culture medium combinations. However, there are currently no reports on organoids for adult anaplastic ependymoma.

[0004] Therefore, developing organoid models of adult anaplastic ependymoma is not only crucial for a deeper understanding of the pathogenesis of anaplastic ependymoma, but also has significant scientific value for optimizing clinical treatment plans and improving treatment outcomes. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a method for constructing organoids of adult anaplastic ependymoma and a culture medium formulation suitable for adult anaplastic ependymoma organoids. This aims to provide an in vitro culture model that more closely reflects the biological characteristics of ependymoma in vivo for studying the pathogenesis of adult anaplastic ependymoma, screening anti-tumor drugs, and developing personalized treatment plans.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] The first aspect of the present invention provides a culture medium for ependymoma organoids.

[0008] Furthermore, the culture medium includes a basal culture medium and specific additives.

[0009] Furthermore, the basal culture medium is NeuroCult™ NS-A Basal Medium.

[0010] Furthermore, the specific additives include B-27 additive, N2 additive, BSA additive, GlutaMAX additive, EGF2, bFGF2, decellularized brain matrix, and penicillin-streptomycin.

[0011] Furthermore, the final concentration of the NeuroCult™ NS-A Basal Medium is 1×.

[0012] Furthermore, the final concentration of the B-27 additive is 50×; the final concentration of the N2 additive is 100×; the final concentration of the BSA additive is 300 μg / mL; the final concentration of the GlutaMAX additive is 100×; the final concentration of the EGF2 is 10 ng / mL; the final concentration of the bFGF2 is 10 ng / mL; the final concentration of the decellularized brain matrix is ​​1 mg / mL; and the final concentration of the penicillin-streptomycin is 100×.

[0013] Furthermore, the ependymomas include anaplastic ependymomas and myxoplasmic ependymomas.

[0014] Furthermore, the ependymoma is selected from anaplastic ependymoma.

[0015] In this invention, organoids refer to cell clusters with a 3D structure, and are miniaturized and simplified versions of organs prepared through artificial culture processes. The source of the cells constituting the organoids is unrestricted. Organoids can be derived from tissues or stem cells. Organoids may possess an environment that allows interaction with the surrounding environment during cell growth. Therefore, the 3D organoids of this invention almost completely mimic the interactions of organs in vivo, and can serve as excellent models for observing the development of disease therapeutics. They can similarly replicate the physiological functions of the human body; by constructing organ analogs from patient tissues, disease modeling can be performed based on the patient's genetic information, and drug screening can be conducted through repeated testing.

[0016] In a specific embodiment of the present invention, the organoids are derived from adult anaplastic ependymoma tissue.

[0017] In some embodiments, the basal culture medium includes, but is not limited to, DMEM / F-12, Neurobasal, Dulbecco modified Eagle medium (DMEM), basal medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow basal medium (G-MEM), Eagle basal medium (BME), Iscov modified Dulbecco medium, Ham's F-10, Ham's F-12, 199 medium, RPMI 1640 medium, and NeuroCult™ NS-A Basal Medium.

[0018] In a specific embodiment of the present invention, the basal culture medium is selected from NeuroCult™ NS-A BasalMedium.

[0019] In this invention, the organoid 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.

[0020] In this invention, the organoid 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.

[0021] In this invention, the organoid culture medium may further 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 their ions.

[0022] A second aspect of the present invention provides a method for culturing ependymoma organoids.

[0023] Furthermore, the method includes culturing ependymoma tissue using the culture medium described in the first aspect of the present invention.

[0024] Furthermore, the method also includes mincing the ependymoma tissue before culturing.

[0025] Furthermore, the ependymoma tissue is cut into fragments with a diameter of approximately 0.5-1.0 mm.

[0026] Furthermore, the specific conditions for cultivation are: shaking cultivation at 37°C, 5% CO2, and 120 rpm / min.

[0027] Furthermore, the ependymomas include anaplastic ependymomas and myxoplasmic ependymomas.

[0028] Furthermore, the ependymoma is selected from anaplastic ependymoma.

[0029] A third aspect of the present invention provides an ependymoma organoid.

[0030] Furthermore, the organoids are cultured using the culture medium described in the first aspect of the present invention or obtained by the culture method described in the second aspect of the present invention.

[0031] Furthermore, the ependymomas include anaplastic ependymomas and myxoplasmic ependymomas.

[0032] Furthermore, the ependymoma is selected from anaplastic ependymoma.

[0033] The fourth aspect of this invention provides an in vitro drug screening model for ependymoma.

[0034] Furthermore, the model includes the organoids described in the third aspect of the present invention.

[0035] Furthermore, the ependymomas include anaplastic ependymomas and myxoplasmic ependymomas.

[0036] Furthermore, the ependymoma is selected from anaplastic ependymoma.

[0037] The fifth aspect of this invention provides any of the following methods:

[0038] 1) A method for predicting drug sensitivity in ependymoma, the method comprising contacting an organoid model according to the third aspect of the present invention and a model according to the fourth aspect of the present invention with a test drug;

[0039] 2) A method for screening drugs for the prevention and / or treatment of ependymoma, the method comprising contacting a candidate drug with an organoid of the third aspect of the present invention or a model of the fourth aspect of the present invention;

[0040] 3) A method for constructing an animal disease model, the method comprising transplanting the organoids described in the third aspect of the present invention into the animal to obtain the animal disease model;

[0041] 4) A method for constructing an ependymoma organoid bank, the method comprising: obtaining ependymoma organoids by the culture method according to the second aspect of the present invention; subculturing the ependymoma organoids to obtain subcultured ependymoma organoids; and cryopreserving the subcultured ependymoma organoids to obtain the ependymoma organoid bank.

[0042] Furthermore, the drugs mentioned in 1) include targeted drugs and chemotherapy drugs.

[0043] Furthermore, the method described in 1) also includes exposing the organoid to radiation.

[0044] Furthermore, the method described in 1) also includes measuring the organoids in contact with the test drug and determining the sensitivity of the test drug based on morphological changes in the organoids. Morphological changes in the organoids include a decrease in the number of organoids or a change in the size of the organoids. If the number of organoids decreases or the growth of organoids is inhibited, it indicates that the organoids are sensitive at the test drug concentration.

[0045] In this invention, contacting the organoids with the test drug means placing the organoids and the test drug in a contactable state. Contacting the organoids with the test drug can, for example, involve adding the test drug to a solution containing the organoids. 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, for example, be an agent for treating diseases or cancers other than liver cancer or brain metastases. The test drug can, for example, be a mixture of one or more substances. Preferably, the test drug is a single substance.

[0046] Furthermore, the method described in 2) further includes measuring organoids or models exposed to the candidate substance; and comparing the measured values ​​of organoids or models exposed to the candidate drug with control values. In one embodiment, the method includes determining, based on the comparison results, whether the candidate substance is a drug capable of preventing and / or treating ependymoma.

[0047] In this invention, contacting the candidate drug with the organoid or model means placing the organoid or model and the candidate drug in a contactable state. Contacting the organoid or model with the candidate drug can, for example, be done by adding the candidate drug to a solution containing the organoid or model.

[0048] In this invention, the determination of organoids or models after contact with the aforementioned candidate substances includes reductions in organoid viability, decreases in organoid proliferation, increases in organoid death, changes in organoid size, the number of organoids, and the expression of marker proteins of the cells constituting the organoids. Changes in organoid size can be measured, for example, by measuring the organoid dimensions, which can be determined using known devices (e.g., microscopes or FACS). The organoid dimensions can be, for example, the circumference, diameter (e.g., major and minor axes), or area of ​​the organoid. The expression of marker proteins of the cells constituting the organoids can be determined using known devices (e.g., microscopes or FACS) and known reagents (e.g., antibodies labeled with fluorescent substances). The expression of marker proteins of the cells constituting the organoids can be, for example, the fluorescence intensity corresponding to the amount of marker protein expressed per unit area of ​​the organoid. The measured marker proteins can be one or a combination of two or more. Preferably, the measured marker proteins are a combination of two or more.

[0049] In this invention, the measured value of an organoid or model can be, for example, a single measured value, the average of multiple measurements, or the average of measured values ​​from multiple organoids. The measured value of an organoid can be, for example, the change (e.g., difference or multiple) before and after exposure to the test substance. The control value can be, for example, the measured value of a control organoid before or without exposure to the candidate drug, when using the value after exposure to the candidate drug as the measured value of the organoid.

[0050] Furthermore, the ependymomas include anaplastic ependymomas and myxoplasmic ependymomas.

[0051] Furthermore, the ependymoma is selected from anaplastic ependymoma.

[0052] The sixth aspect of the present invention provides for any of the following applications:

[0053] 1) The application of the culture medium described in the first aspect of the present invention in the culture of ependymoma organoids;

[0054] 2) The use of the organoids described in the third aspect of this invention in screening drugs for the prevention and / or treatment of ependymoma;

[0055] 3) The application of the organoids described in the third aspect of this invention in establishing a drug sensitivity model for ependymoma;

[0056] 4) The application of the organoids described in the third aspect of this invention in the study of the pathogenesis or pathogenic factors of ependymoma;

[0057] 5) The application of the organoids described in the third aspect of this invention in constructing animal disease models.

[0058] 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.

[0059] Advantages and beneficial effects of the present invention:

[0060] (1) This invention is the first to construct organoids of adult anaplastic ependymoma that can grow rapidly. These anaplastic ependymoma organoids can be passaged every 5 to 7 days, and the passaged anaplastic ependymoma organoids can maintain their proliferative activity and morphological characteristics, maintaining the original state of tumor cells for a long time. This feature promotes the research and drug screening of adult anaplastic ependymoma.

[0061] (2) This invention can construct organoids of adult intercalary ependymoma, and the organoid culture medium used can effectively promote the formation of ideal organoid morphology. Compared with other culture media, organoids cultured in this medium exhibit more regular round or elliptical structures with clear and regular edges, and each organ exists independently without mutual adhesion. This culture medium significantly improves the morphological stability and consistency of organoids, providing a reliable basis for further research and application. Attached Figure Description

[0062] Figure 1 Microscopic images of anaplastic ependymoma organoids after 7, 10, and 14 days of culture in the culture medium provided by this invention;

[0063] Figure 2 Microscopic images of anaplastic ependymoma organoids after culturing in comparative culture medium for 7, 10, and 14 days. Detailed Implementation

[0064] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0065] Furthermore, it should be noted in the description of this invention 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 products.

[0066] The method for preparing decellularized brain matrix refers to patent document CN110257335B, which includes: (1) alternating freeze-thaw cycles of brain tissue at -80℃ and room temperature; (2) treating the freeze-thawed brain tissue with deionized water on a shaker; and (3) treating the deionized water-treated brain tissue with 0.5-3% HCl on a shaker. Triton-X100 treatment yielded preliminary decellularized brain tissue; (4) on a shaker, the preliminary decellularized brain tissue was treated with deionized water; (5) on a shaker, the brain tissue treated with deionized water was treated with 2-6% sodium deoxycholate to obtain decellularized brain tissue; the specific preparation process included: 32-week-old female SD rats were perfused with 200ml of physiological saline in the abdominal aorta for a total of 5 minutes until the brain tissue turned white, and then the brain was removed by craniotomy; after removing the cerebellum from the obtained SD rat brain, it was placed at -80℃ for 30 minutes, and then placed at room temperature for 30 minutes, and this was repeated 4 times with alternating freeze-thaw cycles; the brain after alternating freeze-thaw cycles was treated with 100ml of deionized water at 60rpm and room temperature for 4 hours. Then it was treated with 50ml of 1% Triton-X100 at 60rpm and 4℃ for 12 hours to obtain preliminary decellularized brain tissue. Then it was washed with deionized water at 60rpm and room temperature 3 times, 10 minutes each time. The brain tissue was then treated with 50 ml of 4% sodium deoxycholate at 60 rpm and room temperature for 12 hours to obtain decellularized brain tissue. It was then washed three times with deionized water at 60 rpm and room temperature, for 10 minutes each time. Next, it was treated with 50 ml of 1M sucrose solution at 60 rpm and room temperature for 15 minutes to remove residual decellularized solution and cellular components. Finally, it was washed three times with deionized water at 60 rpm and room temperature, for 10 minutes each time.

[0067] Example 1: Method for culturing organoids from anaplastic ependymoma

[0068] 1. Culture medium components for anaplastic ependymoma organoids

[0069] Example Culture medium composition: Based on 1× NeuroCult™ NS-A Basal Medium (Human), 50× B-27 additive, 100× N2 additive, 300 μg / mL BSA additive, 100× GlutaMAX additive, 10 ng / mL epidermal growth factor 2 (EGF2), 10 ng / mL basic fibroblast growth factor 2 (bFGF2), 1 mg / mL decellularized brain matrix, and 100× penicillin-streptomycin additives were added.

[0070] Comparative culture medium composition: Compared with the culture medium composition of the example, the basal culture medium was replaced with a 1:1 ratio of DMEM-F12 and Neurobasal, while the additive composition remained unchanged.

[0071] 2. Culture methods for organoids from anaplastic ependymomas

[0072] (1) Fresh tumor tissue was obtained by craniotomy, transferred to pre-cooled medical polyurethane flexible deformable tubes filled with DMEM-F12 medium, and transported to the laboratory on ice for no more than 1 hour. Informed consent was obtained from all relevant personnel for all specimens.

[0073] (2) Perform aseptic operation in a laminar flow hood and pour the specimen into a culture dish. Use autoclaved microscissors and microforceps to remove red blood vessels and black necrotic tissue produced by surgical electrocoagulation from the specimen, and wash three times with 1×PBS buffer.

[0074] (3) Transfer the cleaned tumor tissue to a 1.5mL EP tube sterilized by autoclaving, and carefully cut the tumor tissue into fragments with a diameter of about 0.5-1.0mm using microscissors.

[0075] (4) Transfer the tumor tissue fragments from step (3) to a 50 mL centrifuge tube, add 20 mL of 1× PBS buffer, mix thoroughly, let stand for 2 minutes, and remove poorly active tissues and impurities from the supernatant. Repeat this washing process 3 times.

[0076] (5) After removing the supernatant, add the example culture medium or comparative culture medium to a 50 mL centrifuge tube and mix thoroughly. Use a 1 mL pipette to transfer the resuspended tumor tissue fragments to a six-well plate that has not been treated with TC, and add 4 mL of culture medium to each well.

[0077] (6) Place the untreated six-well plate in an incubator at 37°C and 5% CO2, and set the shaker speed to 120 rpm / min for incubation.

[0078] (7) Change the culture medium every 48 hours, replacing 2 / 3 of the culture medium each time. Observe the growth status of organoids daily and take photos to record the proliferation rate and morphological changes.

[0079] (8) When the diameter of the organoids of anaplastic ependymoma exceeds 500 μm, use ophthalmic scissors sterilized by high pressure steam to passage the organoids with a diameter exceeding 500 μm in two equal parts.

[0080] 3. Experimental Results

[0081] Microscopic images of anaplastic ependymoma organoids cultured for 7, 10, and 14 days using the culture medium provided in this embodiment of the invention are shown below. Figure 1 As shown, the organoids exhibit more regular circular or elliptical structures with clear and well-defined edges, and each organ exists independently without mutual adhesion. Microscopic images of anaplastic ependymoma organoids cultured for 7, 10, and 14 days using the culture medium provided in this invention are shown below. Figure 2 As shown, the organoids cultured in the comparative culture medium exhibited poor growth, irregular structures, and adhesion between different organoids.

[0082] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A culture medium for ependymoma organoids, characterized in that, The culture medium includes a basal culture medium and specific additives; The basal culture medium is NeuroCult™ NS-A Basal Medium; The specific additives include B-27 additive, N2 additive, BSA additive, GlutaMAX additive, EGF2, bFGF2, decellularized brain matrix, and penicillin-streptomycin.

2. The culture medium according to claim 1, characterized in that, The final concentration of the NeuroCult™ NS-A Basal Medium is 1×; The final concentration of the B-27 additive is 50×; the final concentration of the N2 additive is 100×; the final concentration of the BSA additive is 300 μg / mL; the final concentration of the GlutaMAX additive is 100×; the final concentration of the EGF2 is 10 ng / mL; the final concentration of the bFGF2 is 10 ng / mL; the final concentration of the decellularized brain matrix is ​​1 mg / mL; and the final concentration of the penicillin-streptomycin is 100×.

3. A method for culturing ependymoma organoids, characterized in that, The method includes culturing ependymoma tissue using the culture medium according to any one of claims 1-2.

4. The cultivation method according to claim 3, characterized in that, The method also includes mincing the ependymoma tissue before culture; The ependymoma tissue was minced into fragments approximately 0.5-1.0 mm in diameter; The specific conditions for cultivation were: shaking cultivation at 37°C, 5% CO2, and 120 rpm / min.

5. An ependymoma organoid, characterized in that, The organoids are cultured using the culture medium described in any one of claims 1-2 or obtained by the culture method described in any one of claims 3-4.

6. An in vitro drug screening model for ependymoma, characterized in that, The model includes the organoids described in claim 5.

7. Any of the following methods: 1) A method for predicting drug sensitivity in ependymomas, characterized in that, The method includes contacting the organoid of claim 5 or the model of claim 6 with the test drug; 2) A method for screening drugs for the prevention and / or treatment of ependymoma, characterized in that the method comprises contacting a candidate drug with the organoid of claim 5 or the model of claim 6; 3) A method for constructing an animal disease model, characterized in that the method includes transplanting the organoids of claim 5 into the animal to obtain the animal disease model; 4) A method for constructing an ependymoma organoid bank, characterized in that the method comprises: obtaining ependymoma organoids by the culture method according to any one of claims 3-4; subculturing the ependymoma organoids to obtain subcultured ependymoma organoids; and cryopreserving the subcultured ependymoma organoids to obtain the ependymoma organoid bank.

8. Any of the following applications: 1) The use of the culture medium according to any one of claims 1-2 in culturing ependymoma organoids; 2) The use of the organoids of claim 5 in screening drugs for the prevention and / or treatment of ependymoma; 3) The application of the organoids described in claim 5 in establishing a drug sensitivity model for ependymoma; 4) The application of the organoids described in claim 5 in the study of the pathogenesis or pathogenic factors of ependymoma; 5) The application of the organoids described in claim 5 in constructing animal disease models.

9. The culture medium according to any one of claims 1-2, or the culture method according to any one of claims 3-4, or the organoid according to claim 5, or the in vitro drug screening model for ependymoma according to claim 6, or the method according to claim 7, or the application according to claim 8, characterized in that, The ependymomas include anaplastic ependymomas and myxoplasmic ependymomas.

10. The culture medium according to any one of claims 1-2, or the culture method according to any one of claims 3-4, or the organoid according to claim 5, or the in vitro drug screening model for ependymoma according to claim 6, or the method according to claim 7, or the application according to claim 8, characterized in that, The ependymoma was selected from anaplastic ependymoma.

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