A meningioma organoid model without exogenous matrix glue support, a construction method, a special culture medium thereof and applications

Abstract

The application provides a construction method of a meningioma organoid without exogenous matrix glue support, and the construction method comprises primary culture of the meningioma organoid, and the primary culture comprises: inducing culture of a meningioma tissue block to self-assemble and form an organoid under the condition of dynamic mechanical stimulation in a non-adhesion culture system in MOs A culture medium added with collagen type I and collagen type IV, and the MOs A culture medium takes DMEM / F-12 as a basic culture medium. Through addition of collagen type I and collagen type IV into the culture medium and combination of dynamic mechanical stimulation culture, self-assembly and three-dimensional structure reconstruction of the meningioma tissue block are realized.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for culturing tumor organoids, and more particularly to a meningioma organoid model without exogenous matrix gel support, its construction method, its dedicated culture medium, and its applications. Background Technology

[0002] Meningioma is a common central nervous system tumor originating from arachnoid cap cells, accounting for approximately 30%-40% of intracranial tumors. Most meningiomas are benign, but some exhibit aggressive growth or even relapse, transforming into atypical or anaplastic meningiomas. They often occur in areas closely related to the dura mater, commonly found on the cerebral convexity, parasagittal sinus region, and skull base. The development and progression of meningiomas are closely related to genetic alterations (such as NF2 gene mutations), hormone levels, and the local microenvironment. Clinically, the main symptoms include headache, seizures, and neurological deficits. Current treatments for meningiomas primarily include surgical resection and radiotherapy; however, for recurrent or high-grade meningiomas, treatment options are limited, and the prognosis remains unsatisfactory.

[0003] Due to the significant histological and molecular heterogeneity of meningiomas, traditional research based on tumor cell lines and animal models struggles to fully reflect their in vivo biological characteristics, hindering the effective translation of some basic research findings into clinical practice. Patient-derived meningioma organoids (MOs) can effectively preserve the tissue structure and molecular characteristics of the primary tumor in vitro, making them invaluable for studying the mechanisms of meningioma development, drug screening, and personalized treatment. Organoid models not only expand the limited patient sample but also enable in vitro evaluation of different treatment strategies, improving clinical translation efficiency and reducing potential toxic side effects.

[0004] From the perspective of tumor models, tumor cell line models struggle to maintain the spatial structure and microenvironment characteristics of the primary tumor, while animal models suffer from drawbacks such as long development cycles, high costs, and species inconsistencies. In contrast, organoid models, derived from patient tissues, can better preserve the heterogeneity and spatial structure of tumors under in vitro culture conditions, making them an important model system for current tumor research. Organoid models can simulate the three-dimensional structure and complex biological processes of tissues in vitro, providing an important platform for drug screening and mechanism research.

[0005] In cell culture, scaffolds are commonly used to mimic the extracellular matrix (ECM) environment, providing structural support and biochemical signaling for cells. Matricene, an extract from the basement membrane of tumor tissue, is widely used in three-dimensional culture systems. However, Matricene's complex composition and significant batch-to-batch variability, coupled with its animal tissue origin, present challenges such as immunogenicity and uncontrollable composition, limiting its application in standardized research and clinical translation.

[0006] Current methods for constructing meningioma organoids mainly include two-dimensional adherent culture and three-dimensional culture systems relying on matrix gel or scaffolds. Two-dimensional culture struggles to maintain the spatial structure and heterogeneity of tumors, while three-dimensional culture typically relies on exogenous matrix gels or scaffolds, increasing costs and potentially introducing non-physiological components that affect the stability and reproducibility of experimental results. Furthermore, existing systems generally lack dynamic mechanical stimulation, making it difficult to simulate the actual growth environment of meningiomas in vivo, resulting in limited organoid formation efficiency and stability.

[0007] Therefore, there is an urgent need in this field for an organoid construction method that does not require exogenous matrix adhesive, can simulate the specific microenvironment of meningiomas, and has dynamic mechanical regulation, so as to improve the construction efficiency, structural stability, and biological relevance of organoids. Summary of the Invention

[0008] To address the aforementioned shortcomings of existing technologies, this invention aims to provide a meningioma organoid model without exogenous matrix gel support, its construction method, its dedicated culture medium, and its applications. In a non-adhesive culture system, meningioma tissue blocks are induced to self-assemble into organoids in MOs A medium supplemented with type I and type IV collagen under dynamic mechanical stimulation. This invention simulates the in-situ collagen-rich extracellular matrix environment and dynamic mechanical microenvironment of meningiomas without the need for exogenous matrix gel scaffolds, significantly improving the construction efficiency, structural stability, and biological relevance of organoids. This provides an economical, stable, and standardized in vitro model for meningioma mechanism research, drug screening, and personalized treatment.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for constructing meningoma organoids without exogenous matrix gel support. The method includes primary culture of meningoma organoids, wherein the primary culture includes inducing meningoma tissue blocks to self-assemble into organoids in a non-adhesive culture system in MOs A medium supplemented with type I and type IV collagen under dynamic mechanical stimulation. The MOs A medium uses DMEM / F-12 as the basal medium.

[0010] This invention marks the first time that scaffold-free suspension self-assembly of meningioma tissue blocks has been achieved in a non-adhesive culture system using MOs A medium supplemented with type I and type IV collagen. By adding type I and type IV collagen to the culture medium, the microenvironment of the dura mater and basement membrane in situ, which is predominantly collagen-rich, is specifically simulated in meningioma tissue blocks. This enhances the structural stability, cell adhesion, and polarity maintenance of the tissue blocks, thereby significantly promoting the self-assembly efficiency and morphological stability of organoids under gel scaffold-free conditions.

[0011] This invention eliminates the need for commercially available matrix gels such as Matrigel® and Geltrex Flex, avoiding their complex composition, large batch-to-batch variations, unclear origins, and immunogenicity risks. This significantly reduces the cost of organoid construction while improving the controllability, standardization, and reproducibility of the culture system.

[0012] This invention allows meningioma organoids to be cultured in a suspension form in a shaker under non-adhesive conditions. The shaker provides dynamic mechanical stimulation, simulating the hydrodynamic environment of cerebrospinal fluid flow and other factors experienced by meningiomas in vivo. This avoids the organoids from adhering to the culture wall or becoming loose due to static culture, and promotes the stable formation and long-term maintenance of three-dimensional spherical structures.

[0013] Furthermore, the content of type I collagen relative to the basal culture medium is 10-100 μg / mL, and the content of type IV collagen relative to the basal culture medium is 5-50 μg / mL.

[0014] Furthermore, the content of type I collagen relative to the basal culture medium is 50 μg / mL, and the content of type IV collagen relative to the basal culture medium is 25 μg / mL.

[0015] Furthermore, the dynamic mechanical stimulation is provided by a track-mounted rocking table; the rotational speed of the track-mounted rocking table is 80-140 rpm.

[0016] Furthermore, the rotational speed of the track shaking table is 100-120 rpm.

[0017] Furthermore, the meningioma tissue block was derived from the patient's tumor tissue and obtained after mechanical shearing and erythrocyte lysis.

[0018] Furthermore, the mechanical shearing time is 8-12 minutes; the red blood cell lysis treatment includes adding pre-cooled red blood cell lysis buffer, centrifuging at 800-1200 rpm for 5-8 minutes, and then removing the supernatant.

[0019] Furthermore, the non-adhesive culture system uses non-adhesive culture vessels with low or ultra-low cell adhesion as carriers.

[0020] Furthermore, the non-adhesive culture vessel is a culture plate with a low adsorption treatment on its surface; the low adsorption treatment is to coat the surface of the non-adhesive culture vessel with an inert hydrophilic polymer layer or a hydrogel layer.

[0021] Furthermore, the non-adhesive culture vessel is a 6-well ultra-low adsorption culture plate.

[0022] Furthermore, the MOs A culture medium also contains basic fibroblast growth factor, nerve cell culture additive, B27 serum-free additive, epidermal growth factor, penicillin-streptomycin bispecific antibody, and mycoplasma prevention reagent.

[0023] Furthermore, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The content of the basic fibroblast growth factor relative to the basal culture medium is 10~40 ng / mL, preferably 20 ng / mL; The volume content of the neural cell culture additive relative to the basal culture medium is 0.5% to 2%, preferably 1%; The B27 serum-free additive has a volume content of 1% to 3% relative to the basal culture medium, preferably 2%; The epidermal growth factor concentration relative to the basal culture medium is 100-400 ng / mL, preferably 200 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 0.5% to 2% relative to the basal culture medium, preferably 1%; The mycoplasma prevention reagent has a volume content of 0.03% to 0.07% relative to the basal culture medium, preferably 0.05%.

[0024] Furthermore, the construction method also includes the following steps: passage, cryopreservation and thawing of the organoids.

[0025] Furthermore, the primary culture specifically includes: placing meningioma tissue blocks into the MOs A medium added to each well of a non-adhesive culture dish, adding 50-60 meningioma tissue blocks to each well, then placing the culture dish on a track shaker and continuously shaking it in an incubator at 37±1℃, 5-8 vol% CO2, and 85-95% humidity, performing a half-medium change every 40-60 hours, and culturing for 60-80 hours until spherical organoids are formed, which can continue to grow for 5-8 weeks.

[0026] Furthermore, the passage step includes: when the diameter of the organoids obtained from the primary culture reaches 1.5-2.5 mm, thoroughly removing the supernatant from the primary non-adhesive culture vessel, transferring the organoids to a culture vessel containing pre-cooled MOs A medium, and shearing each organoid; then transferring 20-30 organoid tissues obtained after shearing to a new non-adhesive culture vessel containing MOs A medium; and then placing the passaged non-adhesive culture vessel on a track shaker and continuously shaking it at 100-120 rpm in an incubator at 37±1℃, 5-8 vol% CO2, and 85-95% humidity.

[0027] Furthermore, the cryopreservation step includes: collecting organoids obtained from primary culture or passaged culture, aspirating the supernatant from the culture vessel, adding cryopreservation solution, mixing, and then storing at -80±5℃ for 20-28 hours before transferring to liquid nitrogen for long-term storage.

[0028] Furthermore, the cryopreservation solution contains MOs A medium, DMSO, and Y-27632.

[0029] Furthermore, in the cryopreservation solution, based on the total volume of MOs A medium and DMSO as 100%, the volume percentage of MOs A medium is 85-95%, the volume percentage of DMSO is 5-15%, and the concentration of Y-27632 is 5-15 μmol / L.

[0030] Furthermore, the resuscitation steps include: rapidly thawing the cryopreservation tubes containing organoids in a water bath at 37±1℃, with the transfer time from liquid nitrogen to the water bath not exceeding 1 minute, thawing for 1-2 minutes until no ice is present, aspirating the cryopreservation solution, washing twice with MOs A medium, and then transferring the organoids to a culture dish containing MOs A medium that has not undergone tissue culture treatment, and then continuing to culture according to the primary culture method.

[0031] Secondly, the present invention provides a culture medium for constructing meningoma organoids without exogenous matrix gel support, wherein the culture medium is MOs A medium, which uses DMEM / F-12 as the basal medium and also contains type I collagen and type IV collagen as regulatory factors.

[0032] Furthermore, the content of type I collagen relative to the basal culture medium is 10-100 μg / mL, and the content of type IV collagen relative to the basal culture medium is 5-50 μg / mL.

[0033] Furthermore, the content of type I collagen relative to the basal culture medium is 50 μg / mL, and the content of type IV collagen relative to the basal culture medium is 25 μg / mL.

[0034] Furthermore, the MOs A culture medium also contains basic fibroblast growth factor, nerve cell culture additive, B27 serum-free additive, epidermal growth factor, penicillin-streptomycin bispecific antibody, and mycoplasma prevention reagent.

[0035] Furthermore, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The concentration of the basic fibroblast growth factor relative to the basal culture medium is 10~40 ng / mL; The volume content of the neural cell culture additive relative to the basal culture medium is 0.5% to 2%; The B27 serum-free additive has a volume content of 1% to 3% relative to the basal culture medium; The epidermal growth factor concentration relative to the volume of the basal culture medium is 100~400 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 0.5% to 2% relative to the basal culture medium; The mycoplasma prevention reagent has a volume content of 0.03% to 0.07% relative to the basal culture medium.

[0036] Furthermore, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The concentration of the basic fibroblast growth factor relative to the basal culture medium is 20 ng / mL; The nerve cell culture additive has a volume content of 1% relative to the basal culture medium; The B27 serum-free additive has a volume content of 2% relative to the basal culture medium. The epidermal growth factor concentration relative to the basal culture medium is 200 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 1% relative to the basal culture medium; The mycoplasma prevention reagent has a volume content of 0.05% relative to the basal culture medium.

[0037] Thirdly, the present invention provides a meningioma organoid model without exogenous matrix adhesive support, which is constructed by the method described in the first aspect.

[0038] Furthermore, the organoids grow in suspension under non-adhesive conditions, have a three-dimensional spherical structure, and do not contain exogenous matrix adhesive components.

[0039] Fourthly, the present invention provides the use of a meningioma organoid model constructed according to the construction method described in the first aspect, or a meningioma organoid model constructed according to the method described in the third aspect, for screening drugs for treating meningiomas or for studying the mechanism of meningiomas.

[0040] Compared with the prior art, the beneficial effects of the present invention include at least one of the following: 1) Promoting self-assembly by simulating in-situ extracellular matrix signals through type I / IV collagen: This invention is the first to achieve scaffold-free suspension self-assembly of meningioma tissue blocks in a non-adhesive culture system using MOs A medium supplemented with type I and type IV collagen. By adding type I and type IV collagen to the culture medium, the microenvironment of the dura mater and basement membrane in situ, which is mainly composed of collagen, is specifically simulated, enhancing the structural stability, cell adhesion, and polarity maintenance of the tissue blocks. This significantly promotes the self-assembly efficiency and morphological stability of organoids under gel scaffold-free conditions. 2) Eliminating dependence on exogenous matrix gels, reducing costs and improving controllability: This invention eliminates the need to add commercial matrix gels such as Matrigel® and Geltrex Flex, avoiding their complex composition, large batch variations, unclear sources and immunogenicity risks, significantly reducing the cost of organoid construction, while improving the controllability, standardization and reproducibility of the culture system; 3) Combining dynamic mechanical stimulation to more realistically simulate the microenvironment of meningioma: The meningioma organoids of this invention are cultured in a suspension form in a shaker under non-adhesive conditions. The shaker provides dynamic mechanical stimulation to simulate the hydrodynamic environment of cerebrospinal fluid flow and other conditions experienced by meningioma in vivo. This avoids the organoids from adhering to the wall or becoming loose due to static culture and promotes the stable formation and long-term maintenance of the three-dimensional spherical structure. 4) Simplified culture medium composition, improving economy and operability: This invention discloses MOs A culture medium. Compared with previously reported meningioma organoid culture media (which require the addition of various expensive cytokines and matrix gels), the culture medium formulation of this invention is significantly simplified, the preparation steps are convenient, and the cost is significantly reduced. Simultaneously, experiments have confirmed that MOs A-cultured organoids still retain the fluorescence characteristics and histological properties of the primary tumor (see...). Figure 6 This achieves a balance between simplification and functionality retention; 5) A complete system of methods for culture, passage, cryopreservation, and resuscitation has been established: This invention not only provides a method for constructing organoids, but also systematically establishes a complete technical solution for passage, cryopreservation, and resuscitation. In particular, the use of cryopreservation fluid significantly improves the survival rate and structural integrity of resuscitated organoids, which can recover a typical three-dimensional spherical morphology within 24 hours after resuscitation. Figure 4 or Figure 5This provides reliable technical support for the long-term preservation and shared application of meningioma organoids; 6) The constructed organoid model retains the key biological characteristics of the primary tumor: verified by HE staining and immunofluorescence. Figure 6 The meningioma organoids constructed in this invention are highly consistent with the primary tumor tissue in terms of cell morphology, tissue structure, and molecular marker expression. Specifically, the organoids can maintain the expression of the characteristic meningioma marker SSTR2, while retaining CD68-positive cells and COL1A1-positive extracellular matrix components, indicating that they can better maintain the histological characteristics, tumor microenvironment characteristics, and extracellular matrix characteristics of the primary tumor.

[0041] It is noteworthy that the meningioma organoids constructed in this invention can maintain the expression and secretion of endogenous extracellular matrix components without relying on exogenous matrix gel support, thereby avoiding the potential interference of exogenous matrix gel on tumor cell behavior and drug response. Because the culture system does not use complex exogenous matrix gel, but instead uses collagen I and collagen IV for support, it is more conducive to reducing interference from matrix components, more realistically reflecting the biological characteristics of meningiomas, and ensuring the biological authenticity and data reliability of subsequent drug screening, mechanism studies, and personalized treatment evaluation. Attached Figure Description

[0042] Figure 1 This is a micrograph of a meningioma tissue block cultured on day 0 of this invention. Figure 2 This is a photomicrograph of a meningioma organoid initially formed on day 3 of culture in this embodiment of the invention. Figure 3 This is a photomicrograph of a meningioma organoid cultured on day 10 in this embodiment of the invention. Figure 4 This is a photomicrograph taken on day 0 after resuscitation of a meningioma organoid in an embodiment of the present invention. Figure 5 This is a photomicrograph taken on Day 1 after resuscitation of a meningioma organoid in an embodiment of the present invention. Figure 6 The images show the HE staining and fluorescence results of tumor tissue and organoids from the same patient in this embodiment of the invention. Figure 7 These are micrographs of meningioma organoids cultured on day 10 under different culture systems in Comparative Examples 1-3. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be considered as specific limitations thereof. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. Process parameters not specifically specified in the following embodiments are generally performed under conventional conditions.

[0044] The endpoints and any values ​​of the ranges disclosed in this invention are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this invention.

[0045] In a first aspect, the present invention provides a method for constructing meningoma organoids without exogenous matrix gel support. The method includes primary culture of meningoma organoids, wherein the primary culture includes inducing meningoma tissue blocks to self-assemble into organoids in a non-adhesive culture system in MOs A medium supplemented with type I and type IV collagen under dynamic mechanical stimulation. The MOs A medium uses DMEM / F-12 as the basal medium.

[0046] Meningiomas originate from meningeal / arachnoid cap cells. Their growth microenvironment has structural features of being attached to the dura mater and relies on extracellular matrix mainly composed of collagen for support. At the same time, they are in a low-nutrient, low-inflammation and cerebrospinal fluid-like hydrodynamic environment. Traditional matrix glue systems cannot truly simulate their in-situ microenvironment.

[0047] Specifically, meningiomas possess unique microenvironmental characteristics. Their tumor cells typically grow attached to the dura mater, and the extracellular matrix is ​​predominantly collagen, particularly type I and type IV collagen, which play crucial roles in maintaining tissue structure and cell adhesion. Simultaneously, meningiomas exist in a cerebrospinal fluid-associated, low-nutrient, low-inflammatory environment and are subject to certain mechanical stimuli. Traditional three-dimensional culture systems relying on matrix gels struggle to accurately simulate these specific microenvironmental characteristics of meningiomas.

[0048] This invention marks the first time that scaffold-free suspension self-assembly of meningioma tissue blocks has been achieved in a non-adhesive culture system using MOs A medium supplemented with type I and type IV collagen. By adding type I and type IV collagen to the culture medium, the microenvironment of the dura mater and basement membrane in situ, which is predominantly collagen-rich, is specifically simulated in meningioma tissue blocks. This enhances the structural stability, cell adhesion, and polarity maintenance of the tissue blocks, thereby significantly promoting the self-assembly efficiency and morphological stability of organoids under gel scaffold-free conditions.

[0049] This invention eliminates the need for commercially available matrix gels such as Matrigel® and Geltrex Flex, avoiding their complex composition, large batch-to-batch variations, unclear origins, and immunogenicity risks. This significantly reduces the cost of organoid construction while improving the controllability, standardization, and reproducibility of the culture system.

[0050] This invention allows meningioma organoids to be cultured in a suspension form in a shaker under non-adhesive conditions. The shaker provides dynamic mechanical stimulation, simulating the hydrodynamic environment of cerebrospinal fluid flow and other factors experienced by meningiomas in vivo. This avoids the organoids from adhering to the culture wall or becoming loose due to static culture, and promotes the stable formation and long-term maintenance of three-dimensional spherical structures.

[0051] As an optional implementation, the content of type I collagen relative to the basal culture medium is 10-100 μg / mL, for example, it can be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL or 100 μg / mL, and the content of type IV collagen relative to the basal culture medium is 5-50 μg / mL, for example, it can be 5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL or 50 μg / mL.

[0052] As an optional implementation, the content of type I collagen relative to the basal culture medium is 50 μg / mL, and the content of type IV collagen relative to the basal culture medium is 25 μg / mL.

[0053] Further restrictions are needed on the content ranges of type I and type IV collagen to respectively mimic the mechanical support of the dura mater (type I collagen) and the maintenance of basement membrane-like polarity (type IV collagen). Too low a content fails to effectively promote self-assembly, while too high a content may inhibit cell growth or lead to non-specific aggregation. This invention limits the content of type I collagen relative to the basal culture medium to 10-100 μg / mL and the content of type IV collagen relative to the basal culture medium to 5-50 μg / mL. Selecting these ranges significantly enhances the structural stability and cell adhesion of tissue blocks, promotes the maintenance of tumor cell polarity and three-dimensional structural reconstruction, and improves the self-assembly efficiency and morphological consistency of organoids.

[0054] As an optional implementation, the dynamic mechanical stimulation is provided by a track-mounted rocking table; the rotational speed of the track-mounted rocking table is 80-140 rpm, for example, 80 rpm, 90 rpm, 100 rpm, 110 rpm, 120 rpm, 130 rpm or 140 rpm.

[0055] As an optional implementation, the rotational speed of the track shaking table is 100-120 rpm.

[0056] As an alternative implementation, the meningioma tissue block is derived from the patient's tumor tissue and obtained after mechanical shearing and erythrocyte lysis.

[0057] As an optional implementation, the mechanical shearing time is 8-12 minutes, for example, 8 minutes, 9 minutes, 10 minutes, 11 minutes or 12 minutes; the red blood cell lysis treatment includes adding pre-cooled red blood cell lysis buffer, centrifuging at 800-1200 rpm, for example, 800 rpm, 900 rpm, 1000 rpm, 1100 rpm or 1200 rpm for 5-8 minutes, and then removing the supernatant, for example, centrifuging for 5 minutes, 6 minutes, 7 minutes or 8 minutes.

[0058] Static culture easily leads to tissue block adhesion and differentiation or poor organoid growth, and the lack of dynamic mechanical stimulation makes it difficult to simulate the in vivo fluid environment; insufficient or excessive tissue block treatment (such as excessively long or short shearing time) will affect cell viability. During primary and passage cultures, a track-mounted shaker is used to maintain the suspension growth of tissue blocks or organoids, reduce adhesion, and promote oxygen and nutrient exchange, which is beneficial for the formation and maintenance of meningioma organoids. This invention uses a track-mounted shaker to provide dynamic mechanical stimulation at 80-140 rpm (preferably 100-120 rpm), effectively preventing organoid adhesion and promoting self-assembly; mechanical shearing and erythrocyte lysis of patient tumor tissue ensure tissue block viability. The two synergistically improve the success rate of organoid formation and long-term culture stability.

[0059] As an optional implementation, the non-adhesive culture system uses non-adhesive culture vessels with low or ultra-low cell adhesion as carriers.

[0060] The non-adhesive culture system described in this invention refers to a culture system in which meningioma tissue blocks, cell clusters or organoids are difficult to adhere to the surface of the non-adhesive culture vessel by using non-adhesive culture vessels with low or ultra-low cell adhesion, thereby maintaining their three-dimensional suspension growth state.

[0061] As an optional embodiment, the non-adhesive culture vessel is a culture plate with a low adsorption treatment on its surface, including an ultra-low adsorption culture plate or an ultra-low adhesion culture plate; the low adsorption treatment is to coat the surface of the non-adhesive culture vessel with an inert hydrophilic polymer layer or a hydrogel layer.

[0062] The non-adhesive culture vessel of the present invention is a culture plate with a special low-adsorption treatment on its surface, including but not limited to ultra-low adsorption culture plates, ultra-low adhesion culture plates, or other culture vessels that inhibit cell adhesion. The surface of the non-adhesive culture vessel may be coated with an inert hydrophilic polymer layer, a hydrogel layer, or other materials that can reduce the interaction between cells and the surface of the culture vessel, thereby reducing cell adhesion growth and promoting the formation of three-dimensional organoids.

[0063] As an optional implementation, the non-adhesive culture vessel is a 6-well ultra-low adsorption culture plate.

[0064] The non-adhesive culture vessel of the present invention is further preferably a 6-well ultra-low adsorption culture plate, such as the BeyoGold™ Plus ultra-low adsorption 6-well plate, the Costar® ultra-low adhesion 6-well culture plate, or an ultra-low adsorption culture vessel with equivalent functionality. The non-adhesive culture system of the present invention, combined with dynamic culture on a track-shaking incubator, keeps tissue blocks or organoids in suspension by continuously shaking the culture medium, promoting oxygen and nutrient exchange, and improving organoid formation efficiency and long-term culture stability.

[0065] As an optional implementation, the MOs A culture medium also contains basic fibroblast growth factor (bFGF), nerve cell culture additive (N2), serum-free B27 additive (B27), epidermal growth factor (EGF), penicillin-streptomycin bispecific antibody, and mycoplasma prevention reagent.

[0066] As an optional implementation, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The concentration of the basic fibroblast growth factor relative to the basal culture medium is 10~40 ng / mL; The volume content of the neural cell culture additive relative to the basal culture medium is 0.5% to 2%; The B27 serum-free additive has a volume content of 1% to 3% relative to the basal culture medium; The epidermal growth factor concentration relative to the basal culture medium is 100~400 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 0.5% to 2% relative to the basal culture medium; The mycoplasma prevention reagent has a volume content of 0.03% to 0.07% relative to the basal culture medium.

[0067] As an optional implementation, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The concentration of the basic fibroblast growth factor relative to the basal culture medium is 20 ng / mL; The nerve cell culture additive has a volume content of 1% relative to the basal culture medium; The B27 serum-free additive has a volume content of 2% relative to the basal culture medium. The epidermal growth factor concentration relative to the basal culture medium is 200 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 1% relative to the basal culture medium; The mycoplasma prevention reagent has a volume content of 0.05% relative to the basal culture medium.

[0068] Existing culture media for meningioma organoids are complex and expensive (requiring the addition of various growth factors and matrix gels), and there is a lack of simplified, economical, and dedicated culture media suitable for matrix gel-free systems. The MOs A culture medium provided by this invention (containing DMEM / F12, bFGF, N2, B27, EGF, double antibiotics, mycoplasma prophylaxis reagent, and type I and type IV collagen) has a clearly defined composition and a simple formulation. This culture medium simplifies the formulation, reduces preparation costs, and effectively supports the construction and maintenance of meningioma organoids under matrix gel-free conditions.

[0069] As an optional implementation, the construction method further includes the following steps: passage, cryopreservation and thawing of the organoids.

[0070] This invention not only provides a primary culture method for organoids, but also systematically establishes a complete technical solution for passage, cryopreservation, and thawing. The organoids can recover their typical three-dimensional spherical morphology within 24 hours of thawing, providing reliable technical support for the long-term preservation and shared application of meningioma organoids.

[0071] As an optional implementation, the primary culture specifically includes: placing meningioma tissue blocks into each well of a non-adhesive culture dish containing 50-60 meningioma tissue blocks per well, for example, 50, 52, 55, 56, 58, or 60 blocks; then placing the culture dish on a track shaker at 37±1°C, for example, 36°C, 37°C, or 38°C, with 5-8 vol% CO2, for example, 5 vol%, 6 vol%, 7 vol%, or 8 vol%, and 85-95%, for example, 85%, 86%, or 88%. Cultured in an incubator with 90%, 92%, or 95% humidity with continuous shaking. Perform a half-medium change every 40-60 hours (e.g., 40, 42, 45, 48, 50, 55, 58, or 60 hours). Culture for 60-80 hours (e.g., 60, 64, 65, 68, 70, 72, 75, or 80 hours) until spherical organoids form. They can continue to grow for 5-8 weeks (e.g., 5, 6, 7, or 8 weeks).

[0072] As an optional implementation, the passage step includes: when the diameter of the organoids obtained from the primary culture reaches 1.5-2.5 mm, for example, 1.5 mm, 2 mm, or 2.5 mm, thoroughly removing the supernatant from the primary non-adhesive culture dish, transferring the organoids to a culture dish containing pre-cooled MOs A medium, and cutting each organoid: for example, using microsurgical scissors to cut each organoid into 2 or 4 pieces; then transferring 20-30 pieces of the cut organoid tissue, for example, 20, 22, 25, 28, or 30 pieces, to a new non-adhesive culture dish containing MOs A medium; then placing the passaged non-adhesive culture dish on a track shaker and continuously shaking it at 100-120 rpm, for example, 100 rpm, 110 rpm, or 120 rpm, in an incubator at 37±1℃, 5-8 vol% CO2, and 85-95% humidity.

[0073] In the passage culture of this invention, the rotation speed of the track shaker is 100-120 rpm. Too low a speed can easily cause tissue blocks or organoids to settle and adhere to the culture vessel wall, affecting organoid formation and growth; too high a speed can easily cause them to collide with the sidewalls of the culture vessel, increasing losses. Therefore, by controlling an appropriate shaker speed, the stability of meningioma organoid culture can be improved.

[0074] As an optional implementation, the cryopreservation step includes: collecting organoids obtained from primary culture or passaged culture, aspirating the supernatant from the culture vessel, adding cryopreservation solution, mixing, and then storing at -80±5℃, for example, -75℃, -78℃, -80℃, -82℃ or -85℃ for 20-28 hours, for example, 20 hours, 22 hours, 24 hours, 25 hours or 28 hours, and then transferring to liquid nitrogen for long-term storage.

[0075] As an alternative embodiment, the cryopreservation solution comprises MOs A medium, DMSO, and Y-27632.

[0076] As an optional embodiment, in the cryopreservation solution, based on the total volume of MOs A medium and DMSO as 100%, the volume percentage of MOs A medium is 85-95%, for example, 85%, 88%, 90%, 92%, or 95%, and the volume percentage of DMSO is 5-15%, for example, 5%, 8%, 10%, 12%, or 15%. The concentration of Y-27632 is 5-15 μmol / L, for example, 5 μmol / L, 8 μmol / L, 10 μmol / L, 12 μmol / L, or 15 μmol / L.

[0077] As an optional implementation, the resuscitation step includes: rapidly thawing the cryopreservation tubes containing organoids in a water bath at 37±1°C, for example, 36°C, 37°C, or 38°C, with the transfer time from liquid nitrogen to the water bath not exceeding 1 minute, thawing for 1-2 minutes until no ice is present, aspirating the cryopreservation solution, washing twice with MOs A medium, and then transferring the organoids to a culture dish containing MOs A medium that has not undergone tissue culture treatment, for example, a 6-well flat-bottomed culture plate that has not undergone tissue culture treatment, and then continuing to culture according to the primary culture method.

[0078] Secondly, the present invention provides a culture medium for constructing meningoma organoids without exogenous matrix gel support, wherein the culture medium is MOs A medium, which uses DMEM / F-12 as the basal medium and also contains type I collagen and type IV collagen as regulatory factors.

[0079] As an optional implementation, the content of type I collagen relative to the basal culture medium is 10-100 μg / mL, and the content of type IV collagen relative to the basal culture medium is 5-50 μg / mL.

[0080] As an optional implementation, the content of type I collagen relative to the basal culture medium is 50 μg / mL, and the content of type IV collagen relative to the basal culture medium is 25 μg / mL.

[0081] As an optional implementation, the MOs A culture medium also contains basic fibroblast growth factor, nerve cell culture additive, B27 serum-free additive, epidermal growth factor, penicillin-streptomycin dual antibody, and mycoplasma prevention reagent.

[0082] As an optional implementation, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The concentration of the basic fibroblast growth factor relative to the basal culture medium is 10~40 ng / mL; The volume content of the neural cell culture additive relative to the basal culture medium is 0.5% to 2%; The B27 serum-free additive has a volume content of 1% to 3% relative to the basal culture medium; The epidermal growth factor concentration relative to the basal culture medium is 100~400 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 0.5% to 2% relative to the basal culture medium; The mycoplasma prevention reagent has a volume content of 0.03% to 0.07% relative to the basal culture medium.

[0083] As an optional implementation, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The concentration of the basic fibroblast growth factor relative to the basal culture medium is 20 ng / mL; The nerve cell culture additive has a volume content of 1% relative to the basal culture medium; The B27 serum-free additive has a volume content of 2% relative to the basal culture medium. The epidermal growth factor concentration relative to the basal culture medium is 200 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 1% relative to the basal culture medium; The mycoplasma prevention reagent has a volume content of 0.05% relative to the basal culture medium.

[0084] Thirdly, the present invention provides a meningioma organoid model without exogenous matrix adhesive support, which is constructed by the method described in the first aspect.

[0085] As an optional implementation, the organoid grows in suspension under non-adhesive conditions, possesses a three-dimensional spherical structure, and does not contain exogenous matrix gel components. The organoids of this invention can be primary cultured organoids or passaged cultured organoids, and can be used as organoid models for subsequent drug screening and research. The preferred diameter is 1.5 mm to 2.5 mm.

[0086] Fourthly, the present invention also provides the use of the meningioma organoid model constructed by the construction method described in the first aspect above, or the meningioma organoid model constructed by the construction method described in the third aspect above, for screening drugs for treating meningiomas or for studying the mechanism of meningiomas.

[0087] The meningioma organoid model constructed in this invention, which is free from exogenous matrix gel support, can be stably cultured for a long time and maintain the histological and molecular biological characteristics of meningiomas. Therefore, it can be used as a platform for gene function research. For example, genes can be introduced, knocked out, or overexpressed into organoids through lentiviruses, adenoviruses, adeno-associated viruses (AAVs), or CRISPR / Cas systems to study the role of specific genes in tumor development and progression, i.e., for meningioma mechanism research.

[0088] The present invention will now be described in further detail with reference to specific embodiments and comparative examples.

[0089] DMEM / F-12 medium was purchased from Gibco (Thermo Fisher Scientific, USA). Basic fibroblast growth factor (bFGF) was purchased from PeproTech (USA); Epidermal growth factor (EGF) was purchased from PeproTech (USA). N2 nerve cell culture additive (N2 Supplement) was purchased from Gibco (Thermo Fisher Scientific, USA). B27 Supplement Serum-Free was purchased from Gibco (Thermo Fisher Scientific, USA). Type I collagen was purchased from Corning (USA); Type IV collagen (Collagen IV) was purchased from Sigma-Aldrich (Merck Group, USA); Penicillin-streptomycin was purchased from Gibco (Thermo Fisher Scientific, USA). MycAway® Prophylactic Mycoplasma Prevention Kit was purchased from Yeasen (Yesen Biotechnology, China). Red blood cell lysis buffer was purchased from Beyotime (Beyotime, China). PBS buffer was purchased from Gibco (Thermo Fisher Scientific, USA). The Matrigel® matrix adhesive was purchased from Corning (Corning, USA).

[0090] Example 1. Primary culture of meningioma organoids (MOs) Step A1: Tissue Collection and Transportation Patient-derived meningioma tumor tissue was preserved using sterile cryovials or 15 mL centrifuge tubes without any additives and transported to the laboratory on ice.

[0091] Step A2: Instrument disinfection Ophthalmic forceps, ophthalmic scissors, and other instruments were placed in a sand bath and sterilized by dry heat at 300°C for 30 minutes. After sterilization, the instruments were rapidly cooled with pre-cooled PBS to prevent overheating and damage to tumor tissue.

[0092] Step A3: Tissue Processing The tumor tissue was washed twice with pre-cooled PBS. The tissue was then minced as finely as possible using ophthalmic forceps and scissors, with the mincing time controlled to be around 10 minutes, to ensure thorough mincing while maintaining the success rate of organoid formation and cell viability.

[0093] Step A4: Red blood cell lysis Add an appropriate amount of pre-cooled erythrocyte lysis buffer (5 mL per 100 mg of tissue) according to the amount of tissue block, then centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0094] Step A5: Tissue block screening The tissue blocks were resuspended in PBS and filtered through a cell sieve (100 μm pore size) and a 50 mL sterile centrifuge tube. The tissue blocks at the top of the cell sieve are the tissue blocks for organoid culture; the cell suspension at the bottom can be centrifuged and plated for the culture of primary meningioma cells.

[0095] Step A6: Laying out the boards Using MOs A medium (formula shown in Table 1), seed the tissue into 6-well ultra-low adsorption plates (e.g., BeyoGold™ Plus ultra-low adsorption 6-well plates, flat bottom), adding 4 mL of medium to each well. Use ophthalmic forceps to pick up 50-60 tissue samples per well (see Table 1). Figure 1 No exogenous matrix adhesives (such as Matrigel®, Geltrex Flex, etc.) are added.

[0096] Step A7: Dynamic mechanical stimulation culture Place the six-well plate on a track shaker in a cell culture incubator (37 ℃, 5 vol% CO2, 90% humidity) and culture continuously at 100 rpm. Perform a half-medium medium change every 48 hours (replacing 2 mL of fresh MOs A medium in each well). After approximately 72 hours of culture, spherical MOs cells will appear (see...). Figure 2 After 10 days of cultivation, MOs showed a continuous increase in size (see...). Figure 3 It can grow continuously for 6 weeks.

[0097] Table 1 MOs A medium formulation 2. Passaging of meningioma organoids (MOs) Step B1: Cutting When the diameter of the organoids obtained from the primary culture reaches about 2 mm, the supernatant in the primary non-adhesive culture dish is completely removed. The MOs are then transferred to a 60 mm culture dish containing pre-cooled MOs A medium using a 1 mL pipette tip. Each MOs sphere is then cut into 2 or 4 pieces using microsurgical scissors.

[0098] Step B2: Re-lay the boards After cutting, 20-30 organoid tissue blocks were transferred to a 6-well plate (such as BeyoGold™ Plus 6-well plate with flat bottom) containing 4 mL of MOs A culture medium per well.

[0099] Step B3: Continue dynamic cultivation The passaged non-adhesive culture dishes were placed on a track shaker and cultured in an incubator at 37°C, 5 vol% CO2, and 90% humidity with continuous shaking at 100 rpm. A half-medium medium change (2 mL of fresh passaged medium per well) was performed every 48 hours. The passaged organoids could be cultured for a total of 8 weeks.

[0100] 3. Cryopreservation of meningioma organoids (MOs) Step C1: Collection Collect organoids obtained from primary culture or passaged culture. Tilt the six-well plate and transfer the organoids and culture medium into a 15 mL centrifuge tube, allowing the MOs to settle naturally to the bottom.

[0101] Step C2: Quantification Completely aspirate the supernatant from the culture dish to ensure that each cryovial contains 30-40 organoid spheroids.

[0102] Step C3: Add cryopreservation solution Add cryopreservation solution to the cryovials, which comprises MOs A medium, DMSO, and Y-27632 (from MedChemExpress (MCE), Cat. No.: HY-10071). The total volume of MOs A medium and DMSO is 100%, the volume percentage of MOs A medium is 90%, the volume percentage of DMSO is 10%, and the concentration of Y-27632 is 10 μmol / L. Mix thoroughly (see Table 2 for cryopreservation solution formulation).

[0103] Step C4: Freezing Place the cryovials in a slow-freezing box and store them at -80°C for 24 hours, then transfer them to liquid nitrogen for long-term storage.

[0104] Table 2 MOs cryopreservation solution formulation 4. Resuscitation of meningioma organoids (MOs) Step D1: Thawing Remove the cryovials containing organoids from liquid nitrogen and quickly place them in a 37°C water bath to thaw. The time between transferring them from liquid nitrogen to the water bath should not exceed 1 minute. Thaw for 1-2 minutes until there are no visible ice particles inside the cryovials.

[0105] Step D2: Cleaning Completely aspirate the cryopreservation solution and wash the cryovial twice with MOs A medium.

[0106] Step D3: Restore Culture The organoids were transferred to untreated 6-well flat-bottomed culture plates containing MOs A medium and then cultured using the primary culture method (same as step A7). Day 0 post-resuscitation ( Figure 4 ) and Day 1 ( Figure 5 Microscopic images show that the organoid morphology has been well restored.

[0107] To verify whether the meningioma organoids constructed in this invention retain the histological structure, tumor microenvironment characteristics, and expression of meningioma-specific markers of the primary tumor, HE staining and immunofluorescence detection were performed on the patient's primary tumor tissue and corresponding organoids in this example. The results are shown in Figure 6.

[0108] The left side shows the primary meningioma tissue (Tumor), and the right side shows the corresponding constructed meningioma organoid. HE staining results show that the organoid can form a stable three-dimensional structure and retain a histological morphology similar to the primary tumor.

[0109] The results of CD68 / COL1A1 dual immunofluorescence staining showed that both CD68 (green) positive cells and COL1A1 (yellow) positive extracellular matrix components were detectable in the organoids, and their expression patterns were similar to those of the corresponding primary tumor tissue. This indicates that the organoids retained immune cell components and extracellular matrix characteristics.

[0110] Immunofluorescence staining of SSTR2 showed that the meningioma marker SSTR2 (yellow) was positively expressed in both the primary tumor tissue and the corresponding organoid. Cell nuclei were counterstained with DAPI (blue). This result indicates that the organoid maintained the meningioma-specific molecular phenotype. Therefore, the meningioma organoid model constructed by the method of this invention is successful and exhibits good tumor simulation.

[0111] Comparative Example 1: A method for constructing meningoma organoids relying on traditional matrix gels and complex culture media This comparative example references published literature (Yamazaki S, Ohka F, Hirano M, ...). et al. Newly established patient-derived organoid model of intracranial meningioma. Neuro Oncol The method for culturing meningoma organoids described in (2021; 23: 1936-48.) was combined with the traditional matrix gel-dependent three-dimensional culture system to construct meningoma organoids.

[0112] Build steps: 1. Tissue processing: According to steps A1-A5 in the embodiments of the present invention, processed tissue blocks are obtained from the patient's meningioma tissue.

[0113] 2. Matrigel Resuspension: Mix the obtained tissue block with pre-chilled Matrigel® Corning gel until fully embedded. Then, drop the Matrigel containing the tissue block into the center of a culture plate and incubate at 37°C for 15-30 min to allow gelation. Add culture medium and continue culturing. The Matrigel® used should be the manufacturer's stock solution, and Matrigel® should be used at the working concentration recommended in the product instructions (e.g., diluted to 8-12 mg / mL).

[0114] 3. Plating and gelation: Add approximately 200-300 μL of the matrix gel mixture containing tissue blocks obtained in step 2 to the center of each well of a preheated 6-well plate. Place the culture plate in a 37°C incubator and let it stand for 20-30 minutes to allow the matrix gel to completely solidify into droplets.

[0115] 4. Incubation: After the matrix gel has solidified, add 4 ml of meningoma organoid culture medium to each well. The composition of the culture medium is shown in Table 3 below: Table 3. Culture medium formulation for meningoma organoids in Comparative Example 1 Then, it was placed in an incubator at 37°C, 5% CO2, and 90% humidity for static culture (without dynamic stimulation by shaking). The culture medium was replaced with fresh medium every 48 hours.

[0116] 5. Subculturing: When the organoids reach a diameter of approximately 2 mm, mechanically disperse the matrix gel droplets containing the organoids using a pipette tip to release them. After washing with pre-cooled PBS, re-embed the organoids in a new matrix gel and subculture them, following step 2.

[0117] Comparative Example 2: Construction method of collagen regulatory factors without exogenous matrix gel. This comparative example is basically the same as that of the present invention (Example 1), except that type I collagen (Collagen I) and type IV collagen (Collagen IV) are not added to the MOs A culture medium used.

[0118] Build steps: 1. Refer to steps A1-A7 of Example 1 of the present invention, but the culture medium used in step A6 is a modified MOs A medium without Collagen I and Collagen IV (the other components are exactly the same as in Table 1).

[0119] 2. The suspension culture was also carried out using a dynamic shaker (100 rpm).

[0120] 3. The steps for passage, cryopreservation, and revival of the organoids are the same as in the embodiments.

[0121] Comparative Example 3: Construction method without exogenous matrix gel but using static culture This comparative example is basically the same as that of the present invention (Example 1), except that: instead of using a track shaker to provide dynamic mechanical stimulation, the six-well plate is statically cultured in a cell culture incubator.

[0122] Build steps: 1. Refer to steps A1-A6 of Example 1 of this invention, using the exact same MOs A culture medium (containing Collagen I and Collagen IV).

[0123] 2. Culture conditions: Place the six-well plates containing tissue blocks directly into a cell culture incubator (37℃, 5% CO2, 90% humidity), without placing them on a rocker, for static suspension culture. Perform a half-medium change every 48 hours.

[0124] 3. The steps for passage (static suspension culture), cryopreservation and thawing of the organoids are the same as in the example.

[0125] Comparative Examples 1-3 and the original meningioma tissue used in the embodiments of the present invention were extracted from the same batch from the same patient. Comparative Examples 1-3 were control examples set up during the embodiments of the present invention. The organoid photographs on day 10 of culture in the embodiments are shown below. Figure 3 For comparison images of organoids cultured on day 10 of Examples 1-3, please refer to... Figure 7 The formation rate and morphological description of meningioma organoids under different culture systems in the examples and comparative examples 1-3 are shown in Table 4.

[0126] Organoid formation rate: The percentage of meningioma organoids with clear boundaries, complete structure and a diameter greater than 100 μm formed by day 3 of culture, out of the initial inoculated tissue blocks.

[0127] Table 4 1Yamazaki S, Ohka F, Hirano M, et al. Newly established patient-derived organoid model of intracranial meningioma. Neuro Oncol 2021; 23:1936-48. 2Caffo M, Caruso G, Galatioto S, et al. Immunohistochemical study of the extracellular matrix proteins laminin, fibronectin and type IV collagenin secretory meningiomas. J Clin Neurosci 2008; 15: 806-11. 3Rooprai HK, Liyanage K, Robinson SF, Kandanearatchi A, Dean AF,Pilkington GJ. Extracellular matrix-modulated differential invasion of humanmeningioma cell lines in vitro. Neurosci Lett 1999; 263: 214-6. 4Takahashi J, Mizutani T, Sugihara HY, et al. Suspension culture in arotating bioreactor for efficient generation of human intestinal organoids. Cell Rep Methods 2022; 2: 100337. The embodiments of this invention, by adding a collagen combination (collagen I and collagen IV) and dynamic mechanical stimulation to a matrix-free system, demonstrate superior organoid formation rate, structural stability, and long-term culture capability compared to Comparative Examples 1-3. While Comparative Example 1 can form organoids, it relies on matrix gel, resulting in batch-to-batch variations and cost issues. Comparative Examples 2 and 3 respectively demonstrate that both collagen and dynamic stimulation are indispensable; the absence of either significantly affects the quality of organoid formation.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing meningoma organoids without exogenous matrix gel support, characterized in that, The construction method includes primary culture of meningoma organoids, wherein the primary culture includes: inducing meningoma tissue blocks to self assemble into organoids in a non-adhesive culture system in MOs A medium supplemented with type I and type IV collagen under dynamic mechanical stimulation, wherein the MOs A medium uses DMEM / F-12 as the basal medium.

2. The construction method according to claim 1, characterized in that, The content of type I collagen relative to the basal culture medium is 10-100 μg / mL, and the content of type IV collagen relative to the basal culture medium is 5-50 μg / mL. Preferably, the content of type I collagen relative to the basal culture medium is 50 μg / mL, and the content of type IV collagen relative to the basal culture medium is 25 μg / mL.

3. The construction method according to claim 1, characterized in that, The dynamic mechanical stimulation is provided by a track-mounted rocking table; the rotational speed of the track-mounted rocking table is 80-140 rpm, preferably 100-120 rpm; And / or, the meningioma tissue block is derived from the patient's tumor tissue and obtained after mechanical shearing and erythrocyte lysis; preferably, the mechanical shearing time is 8-12 minutes; the erythrocyte lysis process includes adding pre-cooled erythrocyte lysis buffer, centrifuging at 800-1200 rpm for 5-8 minutes, and then removing the supernatant; And / or, the non-adhesive culture system uses non-adhesive culture vessels with low or ultra-low cell adhesion as carriers; Preferably, the non-adhesive culture vessel is a culture plate with a low adsorption treatment on its surface; the low adsorption treatment is to coat the surface of the non-adhesive culture vessel with an inert hydrophilic polymer layer or a hydrogel layer. More preferably, the non-adhesive culture vessel is a 6-well ultra-low adsorption culture plate.

4. The construction method according to claim 1, characterized in that, The MOs A culture medium also contains basic fibroblast growth factor, nerve cell culture additive, B27 serum-free additive, epidermal growth factor, penicillin-streptomycin dual antibody and mycoplasma prevention reagent. Preferably, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The content of the basic fibroblast growth factor relative to the basal culture medium is 10~40 ng / mL, preferably 20 ng / mL; The volume content of the neural cell culture additive relative to the basal culture medium is 0.5% to 2%, preferably 1%; The B27 serum-free additive has a volume content of 1% to 3% relative to the basal culture medium, preferably 2%; The epidermal growth factor concentration relative to the basal culture medium is 100-400 ng / mL, preferably 200 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 0.5% to 2% relative to the basal culture medium, preferably 1%; The mycoplasma prevention reagent has a volume content of 0.03% to 0.07% relative to the basal culture medium, preferably 0.05%.

5. The construction method according to any one of claims 1-4, characterized in that, The construction method further includes the following steps: passage, cryopreservation and thawing of the organoids.

6. The construction method according to claim 5, characterized in that, The primary culture specifically includes: placing meningioma tissue blocks into the MOs A medium added to each well of a non-adhesive culture dish, adding 50-60 meningioma tissue blocks to each well, then placing the culture dish on a track shaker and continuously shaking it in an incubator at 37±1℃, 5-8 vol% CO2, and 85-95% humidity, performing a half-medium change every 40-60 hours, and forming spherical organoids after 60-80 hours of culture, which can continue to grow for 5-8 weeks; And / or, the passage step includes: when the diameter of the organoids obtained from the primary culture reaches 1.5-2.5 mm, thoroughly removing the supernatant from the primary non-adhesive culture dish, transferring the organoids to a culture dish containing pre-cooled MOs A medium, and shearing each organoid; then transferring 20-30 organoid tissues obtained after shearing to a new non-adhesive culture dish containing MOs A medium; and then placing the passaged non-adhesive culture dish on a track shaker and continuously shaking it at 100-120 rpm in an incubator at 37±1℃, 5-8 vol% CO2, and 85-95% humidity. And / or, the cryopreservation step includes: collecting organoids obtained from primary culture or passaged culture, aspirating the supernatant from the culture vessel, adding cryopreservation solution, mixing, and storing at -80±5℃ for 20-28h, and then transferring to liquid nitrogen for long-term storage; preferably, the cryopreservation solution contains MOs A medium, DMSO and Y-27632. More preferably, in the cryopreservation solution, based on the total volume of MOs A medium and DMSO as 100%, the volume percentage of MOs A medium is 85-95%, the volume percentage of DMSO is 5-15%, and the concentration of Y-27632 is 5-15 μmol / L. And / or, the resuscitation steps include: rapidly thawing the cryopreservation tubes containing organoids in a water bath at 37±1℃, with the transfer time from liquid nitrogen to the water bath not exceeding 1 minute, thawing for 1-2 minutes until no ice is present, aspirating the cryopreservation solution, washing twice with MOs A medium, and then transferring the organoids to a culture dish containing MOs A medium that has not undergone tissue culture treatment, and then continuing to culture according to the primary culture method.

7. A culture medium for constructing meningoma organoids without exogenous matrix gel support, characterized in that, The culture medium is MOs A medium, which uses DMEM / F-12 as the basal medium and also contains type I collagen and type IV collagen as regulatory factors.

8. The culture medium according to claim 7, characterized in that, The content of type I collagen relative to the basal culture medium is 10-100 μg / mL, and the content of type IV collagen relative to the basal culture medium is 5-50 μg / mL; preferably, the content of type I collagen relative to the basal culture medium is 50 μg / mL, and the content of type IV collagen relative to the basal culture medium is 25 μg / mL. And / or, the MOs A medium also contains basic fibroblast growth factor, nerve cell culture additive, B27 serum-free additive, epidermal growth factor, penicillin-streptomycin bispecific antibody and mycoplasma prevention reagent. Preferably, the content of each component in the MOs A culture medium relative to the basal culture medium is as follows: The content of the basic fibroblast growth factor relative to the basal culture medium is 10~40 ng / mL, preferably 20 ng / mL; The volume content of the neural cell culture additive relative to the basal culture medium is 0.5% to 2%, preferably 1%; The B27 serum-free additive has a volume content of 1% to 3% relative to the basal culture medium, preferably 2%; The epidermal growth factor concentration relative to the basal culture medium is 100-400 ng / mL, preferably 200 ng / mL; The penicillin-streptomycin bispecific antibody has a volume content of 0.5% to 2% relative to the basal culture medium, preferably 1%; The mycoplasma prevention reagent has a volume content of 0.03% to 0.07% relative to the basal culture medium, preferably 0.05%.

9. A meningioma organoid model without exogenous matrix adhesive support, characterized in that, It is constructed by the method described in any one of claims 1-6; Preferably, the organoid grows in suspension under non-adhesive conditions, has a three-dimensional spherical structure, and does not contain exogenous matrix adhesive components.

10. The use of the meningioma organoid model without exogenous matrix gel support constructed by the construction method according to any one of claims 1-6, or the meningioma organoid model without exogenous matrix gel support according to claim 9, in screening drugs for the treatment of meningioma or in the study of the mechanism of meningioma.

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