Culture medium and method for constructing auditory neuroma organoid

Abstract

The invention relates to the technical field of bioengineering, in particular to a culture medium and a method for constructing auditory neuroma organs. According to the culture medium, Neurobase Medium and DMEM (Dulbecco Modified Eagle Medium) / F12 are used as a basic culture medium; the feed additive comprises a N2 additive, a B27 additive, L-alanyl-L-glutamine, an antibiotic, HEPES, glucose, beta-mercaptoethanol, a cell growth factor, MEM NEAA and a TGF-beta inhibitor. The cell growth factor is EGF (Epidermal Growth Factor) or FGF-2. The culture medium provided by the invention can effectively improve the construction efficiency of the auditory neuroma organoid, and the constructed auditory neuroma organoid can be used as a reliable in-vitro substitution model, is used for marker detection, drug screening and individualized medical exploration in basic research and clinical transformation research, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a culture medium and method for constructing acoustic neuroma organoids. Background Technology

[0002] Acoustic neuroma, pathologically known as vestibular schwannoma, is a benign intracranial tumor originating from Schwann cells of the vestibulocochlear nerve. It is the most common benign tumor of the cerebellopontine angle, accounting for approximately 80% to 90% of all tumors in this region. Epidemiologically, the vast majority of cases are sporadic and unilateral, with a peak age of onset between 30 and 50 years. Furthermore, approximately 5% to 10% of cases are associated with neurofibromatosis type 2 (NF2), an autosomal dominant genetic disorder. Compared to sporadic cases, NF2-related acoustic neuromas typically present bilaterally, at a younger age, and exhibit more aggressive biological behavior.

[0003] Although acoustic neuromas are pathologically benign, their growth often causes severe and irreversible neurological damage. As the tumor grows, it first compresses the ipsilateral vestibular-cochlear nerve, leading to progressive hearing loss, persistent tinnitus, and vertigo. Subsequently, the tumor may compress the adjacent facial and trigeminal nerves, causing a series of symptoms such as facial numbness, facial muscle weakness, and even facial paralysis. In advanced stages, large tumors can severely compress the brainstem and cerebellum, potentially leading to ataxia, increased intracranial pressure, and even death. Currently, clinical treatment for acoustic neuromas mainly relies on microsurgical resection and stereotactic radiotherapy (such as Gamma Knife). However, these treatments inherently carry the risk of damaging important nerve functions such as the auditory and facial nerves.

[0004] In terms of drug treatment, there are currently no clinically validated targeted drugs or standard chemotherapy regimens that can effectively inhibit the growth of acoustic neuromas. This gap in treatment leaves many patients facing the risks of tumor residue, recurrence, and continued deterioration of neurological function after surgery or radiotherapy, severely impacting their quality of life. Therefore, developing novel research models that can accurately simulate the in vivo growth characteristics and drug responses of acoustic neuromas is of paramount importance for elucidating their pathogenesis, screening effective therapeutic drugs, and promoting personalized precision medicine.

[0005] To delve deeper into the pathogenesis of acoustic neuroma and identify effective drug targets, establishing a research model that accurately reflects its in vivo pathophysiological characteristics is crucial. In the field of tumor research, conventional model construction methods primarily include in vitro cell culture and in vivo animal transplantation. However, directly applying these conventional techniques to acoustic neuroma research presents numerous challenges and inherent limitations due to its unique biological characteristics.

[0006] Specifically, attempts to establish primary tumor cell culture models first encounter technical bottlenecks. Acoustic neuroma cells exhibit extremely low proliferative activity in vitro and have very demanding requirements for the culture environment. This makes it exceptionally difficult to successfully establish stable passaged primary cell lines from clinical samples, with a success rate generally less than 5%. More importantly, even if a few cells are successfully expanded in vitro, prolonged passaged culture can easily induce phenotypic drift, causing the cells to rapidly lose their specific molecular characteristics as acoustic neuroma cells. For example, they may cease expressing the SOX10 protein, a key marker for acoustic neuroma cells, thus losing their representative value as a research model.

[0007] If simplified single-cell models (such as two-dimensional planar culture) are used in research, the problem of oversimplification arises. Such models cannot reproduce the original three-dimensional spatial structure and complex tumor microenvironment (TME) of tumor tissue. In the human body, the growth and development of acoustic neuroma are profoundly influenced by the complex network of interactions between nerve fibers, vascular matrix, fibroblasts, and various immune cells (such as macrophages) inherent in its microenvironment. Two-dimensional culture completely ignores these crucial intercellular and cell-matrix interactions. It is foreseeable that drug sensitivity tests based on such models will have extremely low correlation with actual clinical efficacy in patients, thus severely limiting their application value as a drug screening platform.

[0008] Furthermore, using orthotopic xenograft models in rodents also presents significant limitations. First, due to the indolent growth characteristics of acoustic neuroma cells, establishing xenograft models in animals faces the dual challenges of long tumor formation cycles and high experimental costs. Second, there are significant differences in the anatomical structure of the auditory nerve between humans and rodents, making it impossible for this model to realistically simulate the pathophysiological processes of human acoustic neuromas and their invasion of surrounding delicate neural structures (such as the facial nerve). More importantly, biological differences between species are not only reflected at the macroscopic level but also at the microscopic level of molecular signaling pathways. For example, the mTOR signaling pathway, which plays a central role in regulating cell growth and proliferation, exhibits significant differences in its specific regulatory mechanisms across different species, greatly reducing the clinical translational value of cross-species research.

[0009] In summary, due to the biological inertia of acoustic neuroma cells, their dependence on complex microenvironments, and the species differences between animal models and humans, conventional techniques in this field have yielded limited effectiveness in constructing acoustic neuroma research models. These potential obstacles, including low success rates in establishing cell lines, inability to maintain tumor molecular characteristics, inability to simulate complex tumor microenvironments, disconnect between drug screening results and clinical applications, and low translational value, collectively constitute technical barriers to the development of new drugs and the study of mechanisms in acoustic neuroma. Therefore, there is an urgent need in this field for a novel, efficient, and reliable research model that can overcome the above-mentioned deficiencies, maintain the original biological characteristics of acoustic neuroma cells, and highly simulate their microenvironment in the human body, thereby providing a key technical platform and tool for disease mechanism research and innovative drug development in this field. Summary of the Invention

[0010] To address the problems existing in the prior art, this invention provides a culture medium and method for constructing acoustic neuroma organoids. The culture medium, composed of specific components, significantly improves the success rate of acoustic neuroma organoid construction, and the constructed organoids can serve as reliable in vitro replacement models.

[0011] In a first aspect, the present invention provides a culture medium for culturing acoustic neuroma organoids, said culture medium being based on Neurobasal Medium and DMEM / F12; and comprising: N2 additive (100×) 10 mL / L, B27 additive (50×) 20 mL / L, L-alanyl-L-glutamine 1~4 mM, antibiotic 6~12 mL / L, HEPES 3~6 mM, glucose 0.2~0.8 g / L, β-mercaptoethanol 0.025~0.1 mM, cytokine 30 ng / mL, MEM NEAA 6~12 mL / L and TGF-β inhibitor 300~800 nM; the cytokine is EGF or FGF-2.

[0012] Organoid modeling techniques for vestibular schwannomas (acoustic neuromas) have not been publicly reported to date. In previous studies, this invention found that organoid construction for acoustic neuromas is quite difficult, for example, the success rate using primary cells is less than 5%. Furthermore, after long-term passage, they rapidly lose tumor-specific molecular characteristics.

[0013] Through extensive research and analysis, this invention provides a culture medium for culturing acoustic neuroma organoids. This medium, composed of the above components, effectively improves the success rate of acoustic neuroma organoid construction. Furthermore, the constructed organoids exhibit high fidelity: preserving the spatial heterogeneity of tumor tissue (including Schwann cells and vascular endothelial cells); high efficiency: requiring only a small amount of biopsy tissue (≥1 mm in diameter), functional organoids can be constructed within 2-3 weeks with a success rate >85%, far exceeding that of primary cell culture (<5%); and high throughput adaptability: supporting multi-dimensional screening of targeted drugs, radiosensitizers, and gene editing therapies, with improved preclinical prediction accuracy compared to traditional models.

[0014] In a preferred embodiment, the present invention provides a culture medium for constructing acoustic neuroma organoids. The culture medium is based on Neurobasal Medium and DMEM / F12 and consists of the following components: N2 additive (100×) 10 mL / L, B27 additive (50×) 20 mL / L, L-alanyl-L-glutamine 1.8–3.7 mM, antibiotic 8–12 mL / L, HEPES 3–5 mM, glucose 0.2–0.6 g / L, β-mercaptoethanol 0.04–0.06 mM, cytokines 30 ng / mL, MEM NEAA 8–12 mL / L, and TGF-β inhibitor 400–600 nM; wherein the cytokines are EGF or FGF-2.

[0015] Furthermore, in the basal culture medium, the mass ratio of Neurobasal Medium to DMEM / F12 is (2~3.5):1; Preferably, the mass ratio of Neurobasal Medium to DMEM / F12 is (2.8~3.2):1.

[0016] Under the preferred basal culture medium ratio of this invention, better growth support can be provided for organoids, resulting in more stable organoid structures.

[0017] In a second aspect, the present invention provides a cryopreservation culture medium for acoustic neuroma organoids, comprising: the aforementioned culture medium; preferably, it further comprises: ROCK inhibitor, DMSO, caspase inhibitor and trehalose; More preferably, the cryopreservation culture medium comprises: 10 μM ROCK inhibitor, 5 v / v% DMSO, 5-10 μM caspase inhibitor, and 50-100 μM trehalose.

[0018] More preferably, the ROCK inhibitor is Y-27632 and the caspase inhibitor is Z-VAD-FMK.

[0019] Thirdly, the present invention provides the use of the aforementioned culture medium in any of the following: (1) Culture of acoustic neuroma organoids; (2) Improve the success rate of constructing acoustic neuroma organoids.

[0020] Fourthly, the present invention provides a method for constructing acoustic neuroma organoids, comprising: After pretreatment to obtain tissue fragments from the isolated acoustic neuroma, the fragments were placed in the aforementioned culture medium for hypoxic culture.

[0021] Furthermore, the pretreatment includes: chopping, sieving, removing impurities, and removing residual red blood cells; Preferably, the aperture of the sieve is 1.0 mm; More preferably, the conditions for removing residual red blood cells include: a temperature of 37°C, a CO2 concentration of 5%, a shaker speed of 100-140 rpm, and a time of 10-15 minutes.

[0022] The ex vivo acoustic neuroma described in this invention can be an ex vivo acoustic neuroma specimen obtained by craniotomy or stereotactic biopsy.

[0023] Furthermore, the hypoxic culture includes: (1) Incubate at an oxygen concentration of 0.5-1.5% for 2-4 hours; (2) Incubate at an oxygen concentration of 2.5-3.5% for 40-56 hours; (3) Incubate at an oxygen concentration of 8-12% for 40-56 hours; (4) Incubate at an oxygen concentration of 14-18% for 40-56 hours; (5) The subsequent culture was carried out at a normal oxygen concentration of 21%.

[0024] The hypoxic culture conditions preferred in this invention have significant advantages in promoting the formation of three-dimensional structures of acoustic neuroma organoids, maintaining tissue activity, and enhancing growth capacity. The morphology of the constructed organoids is closer to that of the primary tumor, and the Ki-67 index, SOX10, and S100B expression are more consistent with the original tissue, providing a reliable experimental model for subsequent drug sensitivity testing, molecular mechanism research, and personalized treatment modeling.

[0025] Furthermore, the culture temperature for the hypoxic culture is 37°C, and the shaking speed is 100~160 rpm.

[0026] Fifthly, the present invention provides an acoustic neuroma organoid constructed by the aforementioned method.

[0027] The present invention has the following beneficial effects: This invention provides a culture medium for acoustic neuroma organoids. Based on this culture medium, the construction of acoustic neuroma organoids has a higher success rate and efficiency. The constructed acoustic neuroma organoids have high fidelity and high throughput adaptability, and can serve as an ideal in vitro proliferation research model. It is suitable for various scenarios such as drug screening, tumor growth regulation mechanism research, and personalized treatment response evaluation, and has important application value and practical significance. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0029] Figure 1 This is an operational diagram of constructing an acoustic neuroma organoid provided in Embodiment 2 of the present invention.

[0030] Figure 2 This is a morphological diagram of the successfully constructed acoustic neuroma organoid provided in Embodiment 2 of the present invention.

[0031] Figure 3 This is an evaluation chart of the growth curve of acoustic neuroma organoids provided in Embodiment 2 of the present invention; the left figure is the processing flow, the right figure is the growth curve, and Examples-1 to-5 are repeated experiments from 5 different patients.

[0032] Figure 4 This is a diagram showing the histological verification and morphological comparison analysis results of the acoustic neuroma organoids provided in Embodiment 2 of the present invention.

[0033] Figure 5 This is an image showing the immunohistochemical verification results of SOX10 expression in acoustic neuroma organoids provided in Embodiment 2 of the present invention.

[0034] Figure 6 This is a diagram showing the results of S100B expression verification and staining consistency analysis with the original tissue in acoustic neuroma organoids provided in Embodiment 2 of the present invention.

[0035] Figure 7 This is a diagram showing the phenotypic verification and tissue consistency evaluation results of the immune microenvironment of acoustic neuroma organoids provided in Embodiment 2 of the present invention.

[0036] Figure 8 This is a diagram showing the Ki-67 staining verification and tissue consistency analysis results of the proliferative capacity of acoustic neuroma organoids provided in Example 2 of this invention.

[0037] Figure 9This is a graph showing the results of the imbalance in the Neurobasal medium:DMEM / F12 ratio provided in Experimental Example 1 of this invention.

[0038] Figure 10 This is a graph showing the results of continuous addition of EGF / bFGF provided in Experimental Example 1 of this invention.

[0039] Figure 11 This is the result graph of Experiment Example 1 of the present invention without the addition of the TGF-β inhibitor SB431542.

[0040] Figure 12 This is a diagram showing the results of normoxic culture provided in Experimental Example 1 of this invention.

[0041] Figure 13 The growth curves of acoustic neuroma organoids in the classic neurotumor organoid culture medium provided in Experimental Example 1 of this invention are shown. Examples 6-10 are replicate experiments from 5 different patient sources. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0043] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0044] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0045] In the following examples, Neurobasal Medium is commercially available, for example, from Thermo Fisher, product number 21103049.

[0046] In the following embodiments, the DMEM / F12 is commercially available, for example from Gibco, part number C11330500BT.

[0047] In the following examples, L-alanyl-L-glutamine is commercially available, for example, GlutaMAX (100×), Gibco, catalog number 35050061.

[0048] The N2 additives used in the following examples are commercially available, such as Gibco, catalog number 17502-048.

[0049] The B27 additive in the following examples is commercially available, for example, from Gibco, catalog number 12587-010.

[0050] The antibiotics used in the following examples are commercially available, such as penicillin-streptomycin-gentamicin solution, Beyotime, catalog number C0223.

[0051] In the following examples, HEPES is commercially available, for example, Beyotime, product number ST090-100g.

[0052] In the following examples, glucose is commercially available, for example, from Sangon Biotech, product number A610498.

[0053] In the following examples, β-mercaptoethanol is commercially available, for example, from Sigma, catalog number M3148.

[0054] In the following examples, EGF is commercially available, such as Novoprotein, catalog number C029-500μg.

[0055] In the following examples, FGF-2 is commercially available, for example, Origene, catalog number TP750002.

[0056] In the following examples, MEM NEAA is commercially available, for example, Gibco, product number 11140050.

[0057] In the following examples, SB431542 is commercially available, for example, MCE, part number HY-10431.

[0058] In the following examples, Y-27632 is commercially available, for example, MCE, part number HY-10071.

[0059] In the following examples, DMSO is commercially available, for example, MCE, product number HY-Y0320.

[0060] In the following examples, Z-VAD-FMK is commercially available, for example, MCE, product number HY-16658B.

[0061] In the following examples, D-(+)-Trehalose is commercially available, for example, MCE, product number HY-N1132.

[0062] The DMEM / F12 (1:1) liquid culture medium (containing sodium pyruvate and HEPES) used in the following examples is commercially available, for example, Biosharp, catalog number BL305A.

[0063] The Y27632 in the following examples is commercially available, for example, MCE, part number HY-10071.

[0064] The D-(+)-Trehalose used in the following examples is commercially available, for example, MCE, product number HY-N1132.

[0065] The artificial cerebrospinal fluid (aCSF) used in the following examples is commercially available, such as Bio-Rad, product number GL0304.

[0066] Example 1 1. In this embodiment, a culture medium specifically for acoustic neuroma organoids comprises the following components: Basic culture medium: Neurobasal Medium, a combination of DMEM / F12 (optimized ratio 3:1).

[0067] Cellular metabolic support components: L-alanyl-L-glutamine, N2 additive, B27 additive, β-mercaptoethanol (β-ME), MEM NEAA (100X).

[0068] Functional modulators: cell growth factors (epidermal growth factor EGF or basic fibroblast growth factor bFGF, in actual use, EGF is added on days 1-3 and bFGF is added on days 4-5), SB431542 (TGF-β inhibitor), antibiotics, HEPES (2M), glucose (20%).

[0069] Table 1. Components of culture medium for acoustic neuroma organoids

[0070] 2. In this embodiment, a cryopreservation solution specifically for neurotumor organoids comprises the following components: Add 10 μmol / L Y-27632 and 10% DMSO (to the final cryopreservation volume) to the above-mentioned special culture medium components, Z-VAD-FMK (20 μM) (a broad-spectrum Caspase inhibitor), and D-(+)-Trehalose (50 mM, a protein stabilizer that forms a glassy protective layer and reduces ice crystal damage).

[0071] Table 2. Cryopreservation culture medium for acoustic neuroma organoids

[0072] 3. In this embodiment, a transport storage solution for organoid tissue derived from acoustic neuroma comprises the following components: DMEM / F12 (1:1) liquid culture medium (containing sodium pyruvate and HEPES), Y27632 (ROCK inhibitor, 10 μM), D-(+)-Trehalose (50 μM) (to cope with temperature fluctuations, maintain pH 7.2±0.2 and 290 mOsm / kg osmotic pressure), antibiotics.

[0073] Table 3. Culture medium for transporting organoids from acoustic neuroma

[0074] Example 2 In this embodiment, a method for constructing acoustic neuroma organoids includes the following steps: (a) Experimental materials.

[0075] Acoustic neuroma specimens obtained through methods such as craniotomy and puncture biopsy.

[0076] (II) Experimental methods.

[0077] 1. Organoid separation procedure for acoustic neuroma (1) Fresh ex vivo acoustic neuroma specimens obtained by craniotomy or stereotactic biopsy are loaded into pre-cooled acoustic neuroma transport storage solution and transported to the laboratory in an ice bath and insulated box.

[0078] (2) Under aseptic conditions, the glioma specimen was poured into a small dish in a clean bench, rinsed repeatedly with pre-cooled PBS 2-3 times, and then soaked in acoustic neuroma transport storage solution.

[0079] (3) Using autoclaved microsurgical scissors and microforceps, and with the aid of an electron microscope (MIXOUT digital microscope, magnification 200x), carefully cut the specimen into small pieces (e.g. Figure 1 (See the top left image). A custom-made sieve with a 1mm aperture is used to screen and obtain tissue particles smaller than 1mm (e.g., ...). Figure 1 (See the upper and middle images). Remove the black necrotic tissue produced by surgical electrocoagulation, and then clean it again with acoustic neuroma transport temporary storage solution to remove impurities.

[0080] (4) Tilt the small dish, the shredded tissue fragments will settle at the bottom, remove the supernatant, add 3 mL of red blood cell lysis buffer (Biosharp, BL503A) to remove the remaining red blood cells. The removal process is carried out on a shaker (Bikman, TM-180) in a 37°C, 5% CO2 incubator. The shaker speed is set to 106 rpm and the removal time is 10 minutes.

[0081] (5) After cleaning, tilt the dish again. The shredded tissue fragments will settle at the bottom. Aspirate the supernatant, add 4 mL of acoustic neuroma organoid culture medium (provided in Example 1), and transfer to a 6-well plate. Place the plate on a shaker in a hypoxic incubator for gradient hypoxia culture (e.g., Figure 1(See the lower left image). Initially, the culture was carried out for 3 hours in an incubator with 1% oxygen, 37°C, and 5% CO2. Then, it was cultured for 48 hours in an incubator with 3% oxygen, 37°C, and 5% CO2. After that, it was cultured for 48 hours in an incubator with 10% oxygen, 37°C, and 5% CO2. After that, it was cultured for 48 hours in an incubator with 16% oxygen, 37°C, and 5% CO2. Finally, it was continuously cultured in an incubator with 21% oxygen (normal oxygen), 37°C, and 5% CO2. The shaking speed was set to 106 rpm.

[0082] (6) Replace 75% of the culture medium every 2 days. The replacement method is to tilt the small dish, let the organoids settle at the bottom, remove the supernatant, and add 3 mL of acoustic neuroma organoid-specific culture medium (replace 3 / 4 of the culture medium).

[0083] (7) After 30 days of culture, organoids can be passaged. Select the larger organoids (with a diameter of 2 mm), soak them in acoustic neuroma transport storage solution, and repeat steps (3)-(6).

[0084] 2. Cryopreservation of acoustic neuroma organoids.

[0085] The organoids obtained in step 1 were transferred to cryopreservation tubes (containing the cryopreservation culture medium of Example 1), with each tube containing 30 organoids. After being cooled to -80°C, they were transferred into liquid nitrogen.

[0086] 3. Resuscitation of acoustic neuroma organoids.

[0087] (1) Thaw the organoids frozen in liquid nitrogen rapidly in a water bath at 37°C. After thawing, remove the cryopreservation medium, rotate and slowly add 4 mL of cryopreservation medium for acoustic neuroma organoids, transfer it into a 6-well plate, place it on a shaker in a 37°C, 5% CO2 incubator, set the shaker speed to 106 rpm / min, and incubate overnight. (2) After 12 hours, remove the supernatant from the previous culture medium, add 4 mL of acoustic neuroma organoid culture medium, and continue culturing.

[0088] 4. Histological verification and morphological comparative analysis of acoustic neuroma organoids.

[0089] The histological structure of acoustic neuroma organoids was systematically evaluated by tissue sections and hematoxylin & eosin (HE) staining.

[0090] 5. Immunohistochemical verification of SOX10 expression in acoustic neuroma organoids.

[0091] To further verify whether the cultured acoustic neuroma organoids retain the original tissue characteristics at the molecular marker level, this invention uses SOX10 immunohistochemical staining to compare and analyze the primary tumor tissue and organoids.

[0092] 6. Validation of S100B expression in acoustic neuroma organoids and analysis of staining consistency with the original tissue.

[0093] To verify the accuracy of the acoustic neuroma organoids constructed in this invention in terms of neural crest-specific marker expression, S100B protein was selected for immunohistochemical staining analysis. S100B, as a classic positive marker for schwannomas, exhibits high sensitivity and specificity in the diagnosis of acoustic neuromas.

[0094] 7. Phenotypic verification and tissue consistency evaluation of the immune microenvironment of acoustic neuroma organoids.

[0095] To verify the effectiveness of the constructed acoustic neuroma organoids in simulating the immune microenvironment, this invention uses a variety of immune cell-related markers for immunofluorescence and immunohistochemical staining, including CD3 (T cells), IBA-1 (microglia / macrophages), CD31 (vascular endothelial cells), and CD68 (macrophages), and compares them with the primary tissue.

[0096] 8. Ki-67 staining verification of the proliferative capacity of acoustic neuroma organoids and analysis of their consistency with tissue.

[0097] To further verify whether the acoustic neuroma organoids constructed in this invention retain the cell proliferation capacity and biological activity of the primary tumor, this invention uses Ki-67, a classic cell proliferation marker, for immunohistochemistry and immunofluorescence staining.

[0098] (III) Experimental Results.

[0099] 1. Organoid morphology.

[0100] Figure 2 Examples 1-5 in the text represent organoid morphologies formed from five different acoustic neuroma tissue samples under the same culture conditions, with a culture time of approximately 8 to 14 days. The tissue masses are spherical or near-spherical with clear edges, relatively uniform size, and smooth surfaces, exhibiting a diameter of approximately 300–600 μm. They demonstrate good three-dimensional structure and aggregation, indicating successful organoid construction.

[0101] Examples 2-1 to 2-5 of Example 2 are magnified images of the organoids from Sample 2 on day 8 of culture, further demonstrating their mature morphology. The organoids in the images are densely structured with high sphericity. Some individuals show a dark core region within the tissue, suggesting the presence of cell proliferation or mild necrosis centers, consistent with the developmental patterns of tumor organoids. Their diameter ranges from approximately 300-500 μm.

[0102] Light microscopy images clearly show that the organoid culture system established in this invention can stably obtain acoustic neuroma organoids with typical morphology, uniform size, and three-dimensional structure, which are suitable for subsequent molecular biological analysis, drug screening, and pathological simulation studies, and have good reproducibility and biological representativeness.

[0103] 2. Evaluation of growth curves for acoustic neuroma organoids.

[0104] Figure 3 The study presents the trend of diameter fold increase over time (unit: culture weeks, W) of organoids from five different patients with acoustic neuromas under the above culture protocol. The results show that all organoids exhibited varying degrees of proliferation from week 1 to week 4; organoids from sample 3 showed the most significant proliferation, with an average diameter increase of nearly 5-fold by week 4; the proliferation folds of the remaining organoids ranged from 2 to 3-fold, demonstrating good scalability. This indicates that the culture strategy of this invention has good versatility, adaptability, and reproducibility, and can be applied to the construction of organoids from multiple patient tissues.

[0105] The above results fully verify that the gradient hypoxia culture system constructed in this invention has significant advantages in promoting the formation of three-dimensional structures of acoustic neuroma organoids, maintaining tissue activity and enhancing growth capacity, and provides a reliable experimental model for subsequent drug sensitivity testing, molecular mechanism research and personalized treatment modeling.

[0106] 3. Histological verification and morphological comparative analysis results of acoustic neuroma organoids.

[0107] The results are as follows Figure 4 As shown: (1) Acoustic neuroma tissue (top row of images).

[0108] HE staining showed that the tumor tissue consisted of densely packed spindle-shaped cells with rounded or oval nuclei, fine chromatin, and sparse cytoplasm. Moderate to strong nucleolus visibility suggests active proliferation.

[0109] In some areas, the cells are arranged in an alternating pattern, exhibiting a palisade-like structure, consistent with the typical histological features of acoustic neuroma.

[0110] It can also identify two types of cell morphological characteristics: Antoni A region (dense arrangement) and Antoni B region (loose arrangement).

[0111] (2) Acoustic neuroma organoids (bottom row of images).

[0112] HE staining results showed: The organoids are spherical or onion-like in shape, with cells arranged in distinct concentric rings, reflecting a highly organized structural remodeling capacity.

[0113] Two typical acoustic neuroma cell arrangements can be clearly observed within the organoids, corresponding to Antoni A and B regions respectively. The cells exhibit alternating distributions of tightly packed and loosely packed areas, highly replicating the histological characteristics of the primary tumor.

[0114] Under high magnification, the morphology of the cell nuclei and the state of the chromatin were consistent with the original tissue, indicating good biological similarity.

[0115] This organoid highly replicates the morphological characteristics of primary acoustic neuroma tissue, particularly its representative onion-like whorl structure and dual-zone morphological features (Antoni A / B). The organoid model of this invention possesses excellent tissue biomimicry capabilities, providing a solid foundation for subsequent personalized drug screening, pathogenesis research, and model translation.

[0116] 4. Immunohistochemical verification results of SOX10 expression in acoustic neuroma organoids.

[0117] The results are as follows Figure 5 As shown: (1) Expression of SOX10 in primary acoustic neuroma tissue (top row of images).

[0118] SOX10 is an important nuclear transcription factor marker in tumors of neural crest origin (such as acoustic neuroma and schwannoma), and it exhibits positive staining of cell nuclei in tumor tissues. The figure shows: Numerous tumor cell nuclei showed brownish-red positive signals, with widespread staining distribution.

[0119] The expression is diffuse or patchy, consistent with the typical immunophenotype of acoustic neuroma tissue.

[0120] Under high magnification, the cell nuclei show strong staining with good specificity, providing a clear diagnostic indication.

[0121] (2) Expression of SOX10 in acoustic neuroma organoids (bottom row of images).

[0122] Immunohistochemical results of organoids showed: The organoid's overall structure is well preserved, and the cells are arranged in an orderly manner.

[0123] The cell nuclei within the organoids also showed significant SOX10 positive expression, with staining localization consistent with that of the tissue.

[0124] At different magnifications, positive staining signals within the nucleus can be observed to be concentrated in the peripheral active area, suggesting that it retains the differentiation lineage characteristic of schwannomas.

[0125] The organoids exhibit a high degree of molecular phenotypic similarity to the original tissues, demonstrating good biological stability and model reliability.

[0126] The acoustic neuroma organoids constructed in this invention fully replicate the immunohistochemical characteristics of the primary tumor tissue at the SOX10 expression level, further validating their high homology and biomimicry in terms of molecular subtyping and tissue origin. This model can serve as a reliable in vitro alternative model for biomarker detection, drug screening, and personalized medicine exploration in basic research and clinical translational studies.

[0127] 5. Validation of S100B expression in acoustic neuroma organoids and analysis of staining consistency with the original tissue.

[0128] The results are as follows Figure 6 As shown: (1) S100B expression in primary acoustic neuroma tissue (top figure).

[0129] Immunohistochemical results showed: The cells in the tumor tissue showed widespread and diffuse S100B positive expression, with staining located in the cytoplasm and around the nucleus; The staining is evenly distributed and of moderate intensity; Under high magnification, spindle-shaped cells were observed arranged along bundles, with clear S100B positive signals and a brownish-yellow color, consistent with the typical immunophenotype of acoustic neuroma tissue.

[0130] (2) S100B expression in acoustic neuroma organoids (see figure below).

[0131] The immunohistochemical features of organoids are basically consistent with those of tissues: The organoids have an intact overall structure, and the cells show strong positive S100B expression with a wide signal distribution.

[0132] The magnified image shows that the cells inside the organoid are tightly packed, and the staining intensity is comparable to that of tissue.

[0133] The staining distribution is arranged in a ring or radial pattern, indicating that the organoids have a good ability to reconstruct tissue structures.

[0134] This indicates that organoids retain key biomarker features of the primary tissue in the neural crest differentiation lineage.

[0135] The acoustic neuroma organoids constructed in this invention exhibit a high degree of consistency with the primary tissue in terms of S100B expression, indicating that the organoids retain the molecular phenotype unique to schwannomas and possess good tissue-derived reproducibility and model stability. This model can be widely applied in various fields such as tumor diagnostic marker validation, disease pathogenesis research, personalized drug screening, and translational medicine exploration, demonstrating significant research and clinical translational potential.

[0136] 6. Phenotypic verification and tissue consistency evaluation results of the immune microenvironment of acoustic neuroma organoids.

[0137] The results are as follows Figure 7 As shown: (1) Multiple immunofluorescence staining (top row).

[0138] The figure shows the expression profiles of tissues and organoids under co-staining with DAPI / CD31 / CD3 / IBA-1 four channels: CD31 (green): Shows that vascular structures are visible in both tissues and organoids, suggesting that some vascular-like structures or endothelial markers are preserved in organoids.

[0139] CD3 (red): T cell infiltration is present in both tissues and organoids, although the number is lower in organoids but the signal distribution is regular.

[0140] IBA-1 (yellow): Microglia / macrophage signaling is clearly expressed in both tissues and organoids, indicating that organoids retain immune-related cellular components.

[0141] (2) Immunohistochemical staining (bottom row).

[0142] CD3 (T lymphocytes): Slightly scattered CD3-positive cells can be seen in the organoids, indicating the presence of T-cell-like infiltration.

[0143] IBA-1 and CD68 (macrophages / microglia): Both can be observed to be positively expressed in organoids. CD68 positive cells are widely distributed and have diverse morphologies, similar to those in tissues.

[0144] CD31 (vascular endothelium): CD31-positive vascular-like structures can be seen in the edge and part of the central region of organoids, suggesting a possible trend of vascular differentiation.

[0145] All markers showed specific localization and positive staining signals in organoids, and were highly similar to the expression patterns from tissue-derived sources.

[0146] The acoustic neuroma organoids constructed in this invention not only reproduce the characteristics of the primary tumor at the morphological and molecular levels, but also highly preserve the distribution and expression profiles of T cells, macrophages, microglia, and endothelial cells in tumor tissue at the level of the immune microenvironment. This model provides an advanced platform for studying tumor immune mechanisms, screening immunotherapy, and conducting personalized immune assessments, possessing excellent biomimetic capabilities and research application value in the immune microenvironment.

[0147] 7. Ki-67 staining verification of the proliferative capacity of acoustic neuroma organoids and results of histopathological analysis.

[0148] The results are as follows Figure 8 As shown: (1) Immunohistochemical staining results (top of the figure).

[0149] Organoid sections showed clear positive nuclear staining signals when stained with Ki-67 antibody.

[0150] The magnified image shows that some cell nuclei exhibit brownish-yellow positive staining, indicating ongoing cell proliferation activity.

[0151] The staining signals exhibit regional distribution within the organoids, with some concentrated in the periphery or active areas, mimicking the spatial distribution pattern of proliferating cells in real tumors.

[0152] The staining intensity was consistent with Ki-67 expression in the primary tumor tissue, demonstrating the biomimicry and representativeness of this model in terms of proliferation capacity.

[0153] (2) Immunofluorescence staining results (see figure below).

[0154] Ki-67 positive cells (red) are clearly visible in the fluorescence image, showing nuclear localization expression.

[0155] DAPI (blue) marks the cell nucleus to aid in the observation of total cell count and location.

[0156] The images showed a significant Ki-67 positivity rate, further demonstrating the presence of an active, proliferating cell population within the organoid structure.

[0157] The fluorescence signal was clear and the background was clean, verifying the specificity and technical stability of the staining.

[0158] The acoustic neuroma organoids constructed in this invention exhibit expression intensity, localization characteristics, and distribution patterns highly consistent with the original tissue at the expression level of the proliferation marker Ki-67, indicating that they possess good tumor biomimicry and biological activity at both structural and functional levels. This organoid system can serve as an ideal in vitro proliferation research model, suitable for various scenarios such as drug screening, research on tumor growth regulation mechanisms, and evaluation of personalized treatment responses.

[0159] Experimental Example 1 1. Based on the method of Example 2, the present invention provides multiple comparative examples for effect verification, as follows: Comparative Example 1: The total amount of basal culture medium remained unchanged, but the ratio of Neurobasalmedium:DMEM / F12 in the special culture medium in Example 2 was adjusted to 1:1.

[0160] Comparative Example 2: The total amount of basal culture medium remained unchanged, but the ratio of Neurobasalmedium:DMEM / F12 in the special culture medium in Example 2 was adjusted to 2:1.

[0161] Comparative Example 3: The total amount of basal culture medium remained unchanged, but the ratio of Neurobasalmedium:DMEM / F12 in the special culture medium in Example 2 was adjusted to 4:1.

[0162] Comparative Example 4: In the culture medium for acoustic neuroma organoids, the pulsed addition of cytokines was adjusted to continuous addition of EGF and bFGF (both added at 30 ng / mL).

[0163] Comparative Example 5: TGF-β inhibitor SB431542 was removed from the culture medium specifically for acoustic neuroma organoids.

[0164] Comparative Example 6: The culture conditions for the acoustic neuroma organoids in step (5) of Example 2 were adjusted to normoxic (21% O2), such as... Figure 1 As shown in the lower right image.

[0165] Comparative Example 7: β-ME was removed from the culture medium specifically for acoustic neuroma organoids.

[0166] Comparative Example 8: MEM-NEAA was removed from the culture medium specifically for acoustic neuroma organoids.

[0167] Comparative Example 9: The brain organoid culture media in the existing literature were used. Specifically, the culture media in various brain organoid literature were screened, and one of the media with the best effect was selected as an example.

[0168] Table 4 Brain organoid culture medium

[0169] 2. Experimental results.

[0170] (1) Basic culture medium ratio.

[0171] like Figure 9 As shown, an imbalanced Neurobasal medium:DMEM / F12 ratio (1:1) resulted in a significant decrease in organoid aggregation rate, structural instability, and a reduction in average diameter, suggesting a significant difference in their support for the growth of nervous system tissues. A ratio of (4:1) yielded similar results, while a 3:1 ratio of Neurobasal medium:DMEM / F12 in the dedicated culture medium resulted in the best organoid construction, followed by 2:1, both of which met the initial requirements for organoid construction.

[0172] (2) Comparison of EGF / bFGF addition methods.

[0173] like Figure 10As shown, the pulsed group (EGF added on days 1-3 and bFGF added on days 4-5) induced a higher proportion of spherical organoids and significantly improved the spatial stratification of organoids compared to continuous addition (Comparative Example 4), indicating that it plays a key regulatory role in morphological remodeling.

[0174] Although Comparative Example 4 could induce clumps, the organoid surface was rough, the internal cavities were obvious, and the proliferation activity was low, indicating that the dynamic regulation of growth factors is particularly important for simulating the tumor microenvironment.

[0175] (3) The presence or absence of a control group for the TGF-β inhibitor SB431542.

[0176] like Figure 11 As shown, although spherical structures can be formed after SB431542 removal, the surface is irregular and the structure is soft, indicating a tendency for EMT and mesenchymal transformation. This phenomenon was also verified by histology and immunohistochemistry. This indicates that SB431542 plays a key role in maintaining the epithelial-like state and stabilizing the structure.

[0177] (4) Comparison of the presence or absence of β-mercaptoethanol (β-ME) and MEM-NEAA.

[0178] The results showed that in the group without β-ME (Comparative Example 7), organoid activity was reduced, central necrosis rate was increased, and survival time was shortened, suggesting that it plays a key role in redox balance and cellular stress protection.

[0179] When MEM-NEAA was missing (Comparative Example 8), the average diameter of organoids decreased and the culture success rate was reduced, suggesting that it is irreplaceable in supporting metabolism and stable growth.

[0180] (5) Comparison of hypoxia gradient vs. normoxic culture.

[0181] like Figure 12 As shown, the acoustic neuroma organoids obtained in Comparative Example 6 exhibited loose and fragmented structures, failing to form typical spherical structures, suggesting that the hypoxic microenvironment is crucial for maintaining spatial structure and activity. In contrast, the organoids constructed in the hypoxia group more closely resembled the primary tumor in morphology, with Ki-67 index, SOX10, and S100B expression showing greater consistency with the original tissue.

[0182] (6) Comparison of classic neurotumor organoid culture media.

[0183] Currently, there is no specific culture medium for acoustic neuroma organoids. During the research process, this invention underwent extensive research and exploration, attempting various culture media for neuroma organoids or brain tissue organoids described in existing literature, but the results were unsatisfactory. The classic neurotumor organoid culture medium in Comparative Example 9 is a relatively better one among the various attempts, but from... Figure 13It can still be seen that the growth rate of acoustic neuroma organoids was significantly poor in this culture medium (and Figure 3 (In comparison). This invention, through extensive research and experimentation, has creatively developed a specialized culture medium for the aforementioned acoustic neuroma organoids, along with an innovative hypoxic gradient culture technique. This medium exhibits excellent acoustic neuroma organoid culture efficiency and is of significant importance.

[0184] 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for culturing acoustic neuroma organoids, characterized by, The medium is based on Neurobasal Medium and DMEM / F12 as base medium; and comprises: N2 supplement (100x) 10 mL / L, B27 supplement (50x) 20 mL / L, L-alanyl-L-glutamine 1-4 mM, antibiotics 6-12 mL / L, HEPES 3-6 mM, glucose 0.2-0.8 g / L, β-mercaptoethanol 0.025-0.1 mM, cytokine 30 ng / mL, MEM NEAA 6-12 mL / L and TGF-β inhibitor 300-800 nM; the cytokine is EGF or FGF-2.

2. The medium of claim 1, wherein, The medium is based on Neurobasal Medium and DMEM / F12 as base medium, and consists of N2 supplement (100x) 10 mL / L, B27 supplement (50x) 20 mL / L, L-alanyl-L-glutamine 1.8-3.7 mM, antibiotics 8-12 mL / L, HEPES 3-5 mM, glucose 0.2-0.6 g / L, β-mercaptoethanol 0.04-0.06 mM, cytokine 30 ng / mL, MEM NEAA 8-12 mL / L and TGF-β inhibitor 400-600 nM; the cytokine is EGF or FGF-2.

3. The medium of claim 1, wherein, In the base medium, the mass ratio of Neurobasal Medium and DMEM / F12 is (2-3.5):

1.

4. A cryopreservation medium for acoustic neuroma organoids, characterized in that, The medium comprises: The medium of any one of claims 1-3; The medium further comprises: ROCK inhibitor, DMSO, caspase inhibitor and trehalose.

5. Use of the medium of any one of claims 1-3 in any one of: (1) culturing an acoustic neuroma organoid; (2) improving the success rate of constructing an acoustic neuroma organoid.

6. A method of constructing an acoustic neuroma organoid, characterized by, The method comprises: After pre-treating an ex vivo acoustic neuroma to obtain tissue fragments, the tissue fragments are placed in the medium of any one of claims 1-3 for hypoxic culture.

7. The method of claim 6, wherein, The pre-treatment comprises: cutting, sieving, removing impurities and removing residual red blood cells; the pore size of the sieving is 1.0 mm.

8. The method of claim 7, wherein, The conditions for removing residual red blood cells comprise: temperature 37℃, 5% CO2 concentration, shaker speed 100-140 rpm and time 10-15 minutes.

9. The method according to any one of claims 6-8, characterized in that, The hypoxic culture comprises: (1) culturing under 0.5-1.5% oxygen concentration for 2-4 hours; (2) culturing under 2.5-3.5% oxygen concentration for 40-56 hours; (3) culturing under 8-12% oxygen concentration for 40-56 hours; (4) culturing under 14-18% oxygen concentration for 40-56 hours; (5) subsequent culturing under 21% normoxic concentration.

10. An acoustic neuroma organoid, characterized in that, The acoustic neuroma organoid is constructed by the method of any one of claims 6-9.

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