Culture medium and method for constructing diffuse endogenous brain bridge glioma organ model and application of model

Abstract

The invention provides a culture medium and a method for constructing a diffuse endogenous brain bridge glioma (DIPG) organ model, the culture medium comprises a DMEM / F12 culture medium, a Neurobasic culture medium, an L-glutamine substitute, a non-essential amino acid supplement, an N2 supplement, a B-27 supplement, 2-mercaptoethanol, penicillin, streptomycin and insulin, and the DIPG organ model is prepared from the DMEM / F12 culture medium, the Neurobasic culture medium, the L-glutamine substitute, the non-essential amino acid supplement, the N2 supplement, the B-27 supplement, the 2-mercaptoethanol, the penicillin, the streptomycin and the insulin. In the method, a DIPG sample is cultured into an organoid by using the culture medium. The invention also provides an application of the culture medium in preparation of a DIPG (Diisopropylidyne) organ-like model, the organ-like model prepared by the method, and an application of the DIPG organ-like model in cancer pathogenesis research, cancer treatment drug screening and development, cancer personalized treatment and / or cancer immunotherapy evaluation.

Description

Technical Field

[0001] This invention belongs to the field of organoid preparation, specifically, it relates to a culture medium and method for constructing a diffuse endophytic pontine glioma organoid model, and also relates to the application of the model. Background Technology

[0002] Diffuse intrinsic pontine glioma (DIPG) is a rare and highly aggressive brainstem tumor in children, typically classified as a WHO grade 3-4 high-grade tumor. It almost exclusively occurs in childhood, with a peak incidence between 5 and 10 years of age, accounting for 10%-20% of all central nervous system tumors in children. Common symptoms include cranial nerve palsy (such as facial asymmetry, diplopia), cerebellar ataxia (such as gait instability, clumsiness), and long tract signs (such as increased muscle tone, positive Babinski sign). DIPG progresses rapidly, with a median survival of only 8-12 months and a 5-year survival rate of less than 1%. Most children die within one year of diagnosis, representing a significant challenge in clinical practice. Current treatment options include surgery, radiotherapy, and chemotherapy. Due to the tumor's diffuse infiltration within the pons, which controls vital centers, surgical resection is technically impossible; forced resection would cause catastrophic neurological damage or even death. Radiotherapy is the primary treatment, but its effects are temporary, and tumor recurrence is almost inevitable. Chemotherapy has limited effectiveness, and no significantly effective chemotherapy drugs have yet been found. Current treatments cannot cure this tumor.

[0003] Given its unresectable growth pattern and lack of long-term survivors, DIPG has historically been diagnosed primarily through imaging, resulting in an extremely limited availability of DIPG tumor tissue for research over the past few decades. However, the rapid development of multimodal neuronavigation technology in recent years has enabled experienced clinicians to safely obtain tumor specimens by selecting appropriate puncture sites, clarifying pathology and molecular diagnosis, and providing possibilities for studying the pathogenesis of DIPG and exploring new treatment strategies. However, the success rate of patient-derived tumor xenograft (PDX) models is currently very limited, and they have a long latency period of 2 to 11 months during tumorigenesis. Furthermore, because DIPG is located within the brainstem, it is highly sensitive to tumor-related inflammation-related neurotoxicity induced by CAR-T therapy, making dose-escalation experiments very difficult in both nude mouse PDX models and clinically recruited patients. Therefore, suitable tumor models are urgently needed to advance DIPG treatment research.

[0004] Organoids are cell clusters with specific structures and functions that are self-induced from human adult stem cells or pluripotent stem cells through three-dimensional in vitro culture. They are highly similar to real human organs in structure and function and can be stably passaged in vitro. Unlike traditional in vitro cell culture, organoids are highly similar to primary tissues in composition and structure, serving as an important bridge between traditional 2D cell culture and in vivo mouse models. As a cancer model, organoid technology has achieved 100% sensitivity and 93% specificity in determining the effectiveness of anticancer drugs. Especially for common cancers such as lung cancer, gastric cancer, liver cancer, and pancreatic cancer, various organoids have been successfully prepared and can be used as corresponding cancer models for research on cancer pathogenesis, screening and development of cancer therapeutic drugs, personalized cancer treatment, evaluation of cancer immunotherapy, and many other aspects.

[0005] In the field of glioma organoids, a method for culturing glioblastoma organoids has been established, allowing for the rapid generation of glioblastoma organoids (GBOs) directly from fresh adult glioblastoma specimens. GBOs retain many key characteristics of their parent tumors and can be used to rapidly test responses to standard and targeted therapies, including those derived from CAR-T cell immunotherapy. For example, patent document 1 (hereinafter referred to as "Patent Document 1"), with patent application number 202410391021.4, publication number CN118126952A, and invention title "A Culture Medium and Method for Brainstem Glioma Organoids," discloses a culture medium and corresponding method specifically for brainstem glioma organoids. By improving the added cell growth factors, small molecule compounds, and nutrients, the method better simulates the tumor's in vivo growth environment, promoting not only the growth of brainstem glioma organoids but also increasing the success rate of organoid culture.

[0006] However, as a specific type of glioma, DIPG tumor tissue is inherently difficult to obtain, and the culture media and methods used for organoids derived from different tissues vary significantly, making cross-referencing difficult. Therefore, there are currently no research reports on DIPG organoids. Consequently, there is an urgent need in this field to construct patient-derived DIPG organoids as disease models for the research and treatment of DIPG.

[0007] Existing technical documents: Patent Document 1: Chinese Patent Application Publication No. CN118126952A. Summary of the Invention

[0008] This invention addresses the aforementioned problems in the prior art, aiming to provide a culture medium and method for constructing a diffuse endophytic pontine glioma (DIPG) organoid model. Using this culture medium and method, patient-derived DIPG organoid models can be constructed. Furthermore, this invention aims to provide applications of this model in various fields, including cancer pathogenesis research, cancer drug screening and development, personalized cancer treatment, and cancer immunotherapy evaluation. In a first aspect of the invention, a culture medium for constructing a diffuse endogenous pontine glioma organoid model is provided, comprising DMEM / F12 medium, Neurobasal medium, L-glutamine substitute, non-essential amino acid supplement, N2 supplement, B-27 supplement, 2-mercaptoethanol, penicillin, streptomycin, and insulin.

[0009] In some preferred embodiments, the ratio of the DMEM / F12 medium to the Neurobasal medium is 2:1 to 1:2.

[0010] In some preferred embodiments, the content of the L-glutamine substitute is 2 to 4 mM.

[0011] In some preferred embodiments, the dosage of the non-essential amino acid supplement, the N2 supplement, and the B-27 supplement is independently 0.5 to 1×.

[0012] In some preferred embodiments, the content of 2-mercaptoethanol is 100–500 μM.

[0013] In some preferred embodiments, the content of penicillin is 50-100 U / mL, and / or the content of streptomycin is 50-100 mg / mL.

[0014] In some preferred embodiments, the insulin content is 1–20 μg / mL.

[0015] In some preferred embodiments, the culture medium does not contain other cytokines.

[0016] In some preferred embodiments, the other cytokines include Wnt-3a.

[0017] In some preferred embodiments, the culture medium comprises the DMEM / F12 medium, the Neurobasal medium, the L-glutamine substitute, the non-essential amino acid supplement, the N2 supplement, the B-27 supplement, the 2-mercaptoethanol, the penicillin, the streptomycin, and the insulin.

[0018] In a second aspect of the invention, a method for constructing an organoid model of diffuse endophytic pontine glioma is provided, comprising the following steps: (1) Collect samples of diffuse engenerative pontine gliomas; (2) Using the culture medium described in the first aspect of the present invention, the diffuse endophytic pontine glioma sample collected in step (1) is cultured into an organoid. (3) Passage and identify the diffuse endophytic pontine glioma organoids obtained in step (2). If the identification is correct and the passage is stable, they can be used as a diffuse endophytic pontine glioma organoid model.

[0019] In some preferred embodiments, as a specific step in collecting diffuse engenerative pontine glioma samples in step (1), during the operation of a patient with diffuse engenerative pontine glioma, the enhancing lesions and cystic areas are avoided, and abnormal signal lesions on the lateral side of the brainstem on T2-FLAIR are selected for puncture. The lesions are removed and immersed in sample preservation solution, which is used as diffuse engenerative pontine glioma samples for step (2).

[0020] In some preferred embodiments, in step (1), the sample preservation solution is pre-cooled, and the lesion is kept under icy conditions until step (2) is started.

[0021] In some preferred embodiments, in step (1), 2 to 5 lesions the size of rice grains are removed by puncture.

[0022] In some preferred embodiments, in step (2), the diffuse endophytic pontine glioma sample is cut into fragments with a diameter of 0.5 to 1 mm, then necrotic or surrounding brain tissue and impurities are removed, and then the culture is performed.

[0023] In some preferred embodiments, in step (2), 75% of the culture medium described in the first aspect of the present invention is replaced every two days for a total of 1 to 2 weeks.

[0024] In some preferred embodiments, in step (2), the culture is carried out at a rotation speed of 100-150 rpm under conditions of 37°C, 5% CO2 and 90% humidity.

[0025] In some preferred embodiments, the passage in step (3) is carried out as follows: the diffuse endophytic pontine glioma organoids obtained in step (2) are cut into fragments with a diameter of 200-500 μm, and the fragments are placed in the culture medium described in the first aspect of the present invention for further culture, so that the fragments regrow to form organoids.

[0026] In some preferred embodiments, the identification in step (3) is performed by hematoxylin-eosin staining and / or immunohistochemical staining.

[0027] In a third aspect of the invention, the application of the culture medium described in the first aspect of the invention is provided in the preparation of a diffuse endogenous pontine glioma organoid model.

[0028] In a fourth aspect of the invention, a diffuse endogenous pontine glioma organoid model prepared by the method described in the second aspect of the invention is provided.

[0029] In a fifth aspect of the invention, the application of the diffuse endogenous pontine glioma organoid model described in the fourth aspect of the invention is provided in the study of cancer pathogenesis, screening and development of cancer therapeutic drugs, personalized cancer treatment, and / or evaluation of cancer immunotherapy.

[0030] This invention overcomes a long-standing technical challenge in the field of glioma organoids, enabling the efficient and stable establishment of diffuse endophytic pontine glioma organoid models. Compared with traditional culture media and methods commonly used in existing technologies, it significantly reduces complexity and operational difficulty, offering excellent convenience and economy, and has broad application prospects. Attached Figure Description

[0031] Figure 1 This is a diagram illustrating the culture process of the DIPG organoid of the present invention.

[0032] Figure 2 This is a diagram illustrating the passage culture process of the DIPG organoids of the present invention.

[0033] Figure 3 This is a diagram showing the results of hematoxylin-eosin staining identification of the DIPG organoids of the present invention.

[0034] Figure 4 This is a figure showing the immunohistochemical staining identification results of the DIPG organoids of the present invention.

[0035] Figure 5 This diagram illustrates the process by which the DIPG organoids of this invention invade brain organoids. In the diagram, red represents DG1 organoids, and blue represents brain organoids.

[0036] Figure 6 This is a figure showing the results of co-culturing DIPG organoids with CAR-T cells according to the present invention. Detailed Implementation

[0037] When a value or range is given, the term “about” as used herein means that the value or range is within 20%, 10%, and 5% of the given value or range.

[0038] The terms “comprising,” “including,” and their equivalents as used herein also include the meanings of “containing” and “composed of,” for example, a composition “containing” X may consist of only X or may contain other substances, such as X+Y.

[0039] In this document, concentrations, contents, percentages, and other values ​​are expressed in range form. It should also be understood that this range form is used for convenience and brevity only, and should be flexibly interpreted to include the values ​​explicitly mentioned at the upper and lower limits of the range, as well as all individual values ​​or subranges included within that range.

[0040] To construct patient-derived DIPG organoid models, this invention provides a culture medium for constructing DIPG organoid models, comprising DMEM / F12 medium, Neurobasal medium, L-glutamine substitute, non-essential amino acid supplement, N2 supplement, B-27 supplement, 2-mercaptoethanol, penicillin, streptomycin, and insulin. The composition of the culture medium of this invention is described in detail below.

[0041] The DMEM / F12 medium described in this invention is a modified version of DMEM and Ham's F-12 medium mixed in a 1:1 ratio. This modified DMEM / F-12 medium is richer in nutrients, contains more trace elements, and is widely used for the culture of various mammalian cells, as well as for the culture of mammalian cells at low serum concentrations and for clonal density culture. Typically, DMEM / F-12 medium can be used to culture MDCK, glial cells, DIPG cells, fibroblasts, human endothelial cells, and mouse fibroblasts.

[0042] The Neurobasal medium described in this invention is a basal culture medium specifically designed for neural cell culture and is widely applicable to most neuronal cell applications. It can maintain the survival of neuronal homologous communities for both long and short periods without the addition of a glial cell feeder layer. Typically, Neurobasal medium can be used for the long-term maintenance and maturation of newly formed and adult brain neurons, as well as for the culture and long-term maintenance of glial-derived cells such as glial cells and DIPG cells.

[0043] The definitions, composition, and usage of the DMEM / F12 medium and the Neurobasal medium are well known to those skilled in the art, and both are readily available as commercially available products. In this invention, the DMEM / F12 medium and the Neurobasal medium can be mixed in any suitable ratio, typically 2:1 to 1:2, preferably 1.5:1 to 1:1.5, and more preferably 1:1.

[0044] The L-glutamine substitute described in this invention is a substitute for L-glutamine. The scientific name of L-glutamine is L-2-aminopentanoic acid, with the molecular formula C5H. 10 N2O3 is a non-essential amino acid and a common culture medium supplement, frequently used in biomanufacturing applications, tissue engineering, hybridoma culture, and other special cell cultures, as well as the culture of common mammalian cells (including nerve cells and glial cells). However, in aqueous solutions such as cell culture media, L-glutamine spontaneously degrades, producing toxic ammonia and pyrrolidine carboxylic acid as byproducts. Therefore, to minimize the cytotoxic effects of L-glutamine degradation, L-glutamine substitutes are commonly used in the art to replace glutamine because they are more stable in aqueous solutions such as cell culture media, do not spontaneously degrade, and are non-cytotoxic. Specific types and uses of L-glutamine substitutes are well known to those skilled in the art and are readily available as commercially available products. In this invention, the content of the L-glutamine substitute is typically 2–4 mM, preferably 2–3 mM, and more preferably about 2 mM. When using commercially available L-glutamine substitutes, they can also be used at a dosage of 1× according to the manufacturer's instructions.

[0045] The non-essential amino acid supplement described in this invention is a mixed solution of seven amino acids, namely L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, glycine, L-proline, and L-serine. It can improve cell growth and activity, effectively improve the cell culture medium ratio, reduce the side effects of cells producing non-essential amino acids during cell culture, and promote cell proliferation and metabolism. It is one of the commonly used additives in cell culture.

[0046] The N2 supplement described in this invention is a serum-free, chemically defined supplement made based on Bottenstein's N2 formulation. It is a serum substitute commonly used in the field of neural cell culture and is suitable for the culture of primary neural cells and neural stem cells, the culture and growth of neuroblastoma and glioma, and the survival and expression of postmitotic neurons in primary cultures of the peripheral nervous system (PNS) and central nervous system (CNS).

[0047] The B-27 supplement described in this invention is an optimized serum-free additive that does not contain vitamin A. It is mainly suitable for short-term or even long-term maintenance culture of neuronal cells. For example, it is often used to support low-density or high-density growth and short-term or long-term survival of embryonic, postnatal and adult hippocampal and other CNS neurons. In addition, it is also commonly used in the culture and growth of brain tumors.

[0048] The usage of non-essential amino acid supplements, N2 supplements, and B-27 supplements is well known to those skilled in the art, and they are all readily available as commercially available products. In this invention, the dosage of the non-essential amino acid supplement, N2 supplement, and B-27 supplement is independently 0.5 to 1×, preferably 1×, according to the manufacturer's instructions.

[0049] The 2-mercaptoethanol described in this invention, also known as β-mercaptoethanol, has the molecular formula C2H6OS. It is a strong reducing organic compound that can neutralize oxygen free radicals accumulated in cell culture media, thereby facilitating in vitro cell proliferation. Therefore, it is frequently added to cell culture media. The use of 2-mercaptoethanol is well known to those skilled in the art and is readily available as a commercially available product. In this invention, the content of 2-mercaptoethanol is typically 100–500 μM, preferably 100–300 μM, and more preferably about 200 μM. When using commercially available 2-mercaptoethanol, it can also be used at a dosage of 1× according to the manufacturer's instructions.

[0050] The penicillin and streptomycin described in this invention are often used in combination in cell culture, commonly referred to as "dual antibiotics," and are the most commonly used antibiotics in in vitro cell culture to prevent microbial contamination. The uses of penicillin and streptomycin are well known to those skilled in the art, and both are readily available as commercially available products. In this invention, the penicillin concentration is typically 50–100 U / mL, preferably about 100 U / mL; the streptomycin concentration is typically 50–100 mg / mL, preferably about 100 mg / mL.

[0051] Insulin, as described in this invention, is a protein hormone secreted by pancreatic β-cells in the pancreas in response to stimulation by endogenous or exogenous substances. It simultaneously promotes the synthesis of glycogen, fat, and protein, playing a crucial role in cellular activity and energy metabolism in mammals. Therefore, insulin has a broad and complex role in cell culture, regulating not only fat and protein metabolism but also promoting cell growth. Insulin mediates its regulatory effect on cellular metabolism through interaction with insulin receptors (IR) on the cell membrane. Simultaneously, insulin binds to insulin-like growth factor receptors (IGF receptors (IG) on the cell membrane, further promoting cell growth. In serum-free cell culture media, the addition of insulin is key to maintaining cell growth, effectively regulating the uptake, utilization, and storage of glucose, amino acids, and fatty acids, while inhibiting the breakdown of glycogen, protein, and fat. However, at least in the culture of brainstem glioma organoids, existing technologies generally consider it unsuitable to add insulin; for example, insulin is not added to the brainstem glioma organoid culture medium described in Patent Document 1.

[0052] This invention overcomes the technical bias present in the prior art. Through inventive effort, it was discovered that, at least in the culture of DIPG organoids—a specific type of brainstem glioma organoid—the formation of organoids is significantly worse without the addition of insulin. Not only does growth become slower, but the shape is also less than ideal, making it difficult to meet the requirements for organoid model construction. Therefore, contrary to the understanding in the prior art, insulin is added as an essential component in the culture medium of this invention, typically at a concentration of 1–20 μg / mL, preferably 1–10 μg / mL, more preferably 2–5 μg / mL, and most preferably about 2.5 μg / mL.

[0053] The source of insulin described in this invention is not particularly limited and can be derived from any mammal, but is preferably derived from humans, i.e., human insulin is preferred.

[0054] Wnt-3a is an important member of the WNT signaling pathway family of proteins, playing a crucial role in maintaining embryonic integrity and tissue development. Wnt-3a expression is primarily concentrated in the dorsal midline structures of the central nervous system, which is significant for the brainstem, located in the midline. Especially for diffuse midline gliomas, a specific subtype of brainstem gliomas, the WNT signaling pathway activated by Wnt-3a plays a vital role. After activation of the WNT signaling pathway, Wnt-3a can inhibit the degradation of β-catenin, suppress GSK3β expression, and thereby activate transcription factor translocation into the nucleus, promoting gene transcription and maintaining cell self-renewal, proliferation, and differentiation. Therefore, at least in the culture of brainstem glioma organoids, existing technologies generally consider the addition of Wnt-3a essential; for example, Wnt-3a is added as a necessary component in the brainstem glioma organoid culture medium described in Patent Document 1.

[0055] This invention overcomes the technical bias present in the prior art. Through inventive effort, it was discovered that, at least in the culture of DIPG organoids—a specific type of brainstem glioma organoid—the addition of Wnt-3a can lead to significant differences in morphology, structure, and function between the culture medium composition and the DIPG tumor microenvironment, potentially rendering the cultured organoids unusable as DIPG tumor models. Therefore, the culture medium of this invention contains no other cytokines besides those that may be naturally present in the aforementioned components, especially not the cytokine Wnt-3a.

[0056] In a preferred embodiment of the present invention, the culture medium of the present invention is composed of the DMEM / F12 culture medium, the Neurobasal culture medium, the L-glutamine substitute, the non-essential amino acid supplement, the N2 supplement, the B-27 supplement, the 2-mercaptoethanol, the penicillin, the streptomycin and the insulin, wherein the content of each component falls within the content range of each component described above.

[0057] Based on the aforementioned inventive work of the inventors, the culture medium of this invention can simulate to the greatest extent the external conditions required for the growth and development of DIPG in the human environment, promoting the growth and differentiation of DIPG organoids, and thus can be well used to construct DIPG organoid models. Therefore, this invention also provides a method for constructing DIPG organoid models, including the following steps: (1) Collect DIPG samples; (2) Using the culture medium described in this invention, the DIPG sample collected in step (1) is cultured into an organoid; (3) Passage and identify the DIPG organoids obtained in step (2). If the identification is correct and they can be stably passaged, they can be used as DIPG organoid models.

[0058] In this invention, there are no particular limitations on the specific steps for collecting DIPG samples in step (1), as long as a sufficient amount of DIPG lesions can be effectively obtained for the culture of DIPG organoids. As an exemplary step, it is preferable to select abnormal signal lesions on the lateral side of the brainstem on T2-FLAIR during the surgical procedure of the DIPG patient, avoiding enhancing lesions and cystic areas, and perform puncture on these lesions. The lesions are then removed and immersed in a sample preservation solution as DIPG samples for use in step (2). Through such an exemplary step, a sufficient amount of puncture biopsy DIPG lesions can be obtained for the culture of DIPG organoids through routine surgical procedures such as puncture, which is more conducive to achieving the purpose of this invention.

[0059] It is worth mentioning that in the medical field, it is generally considered that it is very difficult to culture organoids from puncture biopsy lesions, but the exemplary steps described above in this invention can overcome such difficulties, thereby obtaining DIPG lesions more effectively.

[0060] In step (1) of this invention, the sample preservation solution is preferably pre-cooled, and the lesion is preferably kept under icy conditions until step (2) is initiated. This ensures the freshness and viability of the lesion, which is more conducive to the subsequent culturing of DIPG organoids.

[0061] In step (1) of this invention, it is preferable to remove 2 to 5 lesions the size of a grain of rice by puncture, and more preferably 2 to 3 lesions the size of a grain of rice by puncture. This ensures that a sufficient number of DIPG lesions are obtained for the culture of DIPG organoids.

[0062] In step (2) of this invention, it is preferable to cut the DIPG sample into fragments with a diameter of 0.5 to 1 mm, then remove necrotic or surrounding brain tissue and impurities, and then perform the culture. This ensures the purity of the cell source of the primary cultured DIPG organoids and eliminates interference from other tissues and impurities in the DIPG organoid culture, thereby further facilitating the achievement of the objectives of this invention.

[0063] In step (2) of this invention, it is preferable to replace 75% of the culture medium of this invention every two days for a total of 1 to 2 weeks, and the culture is preferably carried out at a rotation speed of 100 to 150 rpm under conditions of 37°C, 5% CO2, and 90% humidity. The reason for this is that such culture operation method, duration, and conditions are more conducive to the culture of DIPG organoids.

[0064] In step (3) of this invention, the passage is preferably performed as follows: the DIPG organoids obtained in step (2) are cut into fragments with a diameter of 200-500 μm, and the fragments are placed in the culture medium of this invention for further culture, allowing the fragments to regrow and form organoids; the identification is preferably performed by hematoxylin-eosin staining and / or immunohistochemical staining. This is because such passage operation is more conducive to the continuous and stable passage of DIPG organoids in vitro, and such identification operation can more accurately identify DIPG organoids.

[0065] The culture medium and method of this invention can be well used to construct DIPG organoid models. Therefore, this invention also provides the application of the culture medium of this invention in the preparation of DIPG organoid models, as well as DIPG organoid models prepared by the method of this invention. These DIPG organoid models can be well applied to cancer pathogenesis research, cancer drug screening and development, personalized cancer treatment, and / or...

[0066] For example, in cancer pathogenesis research, the DIPG organoids of this invention can simulate the tumor microenvironment, preserving tumor heterogeneity and helping researchers to delve deeper into the pathogenesis, tumor progression, and drug resistance mechanisms of DIPG. In cancer drug screening and development, the DIPG organoids of this invention can be used for high-throughput drug screening to identify potential drug targets and therapeutic compounds. In personalized cancer treatment, this invention, by extracting cells from tumor tissue of DIPG patients and rapidly constructing stable organoid models, can provide patients with a personalized drug testing platform, predicting patient responses to different treatments and thus developing optimal treatment plans. In cancer immunotherapy evaluation, the DIPG organoids of this invention can be used to evaluate the effects of therapies such as CAR-T cell therapy and immune checkpoint inhibitor immunotherapy. In particular, the DIPG organoids of this invention can be co-cultured with CAR-T cells, thereby providing effective assistance for preclinical studies of CAR-T therapy for DIPG. Example

[0067] The technical solutions of the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1: DIPG tumor sample collection Clinical specimens of diffuse engenerative pontine gliomas (DIPG) were obtained from the Department of Neurosurgery at Huashan Hospital, Fudan University, and were surgical specimens provided with informed consent from the patients. This study has been approved by the Ethics Committee of Huashan Hospital, Fudan University.

[0069] Samples were obtained via biopsy during the surgical procedure of DIPG patients. Specifically, during the procedure, avoiding enhancing lesions and cystic areas, abnormal signal lesions on the lateral aspect of the brainstem on T2-FLAIR were selected for biopsy. The lesions were then extracted, immersed in ice-cold Hibernate A (sample preservation solution, Thermo Fisher Scientific, A1247501), and transported to the laboratory on ice as DIPG tumor tissue samples for subsequent steps. A total of 7 DIPG tumor tissue samples were collected.

[0070] Example 2: DIPG tumor tissue samples cultured into organoids ( Figure 1 ) Seven DIPG tumor tissue samples collected in Example 1 were transferred to sterile glass dishes containing Hibernate A supplemented with GlutaMax (an L-glutamine alternative, Gibco, 35050061) and penicillin / streptomycin (Gibco) and dissected under a stereomicroscope in a laminar flow biosafety cabinet. Using fine dissecting scissors, the DIPG tumor tissue samples were cut into fragments approximately 0.5–1 mm in diameter, ensuring the removal of necrotic or surrounding brain tissue. The resulting fragments were then gently rotated in 1×RBC lysis buffer (Gibco, 00-4333-57) for 10 minutes, followed by washing with Hibernate A to obtain processed tumor fragments.

[0071] Subsequently, for histological studies, a portion of the obtained treated tumor fragments was removed and fixed with 4% methanol-free formaldehyde for hematoxylin-eosin staining and immunohistochemical staining. The remaining treated tumor fragments were distributed in ultra-low adhesion 6-well plates (Corning Corporation) containing 4 mL of the organoid culture medium of the present invention. Here, the organoid culture medium of the present invention consists of DMEM / F12 medium (Thermo Fisher Scientific, 10565018), Neurobasal medium (Gibco, 21103049) (used in a 1:1 mixture with DMEM / F12 medium), 1×GlutaMax (L-glutamine alternative, Gibco, 35050061), 1×Non-essential amino acid (NEAA, Gibco, 11140050), 1×PenStrep (a compound antibiotic solution of penicillin and streptomycin, Gibco, 15140122), 1×N2 supplement (Gibco, 17502048), 1×B-27 supplement without vitamin A (Gibco, 17504044), 1×2-mercaptoethanol (Gibco, 21985-023), and 2.5 μg / mL human insulin (Sigma, MSST0064), hereinafter the same. Plates were placed in a sterile incubator at 37°C, 5% CO2, and 90% humidity on an orbital shaker rotating at 120 rpm. Approximately 75% of the culture medium was replaced every two days. After 1–2 weeks of culture, all treated tumor fragments developed into spherical organoids.

[0072] Ultimately, all seven DIPG tumor tissue samples obtained in Example 1 were successfully cultured into organoids. The culture process of these DIPG organoids is shown in [illustration]. Figure 1 .

[0073] Example 3: Passaging of DIPG organoids ( Figure 2 ) For DIPG organoids with a long culture time (more than one month), use fine dissecting scissors to cut them into organoid fragments with a diameter of about 200 to 500 μm. Place the obtained organoid fragments in an ultra-low adhesion 6-well plate (Corning) containing 4 mL of the organoid culture medium of the present invention for further culture. The organoid fragments will continue to grow and reform organoids.

[0074] Ultimately, all seven DIPG organoids obtained in Example 2 were successfully passaged and reformed into organoids. The passage culture process of these DIPG organoids is shown in [Figure / Lesson Name]. Figure 2 .

[0075] Example 4: Identification of DIPG organoids ( Figure 3 , Figure 4 ) The DIPG organoids fixed in Example 2 were resuspended in 100 μL of preheated 3% agarose and placed on ice to solidify. The solidified agarose block containing the DIPG organoids was removed, dehydrated with fractionated ethanol, embedded in a paraffin block, and then cut into 5 μm thick cross-sections.

[0076] To perform hematoxylin-eosin staining, the slides were stained with hematoxylin and eosin for 15 minutes and 1 minute respectively, dehydrated with graded ethanol, cleaned with xylene, and then observed under a microscope.

[0077] For immunohistochemical staining, slides were antigen-retrieved in sodium citrate buffer and infiltrated with 0.5% Triton X-100. After blocking with 10% normal goat / horse serum for one hour at room temperature (RT), tissue sections were incubated overnight at 4°C with primary antibodies Ki-67 and H3K27M. They were then incubated with secondary antibodies for one hour at room temperature. DAB staining solution was added to cover the tissue, followed by hematoxylin staining solution. The sections were incubated at room temperature for 5 minutes and rinsed with tap water until blue reappeared. Finally, the slides were mounted with a mounting medium including glycerol and gelatin and observed under a microscope.

[0078] Finally, the seven DIPG organoids obtained in Example 2 were all identified as highly histologically similar to DIPGs after hematoxylin-eosin staining and immunohistochemical staining. Representative identification results of these DIPG organoids using hematoxylin-eosin staining are shown in [Figure / Table / Insert Table ... Figure 3 Representative identification results of immunohistochemical staining are shown in Figure 4 .

[0079] Example 5: DIPG organoids invade brain organoids ( Figure 5 ) Human embryonic stem cells (hESCs) were dissociated into single cells using trypsin (Gibco, C25300054). Subsequently, 1×10⁻⁶ cells were...4 Cells / well were seeded in ultra-low attachment 96-well plates (Corning) and cultured in hESC medium supplemented with rock inhibitor (50 μM, InvivoChem, V2558) and FGF2 (4 ng / ml, SinoBiological, GMP-10014-HNAE) to promote embryoid formation. On day 6, embryoids were transferred to ultra-low attachment 6-well plates (Corning) containing neural induction medium, a 1:1 mixture of DMEM / F-12 medium (Gibco) and Neurobasal medium (Gibco), supplemented with 0.5×N2 supplement, 0.5×B-27 supplement, 5 μg / mL insulin, 2 mM L-glutamine, 0.5×NEAA, 100 μM 2-mercaptoethanol, 50 U / mL penicillin, and 50 mg / mL streptomycin. On day 11, embryoids were embedded in Matrigel (Corning, 356231) and cultured in 10 cm culture dishes with brain organoid culture medium (B-27 supplemented medium without vitamin A) to form neuroepithelium. On day 15, the brain organoids were transferred to a shaker at 85 rpm. Here, the brain organoid culture medium consisted of a 1:1 mixture of medium supplemented with N2 and medium supplemented with B27 (N2-containing medium: DMEM / F12 medium, 1×GlutaMax, 1×N2 supplement, 5 μg / mL insulin, 1 mM L-glutamine, 1×non-essential amino acids, 100 μM 2-mercaptoethanol; B27-containing medium: Neurobasal medium, 1×B-27 supplement, 200 mM L-glutamine), supplemented with 50 U / mL penicillin and 50 mg / mL streptomycin. The culture medium was changed every two days.

[0080] Cultured brain organoids were labeled with red live fluorescent dye (YEASEN, 40717ES50), while DIPG organoids were labeled with blue live fluorescent dye (YEASEN, 40761ES50). Subsequently, both types of organoids were co-cultured in 96-well U-shaped low-adhesion plates in the DIPG organoid culture medium of this invention. The samples were imaged using a fluorescence microscope, and the imaging results are shown below. Figure 5 .

[0081] Depend on Figure 5 It is known that the DIPG organoid culture medium of the present invention and the DIPG organoids prepared by the method of the present invention can effectively invade brain organoids and exhibit functions highly similar to DIPG, thus making them suitable for the study of the pathogenesis of DIPG.

[0082] Example 6: Co-culture of organoids and CAR-T ( Figure 6 ) In collaboration with Fuzhou Tuoxin Tiancheng Biotechnology Co., Ltd., we produced B7-H3 CAR-T cells for patients.

[0083] Approximately 5×10 4 One CAR-T cell was seeded in each well of a 96-well plate (Corning Corporation) and co-cultured with one DIPG organoid obtained in Example 2 per well. Approximately half of the culture medium was exchanged daily by tilting the plate at 45° and aspirating the medium. An apoptosis-inhibiting staining dye (Yeasen Corporation, Y00001982) was added to the culture medium, and images were captured under a fluorescence microscope. The resulting images are shown below. Figure 6 .

[0084] Depend on Figure 6 It is understood that the DIPG organoid culture medium of the present invention and the DIPG organoids prepared by the method of the present invention can be used to rapidly test the response to CAR-T cell immunotherapy, and are therefore suitable for evaluating the efficacy of CAR-T cell immunotherapy.

[0085] Comparative Example 1: Similar to Example 2, except without the addition of human insulin, an attempt was made to culture the DIPG tumor tissue samples collected in Example 1 into organoids. However, the organoid formation was significantly poor; it grew slowly and had an undesirable shape, ultimately failing to successfully culture organoids that met the requirements for organoid model construction.

[0086] Comparative Example 2: Similar to Example 2, except for the addition of the cytokine Wnt-3a, an attempt was made to culture the DIPG tumor tissue samples collected in Example 1 into organoids. However, the cultured organoids differed significantly in morphology from the primary tumors and could not be used as a DIPG tumor model at all. It should be noted that the combination of the technical features in this invention is not limited to the combination methods described in the claims of this invention or the combination methods described in the specific embodiments. All technical features described in this invention can be freely combined or combined in any way, unless they contradict each other.

[0087] It should also be noted that the embodiments listed above are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and similar changes or modifications made thereto are those that can be directly derived or easily conceived by those skilled in the art from the content disclosed in the present invention, and should all fall within the protection scope of the present invention.

Claims

1. A culture medium for constructing an organoid model of diffuse endophytic pontine glioma, comprising DMEM / F12 medium, Neurobasal medium, L-glutamine substitute, non-essential amino acid supplement, N2 supplement, B-27 supplement, 2-mercaptoethanol, penicillin, streptomycin and insulin.

2. The culture medium as described in claim 1, wherein, The ratio of the DMEM / F12 medium to the Neurobasal medium is 2:1 to 1:

2.

3. The culture medium as described in claim 1, wherein, The content of the L-glutamine substitute is 2 to 4 mM.

4. The culture medium as described in claim 1, wherein, The dosage of the non-essential amino acid supplement, the N2 supplement, and the B-27 supplement is independently 0.5 to 1×.

5. The culture medium as described in claim 1, wherein, The content of the 2-mercaptoethanol is 100–500 μM.

6. The culture medium as described in claim 1, wherein, The content of the penicillin is 50-100 U / mL, and / or the content of the streptomycin is 50-100 mg / mL.

7. The culture medium as described in claim 1, wherein, The insulin content is 1–20 μg / mL.

8. The culture medium as described in claim 1, wherein, The culture medium does not contain other cytokines.

9. The culture medium as described in claim 8, wherein, The other cytokines mentioned include Wnt-3a.

10. The culture medium according to any one of claims 1 to 9, comprising the DMEM / F12 medium, the Neurobasal medium, the L-glutamine substitute, the non-essential amino acid supplement, the N2 supplement, the B-27 supplement, the 2-mercaptoethanol, the penicillin, the streptomycin, and the insulin.

11. A method for constructing an organoid model of diffuse endophytic pontine glioma, comprising the following steps: (1) Collect samples of diffuse engenerative pontine gliomas; (2) Using the culture medium of any one of claims 1 to 10, the diffuse endophytic pontine glioma sample collected in step (1) is cultured into an organoid; (3) Passage and identify the diffuse endophytic pontine glioma organoids obtained in step (2). If the identification is correct and the passage is stable, they can be used as a diffuse endophytic pontine glioma organoid model.

12. The method of claim 11, wherein, As a specific step in collecting diffuse engenerative pontine glioma samples in step (1), during the surgery of patients with diffuse engenerative pontine glioma, the enhancing lesions and cystic areas are avoided, and abnormal signal lesions on the lateral side of the brainstem on T2-FLAIR are selected for puncture. The lesions are removed and immersed in the sample preservation solution, which is used as diffuse engenerative pontine glioma samples for step (2).

13. The method of claim 12, wherein, In step (1), the sample preservation solution is pre-cooled, and the lesion is kept under icy conditions until step (2) is started.

14. The method of claim 12, wherein, In step (1), 2 to 5 lesions the size of rice grains are removed by puncture.

15. The method of claim 11, wherein, In step (2), the diffuse endophytic pontine glioma sample is cut into fragments with a diameter of 0.5 to 1 mm, then necrotic or surrounding brain tissue and impurities are removed, and then the culture is performed.

16. The method of claim 11, wherein, In step (2), 75% of the culture medium according to any one of claims 1 to 10 is replaced every two days for a total of 1 to 2 weeks.

17. The method of claim 11, wherein, In step (2), the culture is carried out at a rotation speed of 100-150 rpm under conditions of 37°C, 5% CO2 and 90% humidity.

18. The method of claim 11, wherein, The passage in step (3) is carried out as follows: the diffuse endophytic pontine glioma organoids obtained in step (2) are cut into fragments with a diameter of 200-500 μm, and the fragments are placed in the culture medium of any one of claims 1-10 for further culture, so that the fragments regrow to form organoids.

19. The method of claim 11, wherein, The identification in step (3) is performed by hematoxylin-eosin staining and / or immunohistochemical staining.

20. The use of the culture medium according to any one of claims 1 to 10 in the preparation of an organoid model of diffuse endogenous pontine glioma.

21. A diffuse endophytic pontine glioma organoid model prepared by the method of any one of claims 11 to 19.

22. The application of the diffuse endophytic pontine glioma organoid model as described in claim 21 in cancer pathogenesis research, cancer drug screening and development, personalized cancer treatment, and / or cancer immunotherapy evaluation.

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