Method for constructing brain glioma organoid

Abstract

The invention relates to the technical field of brain glioma organoid construction, in particular to a method for constructing a brain glioma organoid, which comprises the following steps: step S1, mixing a brain glioma sample and primary brain microvascular endothelial cells according to a cell number ratio of 1: 5, inoculating into a porous structure of a stent after mixing, and singly inoculating the primary brain microvascular endothelial cells into a hollow vascular channel of the stent; s2, the stent in the step S1 is placed in a gas-liquid interface culture device to culture a brain glioma sample, the gas-liquid interface culture device comprises a culture medium, the bottom of the stent is immersed in the culture medium, the top of the stent is exposed to a mixed gas environment, and the brain glioma sample is cultured through the design of the composite stent containing the porous proliferation area and the hollow blood vessel channel. The problems of low medicine permeation efficiency and limited tumor-blood vessel interaction research caused by blood vessel deficiency of a traditional model are solved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organoids, and particularly relates to a method for constructing a brain glioma organoid. BACKGROUND

[0002] An organoid is a three-dimensional cell culture which has highly similar histological characteristics to a corresponding human organ and can reproduce part of the physiological function of the organ in vitro. The emergence of organoids provides researchers with a new in vitro model with unique advantages, which can replicate the complex spatial morphology of differentiated tissues and exhibit the interaction between cells and cells and between cells and matrix. Organoids remove the confusion variables that may be introduced by animal models, but provide higher complexity compared with homogenized 2D cell culture. By combining high levels of physiological relevance with the convenience of in vitro manipulation, organoids have the potential to assist or replace in vitro use of primary cells or immortalized cell lines and animal experiments in many cases. In addition, organoids have high genetic stability in culture, maintaining the genotype and phenotype of the source tissue. Therefore, organoids have wide application prospects in the fields of stem cells and development, regenerative medicine, disease research, drug development and precision medicine.

[0003] Glioma is the most common primary intracranial tumor. In order to better study the pathogenesis and treatment of glioma, scientists construct glioma organoids. The existing brain glioma organoid construction technology has many key defects, which restrict its clinical application value: the static culture mode is difficult to induce the formation of functional blood vessel-like structures, the absence of vascular network not only leads to low drug penetration efficiency, but also cannot simulate the regulation of blood flow shear force on the phenotype of vascular endothelial cells in vivo, which limits the study of tumor-vascular interaction; traditional hydrogel scaffolds lack environmental response ability and cannot dynamically adjust growth factor release according to the acidic characteristics of the tumor microenvironment, resulting in structural necrosis in the core area of the organoid due to nutrient deficiency and metabolic waste accumulation, affecting the integrity of the model; the survival state of immune cells in the traditional co-culture system is poor, especially the immune cells with anti-tumor activity are difficult to maintain functional stability, resulting in a lack of reliable model support for the evaluation of the efficacy of new therapies such as immune checkpoint inhibitors. Lack of real-time monitoring means for the metabolic state of the organoid, the culture parameters cannot be dynamically adjusted according to the physiological needs of the cells, resulting in unstable formation efficiency and poor batch consistency of the organoid. Therefore, a method for constructing a brain glioma organoid is improved and designed. SUMMARY

[0004] In view of the above problems in the prior art, the application provides a method for constructing a brain glioma organoid to solve the problems in the background art.

[0005] In order to solve the above technical problems, the application adopts the following technical solutions: A method for constructing a brain glioma organoid, comprising the steps of, Step S1, mixing brain glioma sample with primary brain microvascular endothelial cells at a cell number ratio of 1:5, and inoculating into the porous structure of the scaffold after mixing, and inoculating the primary brain microvascular endothelial cells into the hollow vascular channel of the scaffold alone; Step S2, placing the scaffold in step S1 into a gas-liquid interface culture device to culture the brain glioma sample, the gas-liquid interface culture device comprises a culture medium, the bottom of the scaffold is immersed in the culture medium, the top of the scaffold is exposed to a mixed gas environment, and a microfluidic pump circulates the culture medium at a flow rate of 0.3-0.5mL / min, and applies a fluid shear force of 1-1.5dyn / cm² to the surface of the scaffold. The scaffold area is irradiated with blue light with a wavelength of 470nm and an intensity of 5mW / cm 2 for 2 hours every day.

[0006] Further, the culture medium comprises a basic medium and an additive, the basic medium is a mixed system of DMEM / F12 and Neurobasal at a volume ratio of 3:2, and the additive comprises, in terms of final concentration: 1×B-27 additive, 1×N-2 additive, 15ng / mL epidermal growth factor, 15ng / mL fibroblast growth factor, 100ng / mL vascular endothelial growth factor, 20ng / mL hepatocyte growth factor, 50μM deferoxamine, 2mM pyruvic acid and 100μM vitamin C.

[0007] Further, the preparation method of the scaffold comprises: mixing 8%w / v methacrylated gelatin, 3%w / v hyaluronic acid and 2%w / v silk fibroin, adding 0.1%w / v carbon nanotubes and 50μM RGD peptide, constructing a porous scaffold with a pore size of 100-150μm by laser-assisted bioprinting technology, and reserving a hollow vascular channel with a diameter of 200μm in the scaffold.

[0008] Further, the culture medium further comprises 1mM 2-hydroxyglutarate.

[0009] Further, the mixed gas environment is a mixed gas environment containing 5% CO2 and 1-5% O2.

[0010] Further, the oxygen concentration of the mixed gas environment can be adjusted: 5% O2 for culturing IDH mutant brain glioma organoids, and 1% O2 for culturing glioblastoma organoids.

[0011] Further, the culture conditions comprise monitoring the glucose concentration and lactic acid concentration in the culture medium every 6 hours, supplementing DFO to 75μM in the culture medium when the glucose is <0.8g / L, and starting 165rpm pulse oscillation of the culture device when the lactic acid is >1.8g / L.

[0012] Further, the conditions of the culture further comprise; wherein the medium replacement mode is to supplement 50% new medium into the culture device every 3 days.

[0013] Further, it further comprises pretreatment of the brain glioma sample; mechanically shearing the surgically resected brain glioma tissue into fragments with a diameter of 0.3-0.5 mm, washing with PBS buffer containing penicillin-streptomycin for 3 times, centrifuging at 1000 rpm for 5 minutes to remove necrotic tissue and impurities, and obtaining the brain glioma sample.

[0014] Further, when constructing the glioblastoma organoids, 1 μM Erlotinib and 5 μg / mL cholesterol are further added to the culture medium, and a periodic pressure of 0.1 kPa is applied to the scaffold and the surrounding medium every day during culture, with a frequency of 0.1 Hz.

[0015] Compared with the prior art, the present application has the following beneficial effects: 1. By designing a composite scaffold containing a porous proliferation area and a hollow blood vessel channel, combined with a 1:5 ratio of tumor cell-endothelial cell co-culture system, the three-dimensional structure of "tumor cells surrounding blood vessel branches" is reproduced, and functional blood vessel networks are directionally formed through the hollow channel, solving the problems of low drug penetration efficiency and limited tumor-blood vessel interaction research caused by the absence of blood vessels in traditional models; 2. Establish a "real-time monitoring-dynamic intervention" metabolic management system, effectively solve the problem of lactic acid accumulation (lactic acid concentration is controlled below 1.8 g / L) and core necrosis caused by high glycolysis of tumors by monitoring glucose / lactic acid concentration every 6 hours, combined with DFO supplement (75 μM) and 165 rpm pulse oscillation, and the survival rate of long-term culture (4 weeks) of organoids is improved; 3. The porous scaffold (pore diameter 100-150 μm) constructed by laser printing, combined with GelMA / hyaluronic acid / fibroin composite matrix and RGD peptide modification, improves cell adhesion rate; carbon nanotube addition enhances mechanical strength and signal transduction, so that the invasiveness phenotype (MMPs expression) of the organoids is similar to that of the in vivo tumor. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The flowchart of the method for constructing a brain glioma organoid of the present application. DETAILED DESCRIPTION

[0017] In order for those skilled in the art to better understand the present application, the technical solutions of the present application are further described below in conjunction with the drawings and examples.

[0018] In the field of brain glioma organoid model construction, the prior art has many key limitations: traditional models mostly use single-structure scaffolds, which are difficult to form functional vascular networks and cannot truly reproduce the interactive microenvironment of tumors and blood vessels; in static culture mode, the gas (such as oxygen partial pressure), nutrient gradient and physiological state deviate significantly from the in-vivo state, which easily leads to abnormal cell phenotype differentiation; and restricts experimental repeatability and clinical translation value.

[0019] The present application realizes high bionics of brain glioma organoids in structure, function, metabolism and drug response through multi-faceted technology integration, and provides a reliable model for tumor mechanism research and precise drug testing.

[0020] The present application provides a method for constructing a brain glioma organoid, comprising the following steps, Step S1, mixing brain glioma samples with primary brain microvascular endothelial cells at a cell number ratio of 1:5, and then inoculating into the multi-well structure of the scaffold, wherein the hollow blood vessel channel of the scaffold is inoculated with primary brain microvascular endothelial cells alone; the density of the primary brain microvascular endothelial cells is 2x10 6 cells / mL, 2x10 6 "2x10 6 cells / mL" refers to that when inoculating primary brain microvascular endothelial cells in the hollow blood vessel channel, there are 2 million endothelial cells per milliliter of cell suspension, and the "hollow blood vessel channel" is a tubular structure with a diameter of 200 μm preset in the scaffold, which simulates the physical spatial morphology of in-vivo brain microvessels. This density can ensure that the endothelial cells form a continuous monolayer covering on the inner wall of the channel (cell spacing is about 10-15 μm), avoiding the leakage of the blood vessel wall gap due to insufficient density (such as <1x10 6 cells / mL), and simulating the physical barrier properties of the blood-brain barrier.

[0021] Step S2, culturing the brain glioma samples in the scaffold in step S1 in a gas-liquid interface culture device, wherein the gas-liquid interface culture device comprises a culture medium, the bottom of the scaffold is immersed in the culture medium, the top of the scaffold is exposed to a mixed gas environment, a microfluidic pump circulates the culture medium at a flow rate of 0.3-0.5 mL / min, applies a fluid shear force of 1-1.5 dyn / cm2 to the surface of the scaffold, and irradiates the scaffold area with blue light with a wavelength of 470 nm and an intensity of 5 mW / cm2 for 2 hours every day.

[0022] The tumor-vascular interaction is reproduced by designing a composite scaffold containing porous proliferation zone and hollow vascular channel, realizing tumor cell-endothelial cell mixed culture and directional vascular formation, respectively; brain glioma cells and primary brain microvascular endothelial cells are co-cultured at a ratio of 1:5, the biological correlation of tumor heterogeneity and vascular functional unit is retained; the physical signals such as air-liquid interface culture (30% immersion), low oxygen-CO2 gas environment (1-5% O2+5% CO2), periodic pressure (0.1 kPa / 0.1 Hz) and pulse oscillation are integrated to simulate the in-vivo mechanical and gas microenvironment; in particular, the slow circulation can make the culture medium flow uniformly through the scaffold and the surface of the organoid, just like the blood slowly flows through the blood vessels in the human body, continuously delivering nutrients (glucose, growth factors, etc.) to the cells, while taking away the metabolic waste (such as lactic acid), avoiding the "hunger" or "waste accumulation injury" of the cells; the shear force can "stimulate" the vascular endothelial cells in the scaffold to arrange into a tubular shape (similar to the structure of real blood vessels), while promoting signal transmission between cells, making the vascular network of the organoid closer to the real state in the body, and also allowing nutrients to penetrate more efficiently into the organoid.

[0023] Porous structure mixed inoculation (1:5 cell ratio): In the brain glioma microenvironment in vivo, about 5 vascular endothelial cells (including pericytes, endothelial cells, etc.) are accompanied around every 1 tumor cell cluster. The 1:5 ratio precisely matches this physiological feature. This ratio can not only ensure the dominant position of tumor cells (maintain malignant proliferation characteristics), but also provide sufficient nutritional support and signal regulation (such as endothelial cell secretion of PDGF to promote tumor cell invasion) through endothelial cells. The porous region of the scaffold (pore size 50-200 μm) provides a three-dimensional proliferation space for mixed cells, simulating the structure of "tumor cells surrounding blood vessel branches" in tumor tissue. Tumor cells and endothelial cells are in close contact, and signal interaction can be achieved through paracrine: tumor cells secrete VEGF, bFGF, and other pro-angiogenic factors to stimulate endothelial cell proliferation and migration; endothelial cells secrete collagen, laminin, and other extracellular matrix through the basement membrane to maintain the stemness characteristics of tumor cells. Hollow vascular channel inoculation alone: construction of a functional vascular "transport trunk" and the necessity of directional vascular network: hollow vascular channels (diameter 200-500 μm) simulate the tubular structure of brain microvessels in vivo. Inoculation of endothelial cells alone can avoid the interference of uncontrolled tumor cell proliferation on vascular formation, ensuring that endothelial cells are arranged in a directional manner along the inner wall of the channel and fuse to form a continuous tubular structure (similar to the "endothelial monolayer barrier" of blood vessels in vivo). This channel serves as a "trunk" for nutrient transport and forms a hierarchical network with the "branching blood vessels" of the porous region: endothelial cells in the channel form a barrier through tight junctions, simulating the selective permeability function of the blood-brain barrier; nutrients (glucose, oxygen) in the blood / culture medium are transported through the channel and then diffuse to tumor cells through the branching blood vessels of the porous region, reproducing the material exchange path of "blood vessels-tumor" in vivo. Avoiding the blood vessel structure disorder caused by "single mixed inoculation" (tumor cells overproliferate and squeeze the blood vessel lumen); overcoming the problem of "simple blood vessel channel" lacking tumor-endothelial direct interaction (unable to simulate paracrine signal regulation). Through the combination of "local interaction in porous region + functional transport in channel region", the organoid simultaneously possesses tumor malignant phenotype (such as invasiveness) and physiological microenvironment characteristics (such as vascular nutrient supply), providing a more realistic model for studying tumor angiogenesis mechanisms and anti-angiogenic drug screening. Specifically, the scaffold (a three-dimensional structure carrying tumor cells and vascular cells) is placed in a specially designed culture device (such as a culture plate with a porous membrane). The bottom of the scaffold is about 30% immersed in the culture medium, while the top of the scaffold is exposed to the gas environment (containing 5% CO2 and a specific concentration of oxygen) in the culture device. The scaffold obtains liquid nutrients and gas at the same time through this "half-immersed and half-exposed" state.

[0024] This "30% immersion" ratio is to balance the two major needs of cells: the scaffold bottom contacts the culture medium, and nutrients such as glucose, growth factors, etc. are transported to the cells in the scaffold (especially deep cells) through liquid diffusion, avoiding "nutrient deficiency" caused by complete non-contact with liquid. Gas exchange: the top of the scaffold is exposed to a gas environment, and cells can obtain oxygen by directly contacting the gas (the oxygen concentration in the tumor microenvironment is usually lower than that in normal tissues, which is simulated here by controlling the gas environment), while CO 2 is discharged to avoid "oxygen deficiency" or "gas accumulation" caused by complete immersion in liquid. Avoid core necrosis: if the scaffold immersion ratio is too high (such as more than 50%), the top cells will appear hypoxic necrosis due to insufficient oxygen; if the immersion ratio is too low (such as less than 20%), the bottom cells will wither due to insufficient nutrient supply. The 30% ratio can make the bottom cells focus on absorbing nutrients and the top cells focus on obtaining oxygen, reducing the core necrosis rate of the organoid (experimental data show that it can be reduced to less than 5%). Simulate the "gradient microenvironment" of the in vivo tumor: in vivo tumor tissue, the area close to the blood vessels is rich in nutrients and oxygen, and the area away from the blood vessels is relatively scarce (forming a "nutrient gradient" and "oxygen gradient"). The partial immersion state of the scaffold can reproduce this gradient in vitro, inducing cells to exhibit similar in vivo distribution characteristics (such as tumor cells proliferating in nutrient-rich areas and vascular cells extending to oxygen-rich areas). Promote cell functional differentiation: vascular endothelial cells are more susceptible to shear force and gas signal stimulation at the "gas-liquid interface", and are more likely to arrange into a tubular structure (similar to real blood vessels); tumor cells are more likely to express invasion-related proteins (such as MMPs) in this gradient environment, simulating the invasion characteristics of in vivo tumors. By controlling the contact ratio of the scaffold with liquid and gas, cells can obtain sufficient nutrients and suitable gas, avoiding "nutrient deficiency" or "oxygen deficiency", and reproducing the in vivo gradient environment, ultimately promoting the formation of a three-dimensional model with complete structure and functional tumor-like functions. This step cooperates with subsequent microfluidic circulation, shear force regulation, etc. to optimize the culture conditions.

[0025] In some embodiments, the scaffold region is irradiated with blue light at a wavelength of 470 nm and an intensity of 5 mW / cm2for 2 hours per day. In addition to activating the light-sensitive channel protein in the cells, the blue light irradiation assists in culturing the organoids by regulating calcium signaling in the cells, enhancing intercellular communication, and further simulating the dynamic characteristics of the tumor microenvironment in vivo. The blue light activates the light-sensitive channel protein (e.g., ChR2) to cause a transient increase in the concentration of intracellular calcium ions (Ca2+). This calcium signal is not limited to a single cell, but is also transmitted to surrounding cells through gap junctions to form a "calcium wave." For brain glioma organoids, this cross-cell signal transmission can: promote the functional coupling of tumor cells and vascular endothelial cells, enhance the response of the vascular network to the metabolic demand of the tumor (e.g., upregulate VEGF expression and optimize vascular permeability); simulate the "cooperative signal" during the invasion of tumor cell clusters in vivo, and induce the edge cells of the organoids to exhibit a more invasive phenotype (e.g., cytoskeletal reorganization and increased secretion of matrix metalloproteinases). In addition, moderate-intensity blue light (5 mW / cm2) can regulate cell metabolism through non-light-sensitive channel pathways: stimulate mitochondrial function: blue light can slightly activate mitochondrial respiratory chain-related enzymes (e.g., cytochrome c oxidase), improve oxidative phosphorylation efficiency, increase ATP production, and alleviate "energy stress" caused by high metabolism in tumor cells; regulate glycolysis balance: inhibit overactive glycolytic enzymes (e.g., hexokinase) through calcium signaling downstream pathways, reduce excessive accumulation of lactic acid, and assist in maintaining stable pH in the culture medium (in cooperation with the metabolic monitoring system). Blue light irradiation has a significant directional regulatory effect on vascular endothelial cells in the scaffold: induces endothelial cell polarization: calcium signaling triggered by blue light can promote the reorganization of cytoskeletal proteins (e.g., actin) in endothelial cells, causing them to arrange in an orderly manner along the direction of blue light irradiation (or the direction of fluid shear force), thereby accelerating the maturation of tubular blood vessel structures; overexpresses tight junction proteins (e.g., occludin and claudin-5) to reduce abnormal vascular permeability and reduce nutrient leakage in the core of the organoid, while simulating the characteristics of "abnormal tumor blood vessels but with functional barriers" in vivo. Brain gliomas often infiltrate neural tissue, and blue light irradiation can indirectly simulate the regulation of tumor cells by neural electrical signals: activates the paracrine function of neural crest cells: neural crest cells co-cultured in the scaffold can secrete neurotrophic factors (e.g., BDNF and NGF) after being stimulated by blue light, promoting the maintenance of tumor cell stemness (increasing the proportion of CD133+ cells by 10-15%); regulates the expression of invasion-related genes (e.g., MMP-2 and Twist) through the activity of calcium signal-mediated epigenetic modification enzymes (e.g., histone deacetylases), making the molecular characteristics of the organoids more similar to those of the primary tumor.Low intensity blue light (5mW / cm²) avoids the cytotoxicity of high intensity light, instead protects cells by the following ways: activating antioxidant pathways: inducing the expression of intracellular glutathione peroxidase, superoxide dismutase (SOD), reducing the damage of reactive oxygen species (ROS) to cell DNA; by up-regulating the expression of anti-apoptotic proteins (such as Bcl-2), reducing the apoptosis of marginal cells due to the difference in nutrient gradient in long-term culture, and improving the overall survival rate of organoids (15%-20% higher than the no blue light group). Blue light irradiation in organoid culture is a "multi-target regulation tool": through multiple mechanisms such as calcium signaling, metabolic regulation, vascular maturation and neural interaction simulation, it helps organoids form structure and function characteristics closer to the real state in vivo. These effects form a synergy with medium nutrition supply, fluid shear force regulation, etc., to improve the bionics and stability of organoids.

[0026] In the present application, the culture medium comprises a basic medium and an additive, the basic medium is a DMEM / F12 and Neurobasal mixed system with a volume ratio of 3:2 (containing glucose 4.5g / L, L-glutamine 2mM); the additive comprises, by final concentration: 1×B-27 additive (without vitamin A), 1×N-2 additive, 15ng / mL epidermal growth factor (EGF), 15ng / mL fibroblast growth factor (FGF), 100ng / mL vascular endothelial growth factor (VEGF), 20ng / mL hepatocyte growth factor (HGF), 50μM deferoxamine (DFO), 2mM pyruvic acid and 100μM vitamin C.

[0027] It is explained that DMEM / F12 and Neurobasal are mixed in a volume ratio of 3:2, the core role of DMEM / F12: contains high concentration of glucose (4.5 g / L), matches the metabolic characteristics of "high glycolysis" of brain glioma cells (tumor cell glucose consumption rate is 2-3 times that of normal cells), while providing rich amino acids and vitamins to support rapid proliferation; the supplementary value of Neurobasal: as a special medium for neural cells, its characteristics of low osmotic pressure and high glutamine stability can maintain the vascular barrier function of brain microvascular endothelial cells (avoid morphological disorder of endothelial cells due to unsuitable environment). Glucose (4.5 g / L): higher than the normal tissue fluid glucose concentration (1-1.5 g / L), simulates the characteristics of "high nutrient supply but rapid consumption" of tumor microenvironment, provides initial reserve for continuous monitoring of glucose metabolism dynamics (0.8-1.2 g / L maintenance target as described above); L-glutamine (2 mM): as a key raw material for cell synthesis of proteins and nucleic acids, it also participates in energy metabolism, meeting the material synthesis needs of tumor cell high proliferation and endothelial cell angiogenesis; pyruvic acid (2 mM): as a glycolysis intermediate, it can replace energy supply when glucose concentration fluctuates, maintain cell metabolic homeostasis, especially when lactic acid accumulation leads to microenvironment acidification, reduce cell energy stress damage. 1×B-27 (without vitamin A): removing vitamin A can avoid its induction of tumor cell differentiation, retain the characteristics of glioma stem cells, and at the same time provide neurotrophic support for neural-derived endothelial cells; 1×N-2 additive: contains insulin, transferrin and other components, enhances the cell's ability to uptake nutrients, and alleviates the "nutrient competition" pressure of tumor microenvironment. EGF (15 ng / mL) + FGF (15 ng / mL): synergistically activate the EGFR / FGF signaling pathway of tumor cells, simulate the proliferation driving mechanism of "autocrine growth factors" of in vivo tumors, maintain the malignant proliferation activity of organoids; VEGF (100 ng / mL) + HGF (20 ng / mL): VEGF specifically promotes endothelial cell proliferation and migration, and HGF enhances the stability of endothelial cell tubular structure, the combination of the two makes the blood vessel passage rate increase by 40%, solving the problem of lack of functional blood vessels in simple tumor cell culture. DFO (50 μM): by chelating free iron ions, inhibiting iron-dependent reactive oxygen species generation, reducing DNA damage when tumor cells are in high metabolic oxidative stress; Vitamin C (100 μM): as an antioxidant to scavenge free radicals, while promoting collagen synthesis, enhancing the strength of endothelial cell interconnection, and maintaining the vascular barrier function.

[0028] In some implementable ways, the preparation method of the scaffold comprises: mixing 8% w / v methacrylated gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin, adding 0.1% w / v carbon nanotubes and 50 μM RGD peptide, and constructing a porous scaffold with a pore size of 100-150 μm by laser-assisted bioprinting technology, and the scaffold is reserved with a hollow blood vessel channel with a diameter of 200 μm.

[0029] It is explained that the material ratio, additive selection and structure parameter design of the scaffold are the results of precise optimization based on the biological characteristics of the brain glioma microenvironment (such as extracellular matrix composition, vascular dependence, and mechanical sensing requirement), and the specific explanations are as follows: 8% w / v methacrylated gelatin (GelMA): as the core matrix, GelMA retains the natural cell adhesion sites (such as RGD sequence) of gelatin through methacrylation modification, and has the characteristics of photo-crosslinking, which can be precisely solidified by laser printing to provide a stable three-dimensional support structure. The concentration of 8% is the optimal value for balancing the “structural strength” and “degradability” - lower than 5% is easy to cause the collapse of the scaffold due to insufficient mechanics, and higher than 10% is too slow to degrade, which hinders the growth and expansion of the organoid in the later stage, and its degradation rate is highly matched with the tumor tissue remodeling period (2-4 weeks). 3% w / v hyaluronic acid (HA): HA is a highly expressed extracellular matrix component in the brain glioma microenvironment, which can bind to tumor cells through CD44 receptors, promote the expression of cell migration and invasion related genes (such as MMP9), and simulate the invasive microenvironment of the tumor in vivo. The concentration of 3% can maintain the hydrophilicity of the scaffold, enhance the diffusion efficiency of the nutritional factors in the culture medium, and at the same time provide natural matrix signals for endothelial cell angiogenesis. 2% w / v silk fibroin (SF): the addition of SF can significantly improve the mechanical strength of the scaffold (the elastic modulus is increased by 20-30%), which can resist the volume expansion pressure during the proliferation of the organoid; its β-sheet structure endows the scaffold with good biocompatibility, and the degradation product has no cytotoxicity, avoiding interference with the activity of tumor cells. The three form a balanced biomimetic matrix network of “adhesion-support-degradation”. 0.1% w / v carbon nanotubes (CNTs): the nanoscale fiber structure of CNTs can simulate the topological morphology of collagen fibers in vivo, guiding the migration of tumor cells along the fiber direction (bionic invasion path); at the same time, its excellent electrical conductivity can enhance the electrical signal transmission between cells, promoting the paracrine interaction between tumor cells and endothelial cells (such as the activation of VEGF signaling pathway); the concentration of 0.1% can avoid aggregation toxicity, while significantly improving the mechanical stability of the scaffold. 50 μM RGD peptide: RGD sequence is the core recognition site of cell adhesion, exogenous addition can make up for the lack of natural adhesion sites of artificial materials, promote the specific adhesion of brain glioma cells (high expression of integrin αvβ3) and brain microvascular endothelial cells, and improve the cell seeding efficiency; 50 μM concentration matches the saturation binding concentration of cell surface integrin receptor, avoiding receptor desensitization caused by too high concentration. 100-150 μm porous structure (laser-assisted bioprinting): This pore size range precisely matches the growth space requirements of brain glioma cells - less than 100 μm will limit cell cluster formation, and greater than 150 μm will easily lead to sparse cell distribution and loose structure; Laser printing technology can achieve pore uniformity (deviation ≤10%), ensuring that nutrients (glucose, growth factors) and oxygen can freely diffuse through the pores to the core of the scaffold, avoiding organoid center necrosis; At the same time, the porous structure provides a channel for cell migration, simulating the invasive growth pattern of tumors in vivo. Reserved diameter 200 μm hollow blood vessel channel: 200 μm is the physiological diameter of brain microvessels (in vivo brain capillary diameter is about 10-20 μm, which is magnified to a functional culture size here), providing directional growth space for primary brain microvascular endothelial cells and guiding them to form tubular vascular structures along the inner wall of the channel; The channel is connected with the porous area, which can diffuse the nutrients transported by the blood vessels to the tumor cell area through the pores, solving the problem of "insufficient nutrient transfer gradient" of traditional scaffolds, and realizing the in vitro replication of "tumor-vascular" functional units.

[0030] In some implementable solutions, the culture medium further comprises 1 mM 2-hydroxyglutarate.

[0031] The addition of 1 mM 2-hydroxyglutarate (2HG) in the medium is a precise biomimetic design targeting the metabolic characteristics of IDH mutant glioma. The core logic is to reproduce the molecular phenotype and pathological characteristics of this subtype of tumor by exogenously supplementing key oncogenic metabolites. The explanation is as follows: Isocitrate dehydrogenase (IDH) mutation is a driving event for about 80% of low-grade gliomas: normal IDH enzyme catalyzes isocitrate to alpha-ketoglutarate (a-KG), while mutant IDH (mIDH) abnormally catalyzes a-KG to 2HG (i.e. 2-hydroxyglutarate). In IDH mutant tumors, 2HG accumulates in large quantities (in vivo concentration can reach 0.5-3 mM), becoming a hallmark "oncometabolite", whose concentration is directly related to the malignancy of the tumor and the prognosis of the patient. The 1 mM 2HG concentration is close to the physiological level of IDH mutant tumors in vivo (clinical sample detection shows that it is mostly 0.8-2 mM), which can specifically inhibit key enzymes such as a-KG-dependent demethylase (such as JHDM, TET family) and proline hydroxylase: leading to abnormal histone and DNA methylation (such as decreased H3K4me3 and increased H3K27me3), maintaining the undifferentiated phenotype of tumor cells; inhibiting the cell hypoxia sensing pathway, promoting the expression of angiogenesis-related genes (such as VEGF), simulating the high proliferation characteristics of blood vessels in tumors in vivo. In vitro culture, primary IDH mutant tumor cells lack 2HG supplementation, are prone to lose metabolic adaptation and mutant phenotype (such as decreased mIDH activity and increased a-KG level). The addition of 1 mM 2HG can maintain the abnormal catalytic activity of mIDH through "metabolic feedback", so that the organoids can stably retain: sensitivity to IDH inhibitors (such as ivosidenib), avoiding model "phenotypic drift". Lower limit protection: below 0.5 mM, 2HG cannot effectively inhibit a-KG-dependent enzymes, leading to a decrease in the difference between epigenetic modification and metabolic characteristics and wild-type tumors, and a decrease in model specificity; upper limit control: above 2 mM, excessive 2HG will cause non-specific cytotoxicity (such as increased oxidative stress and damaged mitochondrial function), leading to a decrease in the survival rate of organoids; 1 mM concentration has been verified through pre-experiments, which can stably reproduce the core characteristics of IDH mutation and maintain the structural integrity of the organoids for long-term culture (≥4 weeks).

[0032] In some implementable schemes, the mixed gas environment is a mixed gas environment containing 5% CO2 and 1-5% O2. It should be noted that the mixed gas environment is a mixed gas environment containing 5% CO2 and 1-5% O2. 2 The design of "5% CO2 and 1-5% O2" is a precise biomimic based on the characteristics of the in vivo microenvironment of glioma, and the core role is as follows: CO 2is the key factor to stabilize the pH in cell culture, which precisely controls the pH of the culture medium in the physiological range of 7.2-7.4 by reacting with the bicarbonate buffer system (such as NaHCO3) in the culture medium (CO2+H2O ⇌ H2CO3 ⇌ H⁺+HCO3⁻). This concentration is the gold standard for in vitro culture of mammalian cells: less than 5% will cause the pH to rise (alkaline environment), affecting enzyme activity and cell signaling pathways; more than 5% will trigger a pH drop (acidic stress), inhibit tumor cell proliferation and induce apoptosis, while 5% CO2 can perfectly match the acid-base adaptation state of brain glioma cells in vivo. Brain glioma is often in a "hypoxic microenvironment" (oxygen partial pressure is much lower than that of normal brain tissue of 2-9%) due to rapid proliferation and disordered angiogenesis, and the design of 1-5% O2 is a simulation of this feature. The specific effects include: hypoxia activates hypoxia-inducible factor (HIF-1α), up-regulates target genes such as VEGF and GLUT1, promotes angiogenesis and glycolytic metabolism, and maintains the malignant characteristics of glioma cells such as invasiveness and drug resistance. Combined with the three-dimensional structure of the scaffold, the 1-5% hypoxic environment can form a gradient from the edge (higher oxygen) to the core (lower oxygen) inside the organoid, reproducing the spatial heterogeneity of the "peripheral proliferation and core necrosis" of the tumor in vivo, while the normoxic environment (21% O2) will destroy this gradient, leading to distortion of the organoid phenotype. Hypoxia can stimulate endothelial cells to secrete angiogenic factors, which synergize with the endothelial cell inoculation in the hollow blood vessel channel of the scaffold to promote the formation of functional blood vessel networks, solving the problem of insufficient angiogenesis in traditional normoxic culture. 5% CO2 ensures the stability of the pH of the culture medium, providing a basis for cell metabolism and factor activity; 1-5% O2 regulates hypoxic signals, enhances the interaction between tumor cells and vascular endothelial cells, and synergizes with DFO (a hypoxic mimic) in the culture medium to strengthen the hypoxic response, ultimately achieving high-fidelity simulation of the in vivo glioma microenvironment in structure, metabolism, and function.

[0033] In particular, the oxygen concentration of the mixed gas environment can be adjusted: 5% O2 for culturing IDH mutant glioma organoids, and 1% O2 for culturing glioblastoma organoids. It is explained that the differential adjustment of this oxygen concentration is based on the accurate design of the in vivo microenvironment characteristics of the two glioma subtypes: IDH mutant glioma is usually low in malignancy, the tumor tissue blood vessels are relatively complete, and the in vivo microenvironment oxygen partial pressure is relatively high (close to 5% O2). The oxygen concentration of 5% can simulate its physiological oxygen environment, maintain the IDH mutation-related metabolic phenotype (such as 2-hydroxyglutarate accumulation) and cell homeostasis, and avoid the distortion of the phenotype caused by the excessive activation of the stress pathway under hypoxia. Glioblastoma (GBM) as a high-grade malignant glioma has the characteristics of rapid proliferation and abnormal vascular disorder, and often forms a severe hypoxic area in vivo (oxygen partial pressure can be as low as 1% O2 or below). The setting of 1% O2 can accurately reproduce its hypoxic microenvironment and induce the activation of HIF-1α and other hypoxia-related pathways, which is closely related to the core biological characteristics of GBM such as invasion and metastasis, angiogenesis, and radiotherapy and chemotherapy resistance. By matching the oxygen environment requirements of different subtypes, it can ensure that the organoids are closer to the in vivo real tumor state in terms of metabolic characteristics, signal pathway activation, and drug response, and improve the pathological representativeness and experimental reliability of the model.

[0034] In some implementable schemes, the culture conditions include monitoring the glucose concentration and lactic acid concentration in the culture medium every 6 hours, supplementing DFO to 75 μM in the culture medium when the glucose <0.8 g / L, and starting the 165 rpm pulse oscillation of the culture device when the lactic acid >1.8 g / L.

[0035] It is explained that in the culture of brain glioma organoids, dynamic monitoring and targeted regulation of glucose concentration and lactate concentration are key measures to maintain the physiological activity and metabolic homeostasis of organoids, as follows: the core significance of monitoring every 6 hours; brain glioma cells have active metabolic characteristics, and their energy supply is highly dependent on glucose uptake, and even in an aerobic environment, they tend to produce energy through glycolysis (Warburg effect), resulting in a large accumulation of lactic acid. High-frequency monitoring every 6 hours can capture the dynamic changes of metabolic substrate consumption and product accumulation in real time, providing a basis for precise regulation and avoiding apoptosis, abnormal differentiation or phenotype deviation of organoids due to metabolic imbalance. II. Mechanism of supplementing DFO to 75 μM when glucose is <0.8 g / L; glucose is the core substrate of energy metabolism of organoids, and when its concentration is lower than 0.8 g / L, it means that the energy supply is insufficient, which may trigger a cellular stress response. DFO (desferrioxamine) as an iron chelator can maintain the stability of organoids through the following ways: under low glucose conditions, cells are prone to oxidative damage, and after DFO chelates free iron, it can reduce the generation of hydroxyl radicals, protecting cell structure. Brain glioma cells have a high demand for iron, and DFO can moderately limit iron supply, inhibit excessive proliferation while maintaining basic metabolic functions. Reduce iron-mediated release of inflammatory factors and avoid disturbance of the microenvironment around the organoids. III. The role of starting 165 rpm pulse oscillation when lactate is >1.8 g / L, lactate is the main end product of glycolysis, and its high concentration (>1.8 g / L) can cause a decrease in the pH of the culture medium, inhibit the activity of cellular metabolic enzymes, and induce a stress response. The core role of starting 165 rpm pulse oscillation includes: pulse oscillation can break the diffusion barrier around the organoids, accelerate the diffusion of lactate to the main body of the culture medium, and reduce the local lactate concentration. Oscillation promotes gas exchange between the culture medium and air, increases the dissolved oxygen level, and partially alleviates the vicious cycle of enhanced glycolysis caused by hypoxia. Through the action of fluid dynamics, local metabolic waste is removed, and the microenvironment physicochemical conditions conducive to the growth of organoids are maintained.

[0036] In some implementable solutions, the culture conditions further include; wherein the medium replacement mode is to supplement 50% new medium into the culture device every 3 days.

[0037] It is explained that brain glioma cells are metabolically active (Warburg effect is significant), consume nutrients such as glucose and amino acids quickly, and continuously produce metabolic waste such as lactic acid and ammonia. Supplementing once every 3 days can intervene in time before metabolic imbalance, avoiding apoptosis of organoids due to nutrient deficiency or accumulation of toxic substances.

[0038] The core advantage of "partial replenishment" instead of "complete replacement" is to retain the functional microenvironment components: the original culture medium contains autocrine / paracrine factors secreted by organoids (such as angiogenic factors, extracellular matrix components), which are essential for maintaining tumor cell heterogeneity and vascular endothelial cell function. Retaining 50% of the old medium can avoid the sudden loss of key signaling molecules and maintain the stability of intercellular communication. Replenishing 50% of the new medium can precisely replenish the consumed glucose (4.5 g / L basic concentration), growth factors (EGF, FGF, etc.), and additives (vitamin C, pyruvic acid), while reducing the concentration of waste such as lactic acid and ammonia through dilution (usually reducing lactic acid levels by 30-40%). This can avoid the dramatic fluctuations in nutrition and physicochemical environment caused by complete replacement (such as sudden changes in pH and osmotic pressure). This replacement method complements "every 6-hour metabolic monitoring", "DFO replenishment", and "pulse oscillation": daily monitoring captures metabolic dynamics in real time, 50% replenishment prevents metabolic imbalance through nutrient replenishment and waste dilution, and DFO intervention or oscillation control in extreme cases addresses specific problems.

[0039] In the present application, the pretreatment of the brain glioma sample is also included; specifically including; mechanically shearing the surgically resected brain glioma tissue into fragments with a diameter of 0.3-0.5 mm, washing with PBS buffer containing penicillin-streptomycin for 3 times, centrifuging at 1000 rpm for 5 minutes to remove necrotic tissue and impurities, and obtaining the brain glioma sample.

[0040] It is explained that the brain glioma sample preparation process is specifically as follows, the fresh brain glioma tissue surgically resected is processed through the process of "mechanical shearing-washing-centrifugal purification", and finally the active sample available for culture is obtained, and the purpose of each step is as follows: Mechanically shearing into 0.3-0.5 mm fragments: the tissue is finely sheared with microscissors in vitro, breaking the dense structure of the tissue block, allowing the tumor cells to partially detach from the interstitium, while retaining local intercellular connections and microenvironment components (such as extracellular matrix fragments), laying the foundation for subsequent cell migration and proliferation. Washing with PBS containing penicillin-streptomycin for 3 times: PBS buffer can remove blood stains, cell fragments and other impurities on the surface of the tissue; penicillin-streptomycin (double antibiotics) can eliminate microbial contamination that may be introduced during the operation by inhibiting bacterial cell wall synthesis and protein synthesis, reducing the risk of culture failure. Centrifuging at 1000 rpm for 5 minutes: this centrifugation condition (relatively low speed and short time) can separate the active tissue fragments from necrotic cells and impurities (necrotic tissue has a lower density and is easily suspended in the supernatant) through density difference, avoiding mechanical damage to active cells caused by high-speed centrifugation, and achieving sample purification and improving cell survival rate in subsequent culture. The 0.3-0.5 mm fragment size is the optimal choice to balance cell activity, nutrient supply and proliferation efficiency, with the following advantages: too large size (e.g. >1 mm) will cause the core area of the fragment to be blocked from the outside nutrient exchange, and oxygen and glucose cannot effectively penetrate to the center, which easily leads to cell anoxic necrosis; too small size (e.g. <0.2 mm) will cause excessive mechanical shear to damage the intercellular junction and cell membrane structure, resulting in loss of cell activity (experiments show that the viable cell rate of <0.2 mm fragments is 20-30% lower than that of 0.3-0.5 mm fragments). The diameter of 0.3-0.5 mm makes the fragment surface area to volume ratio in a reasonable range, which can allow the rapid diffusion of glucose and growth factors (such as EGF, FGF) in the culture medium to the inside of the fragment to meet the cell metabolic demand, and can also retain autocrine signal molecules (such as tumor-related factors) in the local microenvironment to maintain the biological phenotype of the cells. Fragments of this size can serve as "proliferation units", and cells can migrate out of the fragment edges and orderly proliferate and aggregate in the porous structure of the scaffold to eventually form organoids with three-dimensional structure, with higher efficiency than other sizes of fragments, and the morphology of the organoids is more similar to that of in vivo tumor tissue.

[0041] In some embodiments, when constructing glioblastoma organoids, 1 μM Erlotinib and 5 μg / mL cholesterol are also added to the culture medium, and a 0.1 kPa periodic pressure is applied to the scaffold and surrounding medium daily during culture, with a frequency of 0.1 Hz.

[0042] The design of adding 1 μM Erlotinib, 5 μg / mL cholesterol and applying 0.1 kPa cyclic pressure (0.1 Hz) in the construction of glioblastoma organoids is a precise microenvironment regulation strategy targeting its pathological characteristics. The specific mechanism is as follows: Glioblastoma (GBM) often has overexpression or mutation of EGFR (epidermal growth factor receptor), which leads to continuous activation of downstream PI3K / Akt, MAPK and other signaling pathways, driving cell unlimited proliferation and invasion. Erlotinib, as a selective EGFR tyrosine kinase inhibitor, simulates the pathological and drug response environment in the following ways: 1 μM concentration can specifically block EGFR kinase activity, inhibit tumor cell over-proliferation, and at the same time maintain a moderate signal strength to maintain the stem cell characteristics of the organoid (avoiding phenotype differentiation caused by complete inhibition). This concentration is close to the plasma effective concentration (0.5-2 μM) of clinical treatment, which can be used to simulate the in vivo drug exposure environment and provide a physiological relevance basis for subsequent drug sensitivity testing; while inhibiting the high activity subpopulation of EGFR, it does not affect the survival of the low expression subpopulation, preserving the inherent cellular heterogeneity characteristics of GBM. Glioblastoma cells have abnormally active cholesterol metabolism, and their cell membranes are rich in cholesterol to support the synthesis of membrane structure at a high proliferation rate, and cholesterol is a key regulator of Hedgehog and other oncogenic signaling pathways. The effects of adding 5 μg / mL cholesterol include: the ability of GBM cells to synthesize cholesterol is often compensatorily enhanced, and exogenous supplementation can reduce the burden of cell self-synthesis and avoid metabolic exhaustion leading to decreased activity; cholesterol stabilizes cell membrane fluidity, ensuring the correct positioning and function of membrane proteins such as EGFR and integrin, promoting tumor cell adhesion to the scaffold and cell-cell communication; cholesterol is a necessary nutrient for tumor stem cell self-renewal, and this concentration can maintain the proportion of CD133+ and other stem cell subpopulations, reproducing the phenotype related to treatment resistance of GBM. The mechanism of 0.1 kPa cyclic pressure (0.1 Hz) mechanical regulation simulates the in vivo mechanical microenvironment: 0.1 kPa pressure is close to the intracranial normal hydrostatic pressure, and 0.1 Hz frequency (10 second cycle) simulates the low-frequency fluctuations caused by respiration or pulse, which can activate YAP / TAZ and other mechanically sensitive transcription factors, promote the synthesis of extracellular matrix (such as fibronectin), and enhance the integration of the organoid and the scaffold; periodic pressure induces the secretion of MMP-2 / 9 and other matrix metalloproteinases through mechanical stimulation, simulating the invasive growth characteristics of GBM, making the organoid more similar to the invasive behavior of the tumor in vivo; low-frequency pressure fluctuations can promote the flow of medium in the porous structure of the scaffold, reducing local nutrient gradient differences and avoiding necrosis caused by hypoxia and hypoglycemia in the core of the organoid.

[0043] Example 1: IDH-mutant brain glioma organoid construction Sample preparation: IDH-mutant glioma tissues were obtained from surgical resection, mechanically sheared into 0.3-0.5 mm fragments, washed with PBS buffer containing penicillin-streptomycin for 3 times, centrifuged at 1000 rpm for 5 min to remove necrotic tissues and impurities, and obtained glioma samples.

[0044] Scaffold preparation: 8% w / v methacrylated gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin were mixed, 0.1% w / v carbon nanotubes and 50 μM RGD peptide were added, and a porous scaffold with a pore size of 100-150 μm was constructed by laser-assisted bioprinting technology, and a hollow blood vessel channel with a diameter of 200 μm was reserved.

[0045] Cell inoculation: glioma samples were mixed with primary brain microvascular endothelial cells at a cell number ratio of 1:5, inoculated into the porous structure of the scaffold; the hollow blood vessel channel was inoculated with primary brain microvascular endothelial cells alone (density 2 x 10 6 cells / mL).

[0046] Culture conditions: Culture medium: DMEM / F12 mixed with Neurobasal at a volume ratio of 3:2 (containing glucose 4.5 g / L, L-glutamine 2 mM), adding 1 x B-27 supplement (without vitamin A), 1 x N-2 supplement, 15 ng / mL EGF, 15 ng / mL FGF, 100 ng / mL VEGF, 20 ng / mL HGF, 50 μM DFO, 2 mM pyruvic acid, 100 μM vitamin C and 1 mM 2-hydroxyglutarate.

[0047] Gas environment: 5% CO2+5% O2.

[0048] Dynamic regulation: microfluidic pump circulates culture medium at a flow rate of 0.3 mL / min, applies a fluid shear force of 1 dyn / cm2; irradiated with blue light at a wavelength of 470 nm and an intensity of 5 mW / cm2for 2 hours daily; glucose / lactic acid concentration is monitored every 6 hours, DFO is supplemented to 75 μM when glucose <0.8 g / L, and pulse oscillation at 165 rpm is started when lactic acid >1.8 g / L; 50% new culture medium is supplemented every 3 days.

[0049] Example 2: Construction of glioblastoma organoids Sample preparation: same as Example 1, sample is glioblastoma tissue.

[0050] Scaffold preparation: same as Example 1.

[0051] Cell inoculation: same as Example 1.

[0052] Culture conditions: Culture medium: 1 mM Erlotinib and 5 pg / mL cholesterol were added to the culture medium of Example 1.

[0053] Gas environment: 5% CO2+ 1% O2.

[0054] Dynamic regulation: microfluidic pump flow rate 0.5 mL / min, fluid shear stress 1.5 dyn / cm2; 0.1 kPa periodic pressure was applied daily (frequency 0.1 Hz), the rest of the monitoring and regulation was the same as Example 1.

[0055] Comparative Example 1: IDH mutant model without blue light Sample processing, scaffold preparation, cell inoculation: exactly the same as Example 1.

[0056] Culture conditions: except for canceling the daily blue light irradiation step, the rest of the culture medium composition, gas environment, fluid shear stress, metabolic monitoring and regulation mode were consistent with Example 1.

[0057] Comparative Example 2: Glioblastoma model without blue light Sample processing, scaffold preparation, cell inoculation: exactly the same as Example 2.

[0058] Culture conditions: except for canceling the daily blue light irradiation step, the rest of the culture medium composition, gas environment, periodic pressure, metabolic regulation were consistent with Example 2.

[0059] Comparative Example 3: Traditional static model without blue light Sample processing: tissue was cut into 1-2 mm, washed once with PBS, and not centrifuged for purification.

[0060] Scaffold preparation: 10% w / v pure gelatin scaffold was used, without porous structure and hollow blood vessel channel.

[0061] Cell inoculation: only brain glioma cells were inoculated, without the addition of primary brain microvascular endothelial cells.

[0062] Culture conditions: single DMEM medium (containing 10% fetal bovine serum), normoxic environment (5% CO2+ 21% O2), static culture without blue light, fluid shear stress and metabolic monitoring, complete medium replacement every 7 days.

[0063] Data detection method Organoid formation rate (7 days): observed by inverted microscope, counted the proportion of organoids with diameter > 100 pm and complete structure in the total number of inoculated cell clusters, recorded and calculated the data on the 7th day every day.

[0064] Vessel penetration rate (14 days): The proportion of endothelial cell tubular structure penetration length to total length in the vessel channel was observed by confocal microscopy using fluorescently labeled endothelial cells (DiI staining), and the average value of 3 fields was taken.

[0065] Core necrosis rate (21 days): The percentage of necrotic area (red fluorescence) in the total area of the organoid was analyzed by Live / Dead staining (calcein-AM / PI) using ImageJ software.

[0066] Endothelial cell tight junction integrity: The expression of tight junction protein occludin was detected by immunofluorescence, and the proportion of positive expression area to total endothelial cell area was calculated, which was divided into "excellent (> 80%)" and "poor (< 60%)".

[0067] Inter-cell calcium signaling efficiency: Calcium fluorescent probe (Fluo-4AM) was used to label cells, and the speed and range of calcium signal transmission after blue light stimulation were recorded by fluorescence imaging system, which was divided into "high" and "low".

[0068] Survival rate after 4 weeks: The proportion of the number of organoids that remained structurally intact and active after 4 weeks of culture to the initial number of organoids was calculated.

[0069] Table 1 is an experimental data statistical table The 7-day formation rate of Examples 1 and 2 (91%, 89%) was significantly higher than that of Comparative Examples 1 and 2 without blue light (68%, 63%), with a difference of 23%-26%. Blue light activates cell photosensitive channel proteins to trigger calcium signaling, enhancing the adhesion and proliferation synergy of tumor cells and endothelial cells. Comparative Example 3, which lacks blue light and a biomimetic system, has the lowest formation rate (42%). The vessel penetration rate of the blue light-containing group (81%-84%) is about 35% higher than that of the group without blue light (49%-53%), and the endothelial cell tight junction integrity (88%-90%) is much better than that of the group without blue light (51%-55%). This is directly related to the blue light-induced polar arrangement of endothelial cells and the up-regulation of tight junction protein (occludin) expression. Comparative Example 3 has a penetration rate of 0 due to the lack of a vascular construction basis. The core necrosis rate of the blue light group (5%-7%) is significantly lower than that of the group without blue light (19%-21%), and the mechanism is that blue light optimizes energy metabolism by regulating mitochondrial function, reducing acidification damage caused by lactic acid accumulation; the necrosis rate of Comparative Example 3 is as high as 48% due to the nutritional exchange barrier of static culture. Blue light irradiation increases the 4-week survival rate of Example 4 (77%-79%) by about 30% compared to the group without blue light (49%-53%), which is due to the activation of antioxidant pathways (such as increased SOD activity) by blue light to reduce apoptosis, while the survival rate of the traditional model is less than 20% due to multiple defects.

[0070] The preferred embodiments of the present application are described in detail above, but the present application is not limited thereto. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, including that each technical feature is combined in any other suitable manner. These simple modifications and combinations should also be considered as disclosed by the present application and fall within the protection scope of the present application.

Claims

1. A method of constructing a brain glioma organoid, characterized by: The method comprises the following steps, S1, mixing the brain glioma sample with the primary brain microvascular endothelial cells at a cell ratio of 1:5, and inoculating into the porous structure of the scaffold after mixing, wherein the hollow vascular channel of the scaffold is inoculated with the primary brain microvascular endothelial cells alone; Step S2, the scaffold in step S1 is placed in a gas-liquid interface culture device to culture the brain glioma sample, the gas-liquid interface culture device comprises a culture medium, the bottom of the scaffold is immersed in the culture medium, the top of the scaffold is exposed to a mixed gas environment, a microfluidic pump circulates the culture medium at a flow rate of 0.3-0.5mL / min, a fluid shear force of 1-1.5dyn / cm² is applied to the surface of the scaffold, and the scaffold is irradiated with blue light with a wavelength of 470nm and an intensity of 5mW / cm 2 for 2 hours every day.

2. The method of claim 1, wherein: The culture medium comprises a basic medium and an additive, wherein the basic medium is a DMEM / F12 and Neurobasal mixed system at a volume ratio of 3:2, and the additive comprises, in terms of final concentration, 1×B-27 additive, 1×N-2 additive, 15 ng / mL epidermal growth factor, 15 ng / mL fibroblast growth factor, 100 ng / mL vascular endothelial growth factor, 20 ng / mL hepatocyte growth factor, 50 μM deferoxamine, 2 mM pyruvic acid and 100 μM vitamin C. ​ 3. The method for constructing glioma organoids as described in claim 1, characterized in that: The preparation method of the scaffold comprises the following steps: mixing 8% w / v methacrylated gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin, adding 0.1% w / v carbon nanotubes and 50 μM RGD peptide, and constructing a porous scaffold with a pore size of 100-150 μm by laser-assisted bioprinting technology, and the scaffold is provided with a hollow vascular channel with a diameter of 200 μm.

4. The method of claim 2, wherein: The culture medium further comprises 1 mM 2-hydroxyglutarate. ​ 5. The method of claim 1, wherein: The mixed gas environment is a mixed gas environment containing 5% CO2 and 1-5% O2. ​ 6. A method of constructing a brain glioma organoid according to claim 5, wherein: The oxygen concentration of the mixed gas environment can be adjusted: 5% O2 for culturing IDH mutant brain glioma organoids and 1% O2 for culturing glioblastoma organoids.

7. The method of constructing a brain glioma organoid of claim 1, wherein: The culture conditions further comprise monitoring the glucose concentration and lactic acid concentration in the culture medium every 6 hours, and supplementing DFO to 75 μM in the culture medium when the glucose is less than 0.8 g / L, and starting the 165 rpm pulse oscillation of the culture device when the lactic acid is greater than 1.8 g / L.

8. A method of constructing a brain glioma organoid according to claim 7, wherein: The culture conditions further comprise that the culture medium replacement mode is to supplement 50% new culture medium into the culture device every 3 days.

9. The method of constructing a brain glioma organoid of claim 1, wherein: The method further comprises pretreatment of the brain glioma sample, wherein the surgical resection brain glioma tissue is mechanically sheared into fragments with a diameter of 0.3-0.5 mm, washed with a penicillin-streptomycin-containing PBS buffer for 3 times, and centrifuged at 1000 rpm for 5 minutes to remove necrotic tissues and impurities to obtain the brain glioma sample.

10. The method for constructing glioma organoids as described in claim 1, characterized in that; When constructing the glioblastoma organoids, 1 μM Erlotinib and 5 μg / mL cholesterol are further added to the culture medium, and a periodic pressure of 0.1 kPa is applied to the scaffold and the surrounding culture medium every day during the culture, and the frequency is 0.1 Hz.

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