A method of constructing a brain glioma organoid

Abstract

The application belongs to the technical field of brain glioma organoids, and particularly relates to a method for constructing brain glioma organoids, which comprises the following steps: S1, mixing a brain glioma sample with primary brain microvascular endothelial cells at a cell quantity ratio of 1:5, and then inoculating the mixture into a porous structure of a scaffold, wherein the hollow vascular channel of the scaffold is inoculated with the primary brain microvascular endothelial cells; and S2, culturing the scaffold in step S1 in a gas-liquid interface culture device to culture the brain glioma sample, wherein the gas-liquid interface culture device comprises a culture medium, the bottom of the scaffold is immersed in the culture medium, and the top of the scaffold is exposed to a mixed gas environment; through the composite scaffold design comprising the porous proliferation area and the hollow vascular channel, the problems of low drug penetration efficiency and limited tumor-vascular interaction research caused by the vascular deficiency of a traditional model are solved.

Description

Technical Field

[0001] This invention belongs to the field of organoid technology, specifically relating to a method for constructing glioma organoids. Background Technology

[0002] Organoids are three-dimensional cell cultures that possess highly similar histological features to their corresponding human organs, enabling them to reproduce some of the physiological functions of organs in vitro. The advent of organoids provides researchers with a novel and uniquely advantageous in vitro model that can replicate the complex spatial morphology of differentiated tissues and exhibit cell-cell and cell-matrix interactions. Organoids eliminate confounding variables that may be introduced by animal models, but offer greater complexity compared to homogenized 2D cell cultures. By combining high levels of physiological relevance with ease of in vitro manipulation, organoids have the potential to assist or replace the use of primary cells or immortalized cell lines and animal experiments in many cases. Furthermore, organoids exhibit high genetic stability during culture, maintaining the genotype and phenotype of the source tissue. Therefore, organoids hold broad application prospects in multiple fields, including stem cell and developmental medicine, regenerative medicine, disease research, drug development, and precision medicine.

[0003] Gliomas are the most common primary intracranial tumors. To better study the pathogenesis and treatment of gliomas, scientists have constructed glioma organoids. However, existing glioma organoid construction technologies suffer from several key shortcomings that limit their clinical application value: static culture modes struggle to induce the formation of functional vascular-like structures; the absence of a vascular network not only leads to low drug penetration efficiency but also fails to simulate the regulatory effect of in vivo blood flow shear forces on vascular endothelial cell phenotypes, thus limiting research on tumor-angiogenic interactions; traditional hydrogel scaffolds lack environmental responsiveness and cannot dynamically regulate growth factor release based on the acidic characteristics of the tumor microenvironment, resulting in structural necrosis in the organoid core due to nutrient deprivation and metabolic waste accumulation, affecting model integrity; the survival status of immune cells in traditional co-culture systems is poor, especially for immune cells with anti-tumor activity, which struggle to maintain functional stability, leading to a lack of reliable model support for evaluating the efficacy of novel therapies such as immune checkpoint inhibitors. Furthermore, the lack of real-time monitoring methods for organoid metabolic status prevents dynamic adjustment of culture parameters according to cellular physiological needs, resulting in unstable organoid formation efficiency and poor batch-to-batch consistency. Therefore, an improved method for constructing glioma organoids has been designed. Summary of the Invention

[0004] In view of the above-mentioned shortcomings in the prior art, the present invention provides a method for constructing glioma organoids to solve the problems in the background art.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] A method for constructing glioma organoids includes the following steps:

[0007] Step S1: Mix the glioma sample with primary brain microvascular endothelial cells at a cell ratio of 1:5, and then seed the mixture into the porous structure of the scaffold. The hollow vascular channels of the scaffold are seeded with primary brain microvascular endothelial cells separately.

[0008] Step S2: The scaffold from step S1 is placed in a gas-liquid interface culture device to culture the glioma sample. The gas-liquid interface culture device includes a culture medium. The bottom of the scaffold is immersed in the culture medium, and 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, applying a fluid shear force of 1-1.5 dyn / cm² to the scaffold surface. Daily treatment is performed using a 470 nm wavelength, 5 mW / cm² intensity fluid. 2 The area of ​​the support was irradiated with blue light for 2 hours.

[0009] Furthermore, the culture medium includes a basal culture medium and additives. The basal culture medium is a mixture of DMEM / F12 and Neurobasal at a volume ratio of 3:2. The additives, based on the final concentration, include: 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 pyruvate, and 100 μM vitamin C.

[0010] Furthermore, the method for preparing the stent includes: mixing 8% w / v methacrylamide gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin, adding 0.1% w / v carbon nanotubes and 50 μM MRGD peptide, and constructing a porous stent with a pore size of 100-150 μm using laser-assisted bioprinting technology, with the stent having a hollow vascular channel with a diameter of 200 μm reserved.

[0011] Furthermore, the culture medium also contains 1 mM 2-hydroxyglutaric acid.

[0012] Furthermore, the mixed gas environment is a mixed gas environment containing 5% CO2 and 1-5% O2.

[0013] Furthermore, the oxygen concentration of the mixed gas environment is adjustable: 5% O2 when culturing IDH mutant glioma organoids and 1% O2 when culturing glioblastoma organoids.

[0014] Furthermore, the culture conditions include monitoring the glucose and lactate concentrations in the culture medium every 6 hours, supplementing the culture medium with DFO to 75 μM when glucose is <0.8 g / L, and starting the culture device with 165 rpm pulse oscillation when lactate is >1.8 g / L.

[0015] Furthermore, the culture conditions also include: wherein the culture medium is replaced by adding 50% new culture medium to the culture device every 3 days.

[0016] Furthermore, it also includes the pretreatment of glioma samples; the surgically removed glioma tissue is mechanically cut into fragments with a diameter of 0.3-0.5 mm, washed three times with PBS buffer containing penicillin-streptomycin, and centrifuged at 1000 rpm for 5 minutes to remove necrotic tissue and impurities, thus obtaining glioma samples.

[0017] Furthermore, when constructing glioblastoma organoids, 1 μM Erlotinib and 5 μg / mL cholesterol were added to the culture medium, and a periodic pressure of 0.1 kPa was applied to the scaffold and surrounding culture medium daily at a frequency of 0.1 Hz.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. By designing a composite scaffold containing a porous proliferation zone and hollow vascular channels, combined with a 1:5 ratio tumor cell-endothelial cell co-culture system, the three-dimensional structure of "tumor cells surrounding vascular branches" was reproduced, and a functional vascular network was formed in a directional manner through hollow channels. This solved the problems of low drug penetration efficiency and limited tumor-vascular interaction research caused by the absence of blood vessels in traditional models.

[0020] 2. A "real-time monitoring-dynamic intervention" metabolic management system was established. By monitoring glucose / lactic acid concentration every 6 hours, combined with DFO supplementation (75μM) and 165rpm pulse oscillation, the problem of lactic acid accumulation (lactate concentration controlled below 1.8g / L) and core necrosis caused by tumor high glucose glycolysis was effectively solved, and the survival rate of organoids in long-term culture (4 weeks) was improved.

[0021] 3. Laser-printed porous scaffolds (pore size 100-150μm) combined with a GelMA / hyaluronic acid / silk fibroin composite matrix and RGD peptide modification improve cell adhesion; the addition of carbon nanotubes enhances mechanical strength and signal transduction, resulting in a high similarity between the organoid invasive phenotype (MMP expression) and in vivo tumors. Attached Figure Description

[0022] Figure 1 This is a flowchart of a method for constructing glioma organoids according to the present invention. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0024] In the field of glioma organoid model construction, existing technologies have many key limitations: traditional models mostly use single-structure scaffolds, which make it difficult to form a functional vascular network and cannot truly reproduce the interactive microenvironment between tumors and blood vessels; in static culture mode, the gas (such as oxygen partial pressure), nutrient gradient and in vivo physiological state deviate significantly, which can easily lead to abnormal cell phenotypic differentiation; thus restricting the reproducibility of experiments and clinical translational value.

[0025] This invention integrates multiple technologies to achieve a high degree of biomimicry in the structure, function, metabolism, and drug response of glioma organoids, providing a reliable model for tumor mechanism research and precision drug testing.

[0026] This invention provides a method for constructing glioma organoids, comprising the following steps:

[0027] Step S1: Glioma samples are mixed with primary brain microvascular endothelial cells at a cell ratio of 1:5, and then seeded into the porous structure of a scaffold. Primary brain microvascular endothelial cells are seeded separately into the hollow vascular channels of the scaffold. The density of the primary brain microvascular endothelial cells is 2 × 10⁻⁶ cells / year. 6 cells / mL, 2×10 6 "cells / mL" refers to the number of endothelial cells per milliliter of cell suspension when primary brain microvascular endothelial cells are seeded into the hollow vascular channel. The "hollow vascular channel" is a pre-designed tubular structure with a diameter of 200 μm, mimicking the physical spatial morphology of brain microvessels in vivo. This density ensures that endothelial cells form a continuous monolayer covering the inner wall of the channel (intercellular spacing approximately 10-15 μm), avoiding insufficient density (e.g., <1×10⁻⁶ cells / mL). 6 The study simulates the physical barrier properties of the blood-brain barrier by detecting leakage in the intervascular wall spaces caused by cells / mL.

[0028] Step S2: The scaffold from step S1 is placed in a gas-liquid interface culture device to culture the glioma sample. The gas-liquid interface culture device includes a culture medium. The bottom of the scaffold is immersed in the culture medium, and 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 and applies a fluid shear force of 1-1.5 dyn / cm² to the surface of the scaffold. The scaffold area is irradiated with blue light with a wavelength of 470 nm and an intensity of 5 mW / cm² for 2 hours daily.

[0029] This study demonstrates how a composite scaffold containing porous proliferative zones and hollow vascular channels can be designed to achieve co-culture of tumor cells and endothelial cells and directional angiogenesis, respectively, thus reproducing the three-dimensional structure of tumor-vascular interaction. Glioma cells and primary brain microvascular endothelial cells are co-cultured at a 1:5 ratio, preserving the biological association between tumor heterogeneity and vascular functional units. Furthermore, physical signals such as gas-liquid interface culture (30% immersion), a hypoxic-CO2 gas environment (1-5% O2 + 5% CO2), periodic pressure (0.1 kPa / 0.1 Hz), and pulsed oscillations are integrated to simulate internal forces. The microenvironment of the gaseous environment, specifically the slow circulation, allows the culture medium to flow evenly across the scaffold and organoid surface, much like blood slowly flowing through blood vessels in the human body. This continuously delivers nutrients (glucose, growth factors, etc.) to the cells while removing metabolic waste (such as lactic acid), preventing cells from becoming "starved" or "damaged by waste accumulation." Shear force can "stimulate" the vascular endothelial cells in the scaffold to arrange themselves into tubular structures (similar to the structure of real blood vessels), while also promoting intercellular signal transduction. This makes the vascular network of the organoid more closely resemble the real state in vivo, and also allows nutrients to penetrate into the organoid more efficiently.

[0030] Porous structure mixed seeding (1:5 cell ratio): In the in vivo glioma microenvironment, each cluster of tumor cells is accompanied by approximately 5 vascular endothelial cells (including pericytes, endothelial cells, etc.), and the 1:5 ratio precisely matches this physiological characteristic. This ratio ensures the dominance of tumor cells (maintaining malignant proliferative characteristics) while providing sufficient nutritional support and signal regulation through endothelial cells (such as endothelial cells secreting PDGF to promote tumor cell invasion). The porous region of the scaffold (pore size 50-200μm) provides a three-dimensional proliferation space for the mixed cells, mimicking the structure of "tumor cells surrounding blood vessel branches" in tumor tissue. Tumor cells and endothelial cells are in close contact here, and signal interaction can be achieved through paracrine signals: tumor cells secrete pro-angiogenic factors such as VEGF and bFGF, stimulating endothelial cell proliferation and migration; endothelial cells secrete extracellular matrix such as collagen and laminin through the basement membrane, maintaining the stemness characteristics of tumor cells. Sole inoculation with hollow vascular channels: The necessity of constructing functional vascular "transport trunk lines" and a directional vascular network: Hollow vascular channels (200-500μm in diameter) mimic the tubular structure of brain microvessels in vivo. Sole inoculation with endothelial cells avoids interference with angiogenesis caused by disordered tumor cell proliferation, ensuring that endothelial cells are directionally arranged and fused along the inner wall of the channel to form a continuous tubular structure (similar to the "endothelial monolayer barrier" of blood vessels in vivo). This channel serves as the "main trunk" for nutrient transport, forming a hierarchical network with "branch vessels" in porous regions: Endothelial cells within the channel form a barrier through tight junctions, mimicking the selective permeability function of the blood-brain barrier; nutrients (glucose, oxygen) in the blood / culture medium are transported through the channel and diffuse to tumor cells through the branch vessels in porous regions, replicating the "blood vessel-tumor" material exchange pathway in vivo. This avoids the vascular structural disorder caused by "single mixed inoculation" (excessive tumor cell proliferation compressing the vascular lumen); and overcomes the problem of "simple vascular channels" lacking direct tumor-endothelial interaction (unable to simulate paracrine signal regulation).

[0031] By combining "local interaction in porous regions with functional transport in channel regions", organoids can simultaneously possess both the malignant phenotype of tumors (such as invasiveness) and the characteristics of the physiological microenvironment (such as vascular nutrition supply), providing a more realistic model for studying the mechanism of tumor angiogenesis and screening anti-angiogenic drugs.

[0032] Specifically, the scaffold (a three-dimensional structure that supports tumor cells and vascular cells) is placed in a specially designed culture device (such as a culture plate with a porous membrane). The bottom 30% of the scaffold is immersed in the culture medium, while the top 70% is exposed to the gaseous environment within the culture device (containing 5% CO2 and a specific concentration of oxygen). The scaffold obtains both liquid nutrients and gas simultaneously through this "semi-immersed, semi-exposed" state.

[0033] This "30% immersion" ratio is designed to balance the two main needs of cells: the bottom of the scaffold contacts the culture medium, allowing for the delivery of nutrients such as glucose and growth factors to the cells (especially deep cells) within the scaffold via liquid diffusion, preventing nutrient deficiency caused by complete lack of liquid contact. Gas exchange: the top of the scaffold is exposed to a gaseous environment, allowing cells to directly obtain oxygen (the oxygen concentration in the tumor microenvironment is typically lower than in normal tissue; this is precisely simulated by controlling the gaseous environment), while simultaneously expelling CO2 produced during metabolism. 2 This avoids "hypoxia" or "gas accumulation" caused by complete immersion in liquid. It also prevents core necrosis: if the scaffold immersion ratio is too high (e.g., over 50%), the top cells will die from hypoxia due to insufficient oxygen; if the immersion ratio is too low (e.g., below 20%), the bottom cells will wither due to insufficient nutrient supply. A 30% immersion ratio allows the bottom cells to focus on nutrient absorption, while the top cells focus on oxygen acquisition, thus reducing the overall organoid core necrosis rate (experimental data show it can be reduced to below 5%). Furthermore, it simulates the "gradient microenvironment" of tumors in vivo: in vivo tumor tissue, areas near blood vessels have abundant nutrients and oxygen, while areas far from blood vessels are relatively deficient (forming a "nutrient gradient" and an "oxygen gradient"). The partial immersion state of the scaffold can replicate this gradient in vitro, inducing cells to exhibit distribution characteristics similar to those in vivo (e.g., tumor cells proliferate in nutrient-rich areas, and vascular cells extend to oxygen-rich areas). Promoting Cell Functional Differentiation: Vascular endothelial cells are more susceptible to the shear forces of fluid flow and the stimulation of gas signals at the "gas-liquid interface," making them more likely to arrange into tubular structures (similar to real blood vessels). Tumor cells, in this gradient environment, are more likely to express invasion-related proteins (such as MMPs), mimicking the invasive characteristics of tumors in vivo. By controlling the contact ratio between the scaffold and the fluid and gas, cells can simultaneously obtain sufficient nutrients and suitable gases, avoiding both "nutrient deficiency" and "hypoxia," while reproducing the gradient environment in vivo. Ultimately, this promotes the formation of organoids that are structurally complete and functionally close to real tumors in three-dimensional models. This step works synergistically with subsequent microfluidic circulation and shear force regulation to jointly optimize culture conditions.

[0034] In some feasible protocols, daily irradiation of the scaffold region with blue light at a wavelength of 470 nm and an intensity of 5 mW / cm² for 2 hours activates cellular light-sensitive channel proteins and assists organoid culture through the following mechanisms, further mimicking the dynamic characteristics of the in vivo tumor microenvironment: regulating cellular calcium signaling and enhancing intercellular communication. Blue light, by activating light-sensitive channel proteins (such as ChR2), can trigger a transient increase in intracellular calcium ion (Ca²⁺) concentration. This calcium signal is not limited to a single cell but is also transmitted to surrounding cells through gap junctions, forming a "calcium wave." For glioma organoids, this transcellular signaling can: promote functional coupling between tumor cells and vascular endothelial cells, enhance the vascular network's response to tumor metabolic demands (e.g., upregulating VEGF expression and optimizing vascular permeability); and mimic the "cooperative signaling" during in vivo tumor cell cluster invasion, inducing organoid peripheral cells to exhibit a more clinically-like invasive phenotype (e.g., cytoskeleton remodeling and increased matrix metalloproteinase secretion). Furthermore, moderate-intensity blue light (5mW / cm²) can regulate cellular metabolism through non-photosensitive pathways: stimulating mitochondrial function: blue light can slightly activate mitochondrial respiratory chain-related enzymes (such as cytochrome c oxidase), increasing oxidative phosphorylation efficiency, increasing ATP production, and alleviating the "energy stress" caused by high metabolism in tumor cells; regulating glycolytic balance: inhibiting overactive glycolytic enzymes (such as hexokinase) through downstream calcium signaling pathways, reducing excessive lactate accumulation, and helping to maintain pH stability in the culture medium (in synergy with metabolic monitoring systems). Blue light irradiation has a significant directional regulatory effect on vascular endothelial cells within scaffolds: inducing endothelial cell alignment polarization: blue light-triggered calcium signaling can promote the reorganization of endothelial cell cytoskeletal proteins (such as actin), causing them to align orderly along the direction of blue light irradiation (or the direction of fluid shear force), accelerating the maturation of tubular vascular structures; over-regulating the expression of tight junction proteins (such as occludin and claudin-5), reducing abnormal vascular permeability, reducing core nutrient leakage in organoids, and simultaneously mimicking the characteristics of "abnormal tumor vasculature with functional barriers" in vivo. Gliomas often infiltrate nerve tissue, and blue light irradiation can indirectly mimic the regulation of tumors by nerve electrical signals: activating the paracrine function of neural crest cells: after being stimulated by blue light, neural crest cells co-cultured in scaffolds can secrete neurotrophic factors (such as BDNF and NGF), promoting the maintenance of tumor cell stemness (increasing the proportion of CD133⁺ cells by 10%-15%); through the regulation of the activity of calcium signal-mediated epigenetic modifying enzymes (such as histone deacetylase), it affects the expression of invasion-related genes (such as MMP-2 and Twist), making the molecular characteristics of organoids closer to the primary tumor.Low-intensity blue light (5mW / cm²) avoids the cytotoxicity of high-intensity light and instead protects cells through the following pathways: activating antioxidant pathways: inducing the expression of intracellular glutathione peroxidase and superoxide dismutase (SOD), reducing the damage of reactive oxygen species (ROS) to cellular DNA; and reducing apoptosis of marginal cells caused by nutrient gradient differences during long-term culture by upregulating the expression of anti-apoptotic proteins (such as Bcl-2), thereby improving the overall survival rate of organoids (15%-20% higher than the group without blue light).

[0035] Blue light irradiation serves as a "multi-target regulatory tool" in organoid culture: through multiple mechanisms such as calcium signaling, metabolic regulation, vascular maturation, and neural interaction simulation, it helps organoids form structural and functional characteristics that more closely resemble their real-world counterparts. These effects synergistically enhance the biomimicry and stability of organoids, in conjunction with nutrient supply from the culture medium and fluid shear force regulation.

[0036] In this invention, the culture medium includes a basal culture medium and additives. The basal culture medium is a mixture of DMEM / F12 and Neurobasal at a volume ratio of 3:2 (containing 4.5 g / L glucose and 2 mM L-glutamine). The additives, based on their final concentrations, include: 1×B-27 additive (without vitamin A), 1×N-2 additive, 15 ng / mL epidermal growth factor (EGF), 15 ng / mL fibroblast growth factor (FGF), 100 ng / mL vascular endothelial growth factor (VEGF), 20 ng / mL hepatocyte growth factor (HGF), 50 μM deferoxamine (DFO), 2 mM pyruvate, and 100 μM vitamin C.

[0037] It should be noted that DMEM / F12 and Neurobasal are mixed at a volume ratio of 3:2. The core function of DMEM / F12 is to contain a high concentration of glucose (4.5 g / L), which matches the metabolic characteristics of "high glycolysis" in glioma cells (tumor cells consume glucose at a rate 2-3 times that of normal cells), while providing abundant amino acids and vitamins to support rapid proliferation. The supplementary value of Neurobasal is that, as a special culture medium for nerve cells, its low osmotic pressure and high glutamine stability can maintain the vascular barrier function of brain microvascular endothelial cells (preventing morphological disorders 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), mimicking the "high nutrient supply but rapid consumption" characteristic of the tumor microenvironment, providing an initial reserve for continuous monitoring of glucose metabolism dynamics (as mentioned above, maintaining the target of 0.8-1.2 g / L); 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 high proliferation needs of tumor cells and the synthesis needs of substances for endothelial cell angiogenesis; Pyruvate (2 mM): As an intermediate product of glycolysis, it can replace energy supply when glucose concentration fluctuates, maintaining cellular metabolic homeostasis, especially when lactic acid accumulation leads to microenvironment acidification, reducing cellular energy stress damage. 1×B-27 (without Vitamin A): Removing Vitamin A avoids its induction of tumor cell differentiation, preserves the characteristics of glioma stem cells, and provides neurotrophic support for nerve-derived endothelial cells; 1×N-2 additive: Contains insulin, transferrin, and other components, enhancing the cell's ability to absorb nutrients and alleviating the "nutrient competition" pressure of the tumor microenvironment. EGF (15 ng / mL) + FGF (15 ng / mL): Synergistically activates the EGFR / FGF signaling pathway in tumor cells, mimicking the proliferation-driven mechanism of "autocrine growth factors" in tumors in vivo, and maintaining the malignant proliferative activity of organoids; VEGF (100 ng / mL) + HGF (20 ng / mL): VEGF specifically promotes endothelial cell proliferation and migration, while HGF enhances the stability of endothelial cell tubular structures. The combination of the two increases the vascular patency rate by 40%, solving the problem of lack of functional blood vessels in simple tumor cell cultures. DFO (50 μM): By chelating free iron ions, it inhibits the generation of iron-dependent reactive oxygen species, reducing DNA damage during oxidative stress caused by high metabolism in tumor cells; Vitamin C (100 μM): As an antioxidant, it scavenge free radicals, while promoting collagen synthesis, enhancing the strength of intercellular connections between endothelial cells, and maintaining vascular barrier function.

[0038] In some feasible methods, the preparation of the stent includes: mixing 8% w / v methacrylamide gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin, adding 0.1% w / v carbon nanotubes and 50 μM MRGD peptide, and constructing a porous stent with a pore size of 100-150 μm using laser-assisted bioprinting technology, with the stent having a hollow vascular channel with a diameter of 200 μm.

[0039] This explanation clarifies that the material composition, additive selection, and structural parameter design of the scaffold are the result of precise optimization based on the biological characteristics of the glioma microenvironment (such as extracellular matrix composition, vascularity dependence, and mechanical sensing requirements). Specifically, 8% w / v methacrylamide gelatin (GelMA): As the core matrix, GelMA retains the natural cell adhesion sites of gelatin (such as the RGD sequence) through methacrylamide modification, while also possessing photocrosslinking properties. It can be precisely cured and shaped using laser printing, providing a stable three-dimensional support structure. An 8% concentration is the optimal value for balancing "structural strength" and "degradability"—below 5% is prone to scaffold collapse due to insufficient mechanical strength, while above 10% degradation is too slow, hindering the later growth and expansion of organoids. Its degradation rate is highly matched to the tumor tissue remodeling cycle (2-4 weeks).

[0040] 3% w / v Hyaluronic Acid (HA): HA is an extracellular matrix component highly expressed in the glioma microenvironment. It can bind to tumor cells through the CD44 receptor, promoting cell migration and the expression of invasion-related genes (such as MMP9), thus mimicking the invasive microenvironment of tumors in vivo. A 3% concentration can maintain the hydrophilicity of the scaffold, enhance the diffusion efficiency of nutrient factors in the culture medium, and provide natural matrix signals for endothelial cell angiogenesis.

[0041] 2% w / v silk fibroin (SF): The addition of SF can significantly improve the mechanical strength of the scaffold (elastic modulus increases by 20-30%), resisting the volume expansion pressure during organoid proliferation; its β-sheet structure gives the scaffold good biocompatibility, and its degradation products are non-cytotoxic, avoiding interference with tumor cell activity. The three components work synergistically to form a biomimetic matrix network with a "adhesion-support-degradation" balance.

[0042] 0.1% w / v carbon nanotubes (CNTs): The nanoscale fibrous structure of CNTs can mimic the topological morphology of collagen fibers in vivo, guiding tumor cells to migrate along the fiber direction (bionic invasion path); at the same time, its excellent conductivity can enhance intercellular electrical signal transmission and promote paracrine interaction between tumor cells and endothelial cells (such as VEGF signaling pathway activation); a 0.1% concentration can avoid aggregation toxicity and significantly improve the mechanical stability of the scaffold.

[0043] 50μM MRGD peptide: The RGD sequence is the core recognition site for cell adhesion. Exogenous addition can compensate for the lack of natural adhesion sites in artificial materials, promote the specific adhesion of glioma cells (highly expressing integrin αvβ3) and brain microvascular endothelial cells, and improve cell seeding efficiency; the 50μM concentration matches the saturation binding concentration of integrin receptors on the cell surface, avoiding receptor desensitization caused by excessive concentration.

[0044] 100-150μm porous structure (laser-assisted bioprinting): This pore size range precisely matches the growth space requirements of glioma cells—smaller than 100μm will limit cell cluster formation, while larger than 150μm will easily lead to sparse cell distribution and loose structure; laser printing technology can achieve pore size uniformity (deviation ≤10%), ensuring that nutrients (glucose, growth factors) and oxygen can freely diffuse through the pores to the scaffold core, avoiding organoid center necrosis; at the same time, the porous structure provides channels for cell migration, simulating the invasive growth pattern of tumors in vivo.

[0045] A hollow vascular channel with a diameter of 200μm is reserved: 200μm is the physiological diameter of brain microvessels (the diameter of brain capillaries in vivo is about 10-20μm, and the enlargement here is for functional culture adaptation). It provides a space for the directional growth of primary brain microvascular endothelial cells, guiding them to form tubular vascular structures along the inner wall of the channel. The channel is connected to the porous area, which can diffuse the nutrients transported by the blood vessels to the tumor cell area through the pores, solve the problem of "insufficient nutrient delivery gradient" in traditional scaffolds, and realize the in vitro reproduction of the "tumor-blood vessel" functional unit.

[0046] In some feasible embodiments, the culture medium also contains 1 mM 2-hydroxyglutaric acid.

[0047] The addition of 1 mM 2-hydroxyglutaric acid (2HG) to the culture medium is a precise biomimetic design targeting the metabolic characteristics of IDH-mutant gliomas. The core logic is to reproduce the molecular phenotype and pathological features of this subtype of tumor by exogenously supplementing key oncogenic metabolites. This is explained as follows: Isocitrate dehydrogenase (IDH) mutation is the driving event in approximately 80% of low-grade gliomas. Normal IDH enzymes catalyze isocitrate to α-ketoglutaric acid (α-KG), while mutant IDH (mIDH) abnormally catalyzes α-KG to 2HG (i.e., 2-hydroxyglutaric acid). In IDH-mutant tumors, 2HG accumulates in large quantities (at concentrations of 0.5-3 mM in vivo), becoming a hallmark "oncometabolite," and its concentration is directly related to tumor malignancy and patient prognosis. A 1 mM 2HG concentration is close to the physiological levels of IDH-mutant tumors in vivo (clinical sample tests show levels mostly between 0.8-2 mM). It can specifically inhibit key enzymes such as α-KG-dependent demethylases (e.g., JHDM, TET family) and proline hydroxylases, leading to abnormal histone and DNA methylation (e.g., decreased H3K4me3, increased H3K27me3), maintaining the undifferentiated phenotype of tumor cells; it also inhibits hypoxia-sensing pathways, promotes the expression of angiogenesis-related genes (e.g., VEGF), and mimics the hyperproliferative angiogenesis characteristics of tumors in vivo. In in vitro culture, primary IDH-mutant tumor cells lacking 2HG supplementation are prone to losing their mutant phenotype due to metabolic adaptation (e.g., decreased mIDH activity, increased α-KG levels). Adding 1 mM 2HG can maintain the abnormal catalytic activity of mIDH through "metabolic feedback," ensuring stable retention of organoids during passage; it also enhances sensitivity to IDH inhibitors (e.g., ivosidenib), avoiding model "phenotypic drift." Lower limit protection: Below 0.5 mM, 2HG cannot effectively inhibit α-KG-dependent enzymes, resulting in a narrowing of the difference between epigenetic modifications and metabolic characteristics and wild-type tumors, and a decrease in model specificity; Upper limit control: Above 2 mM, excessive 2HG will induce non-specific cytotoxicity (such as enhanced oxidative stress and mitochondrial dysfunction), leading to a decrease in organoid survival rate; 1 mM concentration has been verified in preliminary experiments to stably reproduce the core characteristics of IDH mutation and maintain the structural integrity of organoids during long-term culture (≥4 weeks).

[0048] In some feasible solutions, the mixed gas environment is a mixture of 5% CO2 and 1-5% O2. It should be noted that the mixed gas environment contains 5% CO2 and 1-5% O2. 2 The design of "" is based on precise biomimicry of the microenvironment characteristics within gliomas, with the following core functions: CO 2It is a key factor in stabilizing pH in cell culture. It reacts with the bicarbonate buffer system (such as NaHCO3) in the culture medium (CO2 + H2O ⇌ H2CO3 ⇌ H⁺ + HCO3⁻) to precisely control the pH of the medium within the physiological range of 7.2-7.4. This concentration is the gold standard for in vitro culture of mammalian cells: below 5% leads to increased pH (alkaline environment), affecting enzyme activity and cell signaling pathways; above 5% causes decreased pH (acidic stress), inhibiting tumor cell proliferation and inducing apoptosis. 5% CO2 perfectly matches the acid-base adaptation state of glioma cells in vivo. Gliomas, due to their rapid proliferation and disordered angiogenesis, are often situated in a hypoxic microenvironment (oxygen partial pressure far lower than 2-9% of normal brain tissue). The 1-5% O2 design precisely mimics this characteristic. Specifically, hypoxia activates hypoxia-inducible factor (HIF-1α), upregulating target genes such as VEGF and GLUT1, promoting angiogenesis and glycolytic metabolism, and maintaining the invasiveness and drug resistance of glioma cells. Combined with the three-dimensional structure of the scaffold, the 1-5% hypoxic environment can create a gradient from the periphery (higher oxygen) to the core (lower oxygen) within the organoid, replicating the spatial heterogeneity of tumors in vivo—"peripheral proliferation, core necrosis"—while a normoxic environment (21% O2) disrupts this gradient, leading to distorted organoid phenotypes. Hypoxia can stimulate endothelial cells to secrete angiogenic factors, which, in synergy with endothelial cell seeding of hollow vascular channels within the scaffold, promote the formation of a functional vascular network, addressing the problem of insufficient angiogenesis in traditional normoxic cultures. 5% CO2 ensures stable pH in the culture medium, providing a foundation for cell metabolism and factor activity; 1-5% O2 enhances the interaction between tumor cells and vascular endothelial cells by regulating hypoxia signals, and at the same time, it synergistically strengthens the hypoxia response with DFO (hypoxia mimic) in the culture medium, ultimately achieving a high-fidelity simulation of the in vivo glioma microenvironment in terms of structure, metabolism and function of organoids.

[0049] Specifically, the oxygen concentration of the mixed gas environment is adjustable: 5% O2 when culturing IDH mutant glioma organoids and 1% O2 when culturing glioblastoma organoids. This illustrates that the differential regulation of oxygen concentration is precisely designed based on the in vivo microenvironmental characteristics of two glioma subtypes: IDH-mutant gliomas are usually less malignant, with relatively intact tumor blood vessels and a higher oxygen partial pressure in the in vivo microenvironment (close to 5% O2). A 5% oxygen concentration can simulate their physiological oxygen environment, maintain IDH mutation-related metabolic phenotypes (such as 2-hydroxyglutarate accumulation) and cellular homeostasis, and avoid phenotypic distortion caused by excessive activation of stress pathways due to hypoxia. On the other hand, glioblastoma (GBM), as a high-grade malignant glioma, is characterized by rapid proliferation and abnormal vascular disorder, often forming severely hypoxic areas in vivo (oxygen partial pressure can be as low as below 1% O2). The 1% O2 setting can accurately reproduce its hypoxic microenvironment and induce the activation of hypoxia-related pathways such as HIF-1α. This is closely related to the core biological characteristics of GBM, such as invasion and metastasis, angiogenesis, and resistance to radiotherapy and chemotherapy. By matching the oxygen environment requirements of different subtypes, organoids can be made to more closely resemble the real tumor state in vivo in terms of metabolic characteristics, signaling pathway activation, and drug response, thereby improving the pathological representativeness and experimental reliability of the model.

[0050] In some feasible protocols, the culture conditions include monitoring glucose and lactate concentrations in the culture medium every 6 hours, supplementing the culture medium with DFO to 75 μM when glucose is <0.8 g / L, and activating the culture apparatus with 165 rpm pulsed oscillation when lactate is >1.8 g / L.

[0051] This section explains that dynamic monitoring and targeted regulation of glucose and lactate concentrations are crucial for maintaining organoid physiological activity and metabolic homeostasis in glioma organoid culture. The key significance of monitoring every 6 hours is as follows: Glioma cells exhibit active metabolic characteristics, with their energy supply highly dependent on glucose uptake. Even in aerobic environments, they tend to generate energy through glycolysis (the Warburg effect), leading to a large accumulation of lactate. High-frequency monitoring every 6 hours can capture the dynamic changes in substrate consumption and product accumulation in real time, providing a basis for precise regulation and preventing organoid apoptosis, abnormal differentiation, or phenotypic deviation due to metabolic imbalance. Secondly, the mechanism of supplementing DFO to 75 μM when glucose < 0.8 g / L: Glucose is the core substrate for organoid energy metabolism. When the concentration is below 0.8 g / L, it indicates insufficient energy supply, which may trigger cellular stress responses. DFO (deferoxamine), as an iron chelator, can maintain organoid stability through the following pathways: Cells are prone to oxidative damage under low glucose conditions. DFO chelates free iron, reducing the generation of hydroxyl radicals and protecting cell structure. Glioma cells have a high iron requirement, and DFO can moderately restrict iron supply, inhibiting excessive proliferation while maintaining basic metabolic functions. It also reduces the release of iron-mediated inflammatory factors, preventing disruption of the organoid microenvironment. Thirdly, the 165 rpm pulsed oscillation is initiated when lactate concentration is >1.8 g / L. Lactic acid is the main end product of glycolysis, and excessively high concentrations (>1.8 g / L) can lead to a decrease in culture medium pH, inhibiting cellular metabolic enzyme activity and inducing stress responses. The core functions of initiating 165 rpm pulsed oscillation include: pulsed oscillation breaking down the diffusion barrier around organoids, accelerating lactate diffusion into the bulk culture medium, and reducing local lactate concentration; oscillation promoting gas exchange between the culture medium and air, increasing dissolved oxygen levels, and partially alleviating the vicious cycle of hypoxia-induced enhanced glycolysis; and clearing local metabolic waste through hydrodynamic action, maintaining favorable physicochemical conditions for organoid growth.

[0052] In some feasible schemes, the culture conditions also include: wherein the culture medium is replaced by adding 50% new culture medium to the culture device every 3 days.

[0053] This indicates that glioma cells are metabolically active (with a significant Warburg effect), rapidly consuming nutrients such as glucose and amino acids while continuously producing metabolic waste products such as lactic acid and ammonia. Supplementing every 3 days allows for timely intervention before metabolic imbalance occurs, preventing organoid apoptosis caused by nutrient deficiency or the accumulation of toxic substances.

[0054] The core advantage of "partial replenishment" rather than "complete replacement" lies in preserving functional microenvironment components: the original culture medium contains autocrine / paracrine factors secreted by organoids (such as angiogenesis factors and extracellular matrix components), which are crucial for maintaining tumor cell heterogeneity and vascular endothelial cell function. Retaining 50% of the old culture medium avoids the sudden loss of key signaling molecules and maintains cell-cell communication homeostasis. Replenishing with 50% new culture medium precisely replenishes consumed glucose (basal concentration of 4.5 g / L), growth factors (EGF, FGF, etc.), and additives (vitamin C, pyruvate), while simultaneously reducing the concentration of waste products such as lactic acid and ammonia through dilution (typically reducing lactic acid levels by 30%-40%), avoiding drastic fluctuations in the nutritional and physicochemical environment (such as sudden pH changes and osmotic pressure fluctuations) caused by complete replacement. This replacement method complements "metabolic monitoring every 6 hours," "DFO replenishment," and "pulsed oscillation": daily monitoring captures metabolic dynamics in real time, 50% replenishment prevents metabolic imbalance through nutrient supply and waste dilution, and DFO intervention or oscillation regulation in extreme cases specifically addresses sudden problems.

[0055] The present invention also includes pretreatment of glioma samples; specifically, it includes: mechanically cutting surgically removed glioma tissue into fragments with a diameter of 0.3-0.5 mm, washing three times with PBS buffer containing penicillin-streptomycin, centrifuging at 1000 rpm for 5 minutes to remove necrotic tissue and impurities, and obtaining glioma samples.

[0056] The specific process for preparing glioma samples is as follows: freshly removed glioma tissue is processed through a "mechanical mincing-washing-centrifugation purification" process to finally obtain a viable sample suitable for culture. The purpose of each step is as follows:

[0057] Mechanically shearing to 0.3-0.5mm fragments: Microscopic scissors are used to finely cut the isolated tissue, breaking down the dense structure of the tissue block and allowing tumor cells to partially detach from the stroma, while preserving local intercellular connections and microenvironmental components (such as extracellular matrix fragments), laying the foundation for subsequent cell migration and proliferation.

[0058] Wash three times with PBS containing penicillin-streptomycin: PBS buffer can remove blood, cell debris and other impurities from the tissue surface; penicillin-streptomycin (double antibody) can eliminate microbial contamination that may be introduced during surgery by inhibiting bacterial cell wall synthesis and protein synthesis, thus reducing the risk of culture failure.

[0059] Centrifuge at 1000 rpm for 5 minutes: This centrifugation condition (relatively low speed and short time) can separate viable tissue fragments from necrotic cells and impurities through density differences (necrotic tissue has a lower density and is easily suspended in the supernatant). This avoids mechanical damage to viable cells caused by high-speed centrifugation, achieves sample purification, and improves the survival rate of cells in subsequent culture.

[0060] A fragment size of 0.3-0.5 mm is the optimal choice for balancing cell viability, nutrient supply, and proliferation efficiency. Specific advantages are as follows: Excessively large fragments (e.g., >1 mm) can obstruct nutrient exchange between the core and the external environment, preventing oxygen and glucose from effectively penetrating to the center and easily leading to cell hypoxia and necrosis. Conversely, excessively small fragments (e.g., <0.2 mm) can cause excessive mechanical shearing, damaging intercellular connections and cell membrane structure, resulting in loss of cell viability (experiments show that the viability of <0.2 mm fragments is 20%-30% lower than that of 0.3-0.5 mm fragments). A diameter of 0.3-0.5 mm ensures a reasonable surface area to volume ratio, allowing glucose and growth factors (such as EGF and FGF) in the culture medium to rapidly diffuse into the fragment, meeting cellular metabolic needs, while also preserving autocrine signaling molecules (such as tumor-associated factors) in the local microenvironment, maintaining the cell's biological phenotype. Fragments of this size can serve as "proliferation units." After cells migrate from the edge of the fragment, they can proliferate and aggregate in an orderly manner within the porous structure of the scaffold, eventually forming organoids with three-dimensional structures. Their formation efficiency is higher than that of fragments of other sizes, and the morphology of the organoids is more similar to tumor tissue in vivo.

[0061] In some feasible protocols, when constructing glioblastoma organoids, the culture medium is also supplemented with 1 μM Erlotinib and 5 μg / mL cholesterol, and the scaffold and surrounding culture medium are subjected to a periodic pressure of 0.1 kPa at a frequency of 0.1 Hz daily.

[0062] This indicates that the design of adding 1 μM Erlotinib, 5 μg / mL cholesterol, and applying 0.1 kPa periodic pressure (0.1 Hz) to the culture medium 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) (such as EGFRvⅢ), which leads to the continuous activation of downstream signaling pathways such as PI3K / Akt and MAPK, driving unlimited cell proliferation and invasion. Erlotinib, as a selective EGFR tyrosine kinase inhibitor, mimics the pathological and drug response environment in the following ways: Targeted inhibition of abnormal signals: A concentration of 1 μM specifically blocks EGFR kinase activity, inhibiting excessive tumor cell proliferation while preserving moderate signal intensity to maintain organoid stem cell characteristics (avoiding phenotypic differentiation caused by complete inhibition). This concentration is close to the effective plasma concentration for clinical treatment (0.5-2 μM), and can be used to simulate the in vivo drug exposure environment, providing a physiological basis for subsequent drug sensitivity testing; Inhibiting the highly active EGFR subsets without affecting the survival of the low-expression subsets, preserving the inherent cellular heterogeneity of GBM. Glioblastoma cells exhibit abnormally active cholesterol metabolism; their cell membranes are rich in cholesterol to support membrane structure synthesis under high proliferation rates, and cholesterol is a key regulator of oncogenic signaling pathways such as Hedgehog. The effects of adding 5 μg / mL cholesterol include: GBM cells often have a compensatory increase in cholesterol synthesis capacity, and exogenous supplementation can reduce the cell's own synthetic burden and avoid decreased activity due to metabolic depletion; cholesterol stabilizes cell membrane fluidity, ensures the correct positioning and function of membrane proteins such as EGFR and integrins, and promotes the adhesion of tumor cells to the scaffold and intercellular communication; cholesterol is an essential nutrient for the self-renewal of tumor stem cells, and this concentration can maintain the proportion of stem cell subsets such as CD133+, thus reproducing the treatment resistance-related phenotype of GBM. The mechanical regulation mechanism of 0.1 kPa periodic pressure (0.1 Hz) simulates the in vivo mechanical microenvironment: 0.1 kPa pressure is close to the normal intracranial hydrostatic pressure, and the 0.1 Hz frequency (10-second cycle) simulates the low-frequency fluctuations caused by respiration or pulse, which can activate mechanically sensitive transcription factors such as YAP / TAZ, promote the synthesis of extracellular matrix (such as fibronectin), and enhance the integration of organoids with the scaffold; periodic pressure induces the secretion of matrix metalloproteinases such as MMP-2 / 9 through mechanical stimulation, simulating the invasive growth characteristics of GBM, making the organoids more closely resemble the invasive behavior of tumors in vivo; low-frequency pressure fluctuations can promote the flow of culture medium within the porous structure of the scaffold, reduce local nutrient gradient differences, and avoid necrosis of the organoid core due to hypoxia and hypoglycemia.

[0063] Example 1: Construction of organoids from IDH-mutant gliomas

[0064] Sample pretreatment: IDH mutant glioma tissue was surgically removed, mechanically minced into fragments with a diameter of 0.3-0.5 mm, washed 3 times with PBS buffer containing penicillin-streptomycin, centrifuged at 1000 rpm for 5 minutes to remove necrotic tissue and impurities, and glioma samples were obtained.

[0065] Scaffold preparation: 8% w / v methacrylamide gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin were mixed, and 0.1% w / v carbon nanotubes and 50 μM MRGD peptide were added. A porous scaffold with a pore size of 100-150 μm was constructed by laser-assisted bioprinting technology, with a hollow vascular channel with a diameter of 200 μm reserved.

[0066] Cell seeding: Glioma samples were mixed with primary brain microvascular endothelial cells at a cell ratio of 1:5 and seeded into porous scaffold structures; primary brain microvascular endothelial cells were seeded separately into hollow vascular channels (density 2×10⁶). 6 (cells / mL).

[0067] Cultivation conditions:

[0068] Culture medium: DMEM / F12 and Neurobasal were mixed at a volume ratio of 3:2 (containing 4.5 g / L glucose and 2 mM L-glutamine), and 1×B-27 additive (without vitamin A), 1×N-2 additive, 15 ng / mLEGF, 15 ng / mLFGF, 100 ng / mLVEGF, 20 ng / mLHGF, 50 μM MDF, 2 mM pyruvate, 100 μM vitamin C and 1 mM 2-hydroxyglutarate were added.

[0069] Gas environment: 5% CO2 + 5% O2.

[0070] Dynamic control: The culture medium was circulated at a flow rate of 0.3 mL / min using a microfluidic pump, and a fluid shear force of 1 dyn / cm² was applied; the medium was irradiated with blue light at a wavelength of 470 nm and an intensity of 5 mW / cm² for 2 hours daily; the glucose / lactic acid concentration was monitored every 6 hours, and DFO was supplemented to 75 μM when glucose was <0.8 g / L, and a 165 rpm pulse oscillation was initiated when lactate was >1.8 g / L; 50% fresh culture medium was added every 3 days.

[0071] Example 2: Construction of Glioblastoma Organoids

[0072] Sample pretreatment: Same as in Example 1, the sample was glioblastoma tissue.

[0073] Stent preparation: Same as in Example 1.

[0074] Cell seeding: Same as in Example 1.

[0075] Cultivation conditions:

[0076] Culture medium: 1 μM Erlotinib and 5 μg / mL cholesterol were added to the culture medium of Example 1.

[0077] Gaseous environment: 5% CO2 + 1% O2.

[0078] Dynamic control: microfluidic pump flow rate 0.5 mL / min, fluid shear force 1.5 dyn / cm²; daily application of 0.1 kPa periodic pressure (frequency 0.1 Hz), other monitoring and control are the same as in Example 1.

[0079] Comparative Example 1: Blue Light-Free IDH Mutant Model

[0080] Sample processing, scaffold preparation, and cell seeding: exactly the same as in Example 1.

[0081] Culture conditions: Except for the elimination of the daily blue light irradiation step, the other culture medium components, gas environment, fluid shear force, metabolic monitoring and control methods are the same as in Example 1.

[0082] Comparative Example 2: Glioblastoma model without blue light

[0083] Sample processing, scaffold preparation, and cell seeding: exactly the same as in Example 2.

[0084] Culture conditions: Except for the elimination of the daily blue light irradiation step, the other culture medium components, gas environment, periodic pressure, and metabolic regulation were the same as in Example 2.

[0085] Comparative Example 3: Traditional Blue Light-Free Static Model

[0086] Sample processing: The tissue was minced to 1-2 mm, washed once with PBS, and not purified by centrifugation.

[0087] Stent fabrication: A 10% w / v pure gelatin stent was used, without porous structure or hollow vascular access.

[0088] Cell inoculation: Only glioma cells were inoculated; no primary brain microvascular endothelial cells were added.

[0089] Culture conditions: Single DMEM medium (containing 10% fetal bovine serum), normoxic environment (5% CO2 + 21% O2), static culture without blue light, fluid shear force and metabolic monitoring, and complete replacement of the medium every 7 days.

[0090] Data detection methods

[0091] Organoid formation rate (7 days): The proportion of organoids with a diameter >100μm and complete structure formed was observed by inverted microscope to the total number of seeded cell clusters. The data was recorded and calculated on the 7th day.

[0092] Vascular patency (14 days): Fluorescently labeled endothelial cells (DiI staining) were used to observe the proportion of the length of the tubular structure of endothelial cells in the vascular channel to the total length of the channel using confocal microscopy, and the average value of 3 fields of view was taken.

[0093] Core necrosis rate (21 days): The percentage of the total area of ​​the organoid core necrosis region (red fluorescence) was analyzed using ImageJ software by Live / Dead staining (calcein-AM / PI).

[0094] Endothelial cell tight junction integrity: The expression of the tight junction protein occludin was detected by immunofluorescence, and the proportion of positive expression areas to the total area of ​​vascular endothelial cells was calculated and divided into "excellent (>80%)" and "poor (<60%)" grades.

[0095] Intercellular calcium signal transduction efficiency: Cells were labeled with a calcium fluorescent probe (Fluo-4AM), and the speed and range of calcium signal transduction after blue light stimulation were recorded by a fluorescence imaging system and classified as "high" or "low".

[0096] Weekly survival rate: The proportion of organoids that remain structurally intact and highly active after 4 weeks of culture out of the initial number of organoids formed.

[0097] Table 1 is a statistical table of experimental data.

[0098]

[0099] The 7-day formation rates of Examples 1 and 2 (91%, 89%) were significantly higher than those of Comparative Examples 1 and 2 (68%, 63%) without blue light, with a difference of 23%-26%. Blue light activates cellular light-sensitive channel proteins, triggering calcium signal transduction and enhancing the adhesion and proliferative synergy between tumor cells and endothelial cells. Comparative Example 3, lacking both blue light and a biomimetic system, had the lowest formation rate (42%). The vascular patency rate in the blue light-containing group (81%-84%) was approximately 35% higher than that in the blue light-free group (49%-53%), and the integrity of endothelial cell tight junctions (88%-90%) was far superior to that in the blue light-free group (51%-55%). This is directly related to the blue light-induced polar arrangement of endothelial cells and the upregulation of tight junction protein (occludin) expression. Comparative Example 3, lacking a foundation for vascular construction, had a patency rate of 0%. The core necrosis rate in the blue light group (5%-7%) was significantly lower than that in the non-blue light group (19%-21%). The mechanism is that blue light optimizes energy metabolism by regulating mitochondrial function and reduces acidification damage caused by lactic acid accumulation. In contrast, the necrosis rate in Comparative Example 3 was as high as 48% due to nutrient exchange impairment caused by static culture. Blue light irradiation increased the survival rate of Example 4 (77%-79%) by about 30% compared with the non-blue light group (49%-53%). This is because blue light activates antioxidant pathways (such as enhanced SOD activity) and reduces cell apoptosis, while the survival rate of the traditional model was less than 20% due to multiple defects.

[0100] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for constructing glioma organoids, characterized in that: Includes the following steps, Step S1: Glioma samples are mixed with primary brain microvascular endothelial cells at a cell ratio of 1:5, and then seeded into a porous structure of a scaffold. The hollow vascular channels of the scaffold are separately seeded with primary brain microvascular endothelial cells. The scaffold is prepared by mixing 8% w / v methacrylamide gelatin, 3% w / v hyaluronic acid and 2% w / v silk fibroin, adding 0.1% w / v carbon nanotubes and 50 μM MRGD peptide, and constructing a porous scaffold with a pore size of 100-150 μm using laser-assisted bioprinting technology. The scaffold has a reserved hollow vascular channel with a diameter of 200 μm. Step S2: The scaffold from step S1 is placed in a gas-liquid interface culture device to culture glioma samples. The gas-liquid interface culture device includes a culture medium. The bottom of the scaffold is immersed in the culture medium, and the top of the scaffold is exposed to a mixed gas environment. The mixed gas environment is a mixture containing 5% CO2 and 1-5% O2. When culturing IDH mutant glioma organoids, the O2 concentration is 5%; when culturing glioblastoma organoids, the O2 concentration is 1%. A microfluidic pump circulates the culture medium at a flow rate of 0.3-0.5 mL / min, applying a fluid shear force of 1-1.5 dyn / cm² to the scaffold surface. Daily, a fluid is applied at a wavelength of 470 nm and an intensity of 5 mW / cm². 2 The area of ​​the support was irradiated with blue light for 2 hours.

2. The method for constructing glioma organoids as described in claim 1, characterized in that: The culture medium includes a basal culture medium and additives. The basal culture medium is a mixture of DMEM / F12 and Neurobasal at a volume ratio of 3:

2. The additives, based on the final concentration, include: 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 pyruvate, and 100 μM vitamin C.

3. The method for constructing glioma organoids as described in claim 2, characterized in that: The culture medium also contains 1 mM 2-hydroxyglutaric acid.

4. The method for constructing glioma organoids as described in claim 1, characterized in that: The culture conditions include monitoring the glucose and lactate concentrations in the culture medium every 6 hours, adding DFO to the culture medium to 75 μM when glucose is <0.8 g / L, and starting the culture device with a 165 rpm pulse oscillation when lactate is >1.8 g / L.

5. The method for constructing glioma organoids as described in claim 4, characterized in that: The cultivation conditions also include: wherein the culture medium is replaced by adding 50% new culture medium to the cultivation device every 3 days.

6. The method for constructing glioma organoids as described in claim 1, characterized in that: It also includes the pretreatment of glioma samples; the surgically removed glioma tissue is mechanically cut into fragments with a diameter of 0.3-0.5 mm, washed three times with PBS buffer containing penicillin-streptomycin, and centrifuged at 1000 rpm for 5 minutes to remove necrotic tissue and impurities to obtain glioma samples.

7. The method for constructing glioma organoids as described in claim 1, characterized in that; When constructing glioblastoma organoids, 1 μM Erlotinib and 5 μg / mL cholesterol were added to the culture medium, and a periodic pressure of 0.1 kPa was applied to the scaffold and surrounding culture medium daily at a frequency of 0.1 Hz.

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