A method for constructing a high-aggressiveness glioblastoma mouse orthotopic model

Abstract

This invention relates to a method for constructing a mouse orthotopic model of highly aggregated glioblastoma, belonging to the fields of biomedicine and experimental animal model technology. Glioblastoma cells are resuspended in a composite carrier of a specific ratio of matrix gel and serum-free culture medium, and a low-temperature injection technique is used. Utilizing the thermosensitivity of matrix gel in the mouse intracranial environment (i.e., its phase transition from liquid to gel at a certain temperature), precise colonization and physical locking of tumor cells at target coordinate points are achieved. This method improves upon existing glioblastoma mouse orthotopic model construction techniques that suffer from tumor cell diffusion and loss, low tumor formation rate, irregular tumor morphology, large intragroup variability, and needle reflux due to low viscosity of the inoculation carrier. It provides a good disease model for subsequent screening or evaluation of anti-glioblastoma drugs.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and experimental animal models, and in particular to a method for constructing an orthotopic mouse model of highly aggregated glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is the most common primary malignant tumor of the central nervous system, characterized by extremely high invasiveness and mortality. To further investigate its pathogenesis and evaluate the efficacy of antitumor drugs, establishing stable and reliable mouse orthotopic tumor models is a fundamental approach in neuro-oncology research.

[0003] Currently, existing methods for constructing orthotopic models of glioblastoma primarily involve directly injecting tumor cell suspensions into the mouse brain parenchyma using microinjectors. In this routine procedure, phosphate-buffered saline (PBS) or serum-free culture medium is typically used as the cell transport medium. However, this existing technology suffers from the following major problems in practical applications: First, due to the soft texture of brain tissue and the presence of complex interstitial spaces and cerebrospinal fluid flow, low-viscosity PBS or culture medium carriers undergo rapid physical permeation upon entering the brain. This causes the inoculated tumor cells to often fail to remain at the intended anatomical site, instead dispersing diffusely along the needle tract or interstitial spaces, resulting in cell diffusion and flow cytometry. Second, early cell dispersion greatly increases the likelihood of tumorigenesis, leading to a low tumorigenesis rate. This makes subsequent experiments difficult to proceed; secondly, the early dispersion of cells leads to blurred tumor boundaries and a lack of obvious space-occupying effect. This diffuse growth pattern of tumor cells (i.e., irregular tumor morphology) results in significant errors in subsequent tumor volume measurements, seriously affecting the accuracy and precision of subsequent experimental evaluations; furthermore, the diffusion path of tumor cells injected into the brain of each mouse is random, leading to significant differences in tumor volume, growth rate, and drug response among mice in the same batch, reducing the reproducibility of experimental results; finally, low-viscosity liquids are prone to backflow at the moment of needle withdrawal, causing some cells to be carried out of the brain parenchyma, which not only reduces the actual number of cells injected but may also induce ectopic tumors outside the skull or under the scalp.

[0004] Therefore, there is an urgent need for a scheme to construct an in situ model of glioblastoma that can effectively limit early cell spread, improve cell seeding aggregation, and ensure regular tumor morphology. Summary of the Invention

[0005] The purpose of this invention is to address the technical shortcomings in the existing in situ mouse model of glioblastoma, such as tumor cell diffusion and loss due to low viscosity of the inoculation carrier, low tumor formation rate, irregular tumor morphology, large intragroup variability, and needle tract reflux, by providing a method for constructing a highly aggregated glioblastoma mouse model.

[0006] The first aspect of this invention provides a method for constructing an orthotopic mouse model of highly aggregated glioblastoma, employing the following technical solution:

[0007] A method for constructing a mouse orthotopic model of highly aggregated glioblastoma includes the following steps:

[0008] S1. Pre-cool consumables such as microsyringes, centrifuge tubes, and pipette tips in a low-temperature environment;

[0009] S2. Mix the matrix gel and serum-free DMEM medium evenly in the pre-cooled centrifuge tube from S1 to form a composite carrier;

[0010] S3. Resuspend the glioblastoma cells in the composite carrier prepared in S2 to obtain a cell suspension. The entire process is carried out in an ice box to maintain the liquid state of the composite carrier.

[0011] S4. Using a microsyringe pre-cooled in S1, inject the cell suspension obtained in S3 into the target coordinate point of the mouse brain parenchyma using stereotactic positioning.

[0012] S5. After injection, leave the needle in place until the matrix gel undergoes a physical phase transition to form a gel under the intracranial temperature environment of the mouse, then withdraw the needle.

[0013] By employing the above-mentioned technical solution, glioblastoma cells are resuspended in a composite carrier containing a specific ratio of matrix gel and serum-free culture medium. Combined with a low-temperature injection process, the thermosensitivity of matrix gel in the mouse intracranial environment (i.e., its phase transition from liquid to gel at a certain temperature) enables precise colonization and physical locking of tumor cells at target coordinates. At low temperatures, the composite carrier is liquid with low viscosity. At this temperature, the tumor cells (point-like) are randomly and dispersedly distributed in the inoculation solution, facilitating preparation, sample aspiration, and injection. After injection, upon entering the mouse brain parenchyma, the composite carrier rapidly undergoes a physical phase transition induced by body temperature (approximately 37°C), changing from a liquid to a semi-solid gel. At this point, the gel mesh... The physical anchoring of tumor cells, causing them to form highly aggregated clusters at the target coordinate point, is the core foundation for achieving subsequent regular tumor morphology and a significant reduction in intra-group coefficient of variation (CV). Compared to the diffuse growth caused by traditional liquid carriers, the initial lesions formed by this invention are compact and spherical, simulating the pathological characteristics of single-focal tumor origin and eliminating the randomness of early cell diffusion. The differences in tumor volume and growth rate among mice in the same batch are significantly reduced, and the intra-group differences are greatly reduced, improving the statistical power of subsequent experiments. At the same time, the gel plug formed by the composite carrier at body temperature ensures rapid closure of the needle track after needle withdrawal, effectively preventing tumor cells from leaking into the subscalp or outside the skull, and ensuring the purity of in situ tumor formation.

[0014] Preferably, in the above construction method, in step S1, the low-temperature environment is 4°C and the pre-cooling time is not less than 30 minutes.

[0015] Preferably, in the above construction method, in step S2, the volume ratio of matrix gel to serum-free DMEM is 1:2.

[0016] Preferably, in the above construction method, in step S3, the glioblastoma originates from mouse glioblastoma GL261-Luc cells.

[0017] Preferably, in the above construction method, in step S3, the cell concentration in the cell suspension is 2*102 4 cells / μL.

[0018] Preferably, in the above construction method, the micro-syringe in step S4 is wrapped with a flexible cold trap or a semiconductor flexible cooling sheet to draw up the cell suspension.

[0019] Preferably, in the above construction method, the stereotactic injection rate in step S4 is 0.2 μL / min.

[0020] Preferably, in the above construction method, the in-situ needle retention time in step S5 is 8 minutes.

[0021] By adopting the above technical solution, the specific operational steps and parameters in the construction method are optimized to obtain a better mouse model.

[0022] A second aspect of the present invention provides the application of the mouse orthotopic model of highly aggregated glioblastoma constructed by the above method in screening or evaluating drugs for the treatment of glioblastoma.

[0023] In summary, this application includes at least one of the following beneficial technical effects:

[0024] 1. Through the in-situ solidification of the matrix gel within the skull, tumor cells are physically confined to predetermined inoculation coordinates. Compared to the diffuse growth caused by traditional liquid carriers, the initial lesions formed in this application are compact and spherical, simulating the pathological characteristics of a single-focal origin of a tumor.

[0025] 2. The temperature sensitivity of the matrix gel eliminated the randomness of early cell diffusion, thus significantly reducing the differences in tumor volume and growth rate among mice in the same batch, greatly reducing the differences within the model group, and improving the statistical power of subsequent experiments.

[0026] 3. The gel plug formed by the composite carrier at body temperature ensures rapid closure of the needle tract after needle withdrawal, effectively preventing tumor cells from leaking under the scalp or outside the skull, and ensuring the purity of in situ tumor formation. Attached Figure Description

[0027] Figure 1 This is a schematic diagram showing the physical phase transition and cell distribution of the composite carrier at different temperatures.

[0028] Figure 2 The in vivo imaging dynamic matrix diagram of mice constructed for Example 1;

[0029] Figure 3 The in vivo imaging dynamic matrix diagram of the mouse constructed for Comparative Example 1;

[0030] Figure 4 The in vivo imaging dynamic matrix diagram of the mouse constructed for Comparative Example 2. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should all be covered within the protection scope of this invention.

[0032] Unless otherwise specified, the reagents, instruments and equipment used in the following examples are all commercially available products. Other specific conditions not specified shall be performed according to standard conditions or the manufacturer's recommendations.

[0033] The chemical abbreviations and Chinese definitions used in this application are based on the generally accepted understanding within the industry.

[0034] The sources of some of the raw materials and reagents used in this application are as follows:

[0035] Matrix adhesive: purchased from MCE, part number HY-K6008;

[0036] Serum-free DMEM medium: purchased from Gibco, catalog number C11995500BT;

[0037] GL261-Luc cells, a type of glioblastoma, were purchased from Shanghai Xinzhou Biotechnology Co., Ltd. After resuscitation, GL261-Luc cells were cultured in a 37 ℃, 5% CO2 incubator using dedicated GL261-Luc cell culture medium (also purchased from Shanghai Xinzhou Biotechnology Co., Ltd.). When the cells reached 60%-70% confluence, the old culture medium was removed and replaced with fresh complete culture medium containing 1 μg / mL puromycin. The initial maintenance concentration of puromycin was approximately 1 μg / mL. Treatment continued for 48-72 h. During this period, individual cell death was observed, while strongly positive cells continued to divide and grow. If there were many dead cells, the puromycin-containing culture medium was changed every 2 days to prevent the release of harmful substances from dead cells from affecting living cells. When the cell density reached the passage requirement, the puromycin concentration could be increased, but not exceeding 4 μg / mL. After selection, the puromycin concentration was halved (2 μg / mL) for long-term maintenance culture to reduce the inhibitory effect of the drug on cell growth. When the confluence reaches 80%-90% of the passage, the cells are collected and resuspended for injection.

[0038] Anesthetic: Delivector™ Avertin, purchased from Duowo Biotechnology, catalog number DW3106;

[0039] D-fluorescein potassium salt: purchased from Beyotime, product number ST-196;

[0040] Preparation method of D-fluorescein potassium salt: Use sterile D-PBS (Mg-free) 2+ and Ca 2+ Dissolve and prepare a 15 mg / ml solution of potassium D-luciferin, then filter through a 0.2 μm membrane for sterilization. Use immediately after mixing. Protect from light during use.

[0041] Small animal live imaging system: Berthold Technologies, Germany;

[0042] Ice pack: purchased from Labshark;

[0043] Laboratory mice: C57BL / 6J, 6 weeks old, provided by (Chongqing Enbi Biotechnology Co., Ltd.)

[0044] I. Implementation Examples

[0045] Example 1

[0046] This embodiment discloses a method for constructing a mouse orthotopic model of highly aggregated glioblastoma, including the following steps:

[0047] S1. Pre-cool consumables such as microsyringes, centrifuge tubes, and pipette tips in a low-temperature environment.

[0048] Specifically, the 5μL microsyringe, 1.5mL centrifuge tube and pipette tip are placed at 4°C for 30 minutes. In other feasible embodiments, the pre-cooling time can be any time value exceeding 30 minutes.

[0049] S2. Mix the matrix gel and serum-free DMEM medium evenly in the pre-cooled centrifuge tube from S1 to form a composite carrier.

[0050] Specifically, the matrix gel is melted on ice, and the melted matrix gel is mixed with serum-free DMEM culture medium at a volume ratio of 1:2 in a pre-cooled centrifuge tube to form a fluid composite carrier.

[0051] S3. Resuspend the glioblastoma cells in the composite carrier prepared in S2 to obtain a cell suspension. The entire process is carried out in an ice box to maintain the liquid state of the composite carrier.

[0052] Specifically, GL261-Luc glioblastoma cells in the logarithmic growth phase were taken, resuspended in the composite carrier prepared by S2, and the cell concentration was adjusted to 2*102. 4 The cells / μL were kept in an ice box throughout the operation to maintain the liquid state of the composite carrier.

[0053] S4. Using a microsyringe pre-cooled in S1, inject the cell suspension obtained in S3 into the target coordinate point of the mouse brain parenchyma using stereotactic positioning.

[0054] Specifically, after anesthetizing the experimental animal (mouse), it was fixed on a stereotaxic instrument and the anterior fontanelle was exposed; the coordinates of the right striatum were located as follows: AP +0.5mm, ML +2.0mm, DV -3.0mm; the cell suspension was aspirated using a microsyringe pre-cooled with S1 (the external components may be wrapped with a flexible cold trap, flexible semiconductor cooling pad, semiconductor cooling ring, or other components with continuous low temperature control, depending on the situation) and injected at a constant rate of 0.2 μL / min.

[0055] S5. After injection, leave the needle in place until the matrix gel undergoes a physical phase transition to form a gel under the intracranial temperature environment of the mouse, then withdraw the needle.

[0056] Specifically, the needle was left in place for 8 minutes after injection. During this period, the matrix gel was induced by the mouse's body temperature to change from a liquid state to a semi-solid gel, thus locking the cells.

[0057] The mouse models constructed in this embodiment are numbered 1-X (X is the mouse number).

[0058] Schematic diagram of physical phase transitions and cell distribution of the composite carrier at different temperatures, as shown below. Figure 1 As shown, at 4°C, the carrier is in a liquid state with low viscosity. At this temperature, tumor cells (point-like) are randomly and dispersedly distributed in the inoculation solution. This state corresponds to the in vitro preparation, sample aspiration and injection process.

[0059] After the carrier enters the mouse brain parenchyma, it undergoes a rapid physical phase transition induced by the body temperature (about 37°C), changing from a liquid state to a semi-solid gel. At this point, the gel mesh physically anchors the tumor cells, causing them to form highly aggregated clusters at the target coordinate points. This physical process is the core basis for achieving subsequent tumor morphology regularity and a significant reduction in intragroup coefficient of variation (CV).

[0060] II. Comparative Example

[0061] Comparative Example 1

[0062] A method for constructing an orthotopic mouse model of glioblastoma, differing from Example 1 in that, in this comparative example, an equal amount of PBS is used as the carrier to replace the composite carrier in Example 1, and an equal amount of Luc-labeled mouse glioblastoma GL261-Luc cells are inoculated, i.e., 10 cells are inoculated per mouse. 5 The remaining operations were the same as in Example 1, involving only one cell.

[0063] The mouse models constructed in this comparative example are numbered 2-X (X is the mouse number).

[0064] Comparative Example 2

[0065] A method for constructing an orthotopic mouse model of glioblastoma differs from Example 1 in that, in this comparative example, the volume ratio of matrix gel to serum-free DMEM in the composite carrier is 1:3, while the remaining operations are the same as in Example 1.

[0066] The mouse models constructed in this comparative example are numbered 3-X (X is the mouse number).

[0067] III. Performance Test Experiments and Results

[0068] 1. Experimental Methods

[0069] (1) Weigh the mice and administer an intraperitoneal injection of an anesthetic at a dose of 20 μL / g.

[0070] (2) After the mouse enters a stable anesthesia state, remove the hair from the head area to avoid the hair interfering with the subsequent optical imaging signal.

[0071] (3) Inject luciferin potassium salt solution into the peritoneum of mice at a concentration of 10 μL / g per body weight. After injection, let the mice stand for 10 min until the fluorescence signal reaches the strongest stable plateau period.

[0072] (4) Place the mouse into the in vivo optical imaging system and collect chemiluminescence signals for detection.

[0073] 2. Results

[0074] Mouse in vivo imaging results as follows Figures 2-4 As shown, where, Figure 2 The in vivo imaging dynamic matrix diagram of mice constructed in Example 1. Figure 3 This is a dynamic matrix diagram of in vivo imaging of mice constructed for Comparative Example 1. Figure 4 The in vivo imaging dynamic matrix diagram of the mouse constructed for Comparative Example 2.

[0075] As shown in the figure, the tumor formation rate of mice constructed in Example 1 reached 100% after surgery. Around 17 days after surgery, the fluorescence was highly concentrated at the injection coordinate point and distributed in a spherical shape. The tumors formed on the 27th day had clear boundaries and showed typical blocky growth. The cells were in regular spherical clusters. On the 21st day, the tumor volume of each mouse was similar. This proves that the addition of matrix gel can significantly improve the cell aggregation in the early stage of inoculation and prevent diffusion.

[0076] Comparative Example 1 uses the conventional PBS vector inoculation method. The fluorescence images of mice constructed by this method show that the cells are diffused in a cord-like manner along the needle tract and the intercerebral space about 17 days after surgery. Most mice did not form tumors. The tumors formed on day 27 were irregular in shape and the cells were dispersed along the needle tract. On day 21, the tumor volume of each mouse varied greatly. During the 34-day monitoring period, the tumor formation rate was 40%.

[0077] Comparative Example 3 adjusted the volume ratio of matrix gel to serum-free DMEM. The mice constructed using this method had a tumor formation rate of 80% during the 34-day monitoring period, and the tumor tissue size varied considerably among the mice. This demonstrates that a volume ratio of 1:2 for matrix gel to DMEM is necessary to obtain the best tumor formation rate and reduce intragroup variability.

[0078] In summary, in vivo imaging of mice constructed using this method showed that cells formed regular spherical clusters after surgery; while in mouse models constructed using the PBS carrier method, cells were scattered along the needle tract, and most did not form tumors. Long-term monitoring data showed that the tumor formation rate of the method of this invention reached 100%, and the fluorescence signal was highly concentrated; while the PBS carrier group frequently showed signal loss or extremely low values ​​in the early stage. By day 21 after inoculation, the tumor volume in the mouse model constructed using this method was similar and much larger than that in the PBS carrier group. The results demonstrate that the high aggregation inoculation provided by this method greatly improves the standardization of experimental animal models, significantly improves morphological aggregation and model consistency, and effectively avoids needle tract reflux, providing a good disease model for subsequent screening or evaluation of drugs for the treatment of glioblastoma.

Claims

1. A method for constructing a mouse orthotopic model of highly aggregated glioblastoma, characterized in that: Includes the following steps: S1. Pre-cool consumables such as microsyringes, centrifuge tubes, and pipette tips in a low-temperature environment; S2. Mix the matrix gel and serum-free DMEM medium evenly in the pre-cooled centrifuge tube from S1 to form a composite carrier; S3. Resuspend the glioblastoma cells in the composite carrier prepared in S2 to obtain a cell suspension. The entire process is carried out in an ice box to maintain the liquid state of the composite carrier. S4. Using a microsyringe pre-cooled in S1, inject the cell suspension obtained in S3 into the target coordinate point of the mouse brain parenchyma using stereotactic positioning. S5. After injection, leave the needle in place until the matrix gel undergoes a physical phase transition to form a gel under the intracranial temperature environment of the mouse, then withdraw the needle.

2. The construction method according to claim 1, characterized in that: In S1, the low-temperature environment is 4°C, and the pre-cooling time is not less than 30 minutes.

3. The construction method according to claim 1, characterized in that: In S2, the volume ratio of matrix gel to serum-free DMEM is 1:

2.

4. The construction method according to claim 1, characterized in that: In S3, the glioblastoma originates from mouse glioblastoma GL261-Luc cells.

5. The construction method according to claim 1, characterized in that: In step S3, the cell concentration in the cell suspension is 2*102 4 cells / μL.

6. The construction method according to claim 1, characterized in that: The micro-syringe in S4 is externally wrapped with a flexible cold trap or a semiconductor flexible cooling plate to draw up cell suspension.

7. The construction method according to claim 1, characterized in that: The stereotactic injection rate in S4 is 0.2 μL / min.

8. The construction method according to claim 1, characterized in that: The in-situ needle retention time in S5 is 8 minutes.

9. The application of the mouse orthotopic model of highly aggregated glioblastoma constructed by the method according to any one of claims 1-8 in screening or evaluating drugs for the treatment of glioblastoma.

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