Construction method and application of novel subcutaneous transplantation tumor animal model
By using collagen sponge blocks as carriers, single tumor cell suspension is injected into animals and inoculated into the body of animals, the problems of slow tumor growth rate and high experimental cost in the prior art are solved, and more efficient tumor tumor formation and lower experimental difficulty and cost are achieved.
Patent Information
- Application Number
- CN202510233551.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-06
AI Technical Summary
The existing animal model construction methods for subcutaneous transplant tumors have problems such as slow tumor formation speed, unnatural cell growth, high experimental cost and poor repeatability.
A collagen sponge block was used as a scaffold, and a single tumor cell suspension was injected into the collagen sponge block, and it was inoculated into the animal by subcutaneous puncture to construct an animal model of subcutaneous tumor transplantation.
The tumor tumor formation rate and tumor formation speed are improved, the natural morphology of tumor cells is maintained, the experimental cost and difficulty is reduced, and the experiment is improved.
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Figure CN119925030A_ABST
Abstract
Description
Technical Field
[0001] The present application belongs to the field of biomedicine technology, and more specifically, relates to a method for constructing a novel subcutaneous tumor transplantation animal model and its application. Background Art
[0002] Tumor animal models are important tools for studying the occurrence and development mechanisms of tumors and drug screening. At present, commonly used tumor animal models include spontaneous, induced and transplant models. Among them, the transplant tumor model is to make a group of animals simultaneously bear tumors by inoculating a certain amount of tumor cells or tumor masses in animals. The growth rate is relatively consistent, the survival rate of inoculation is high, and it can be continuously transplanted in animals of the same species or strain for a long time. The test cycle is relatively short. It is the most commonly used model type in the current preclinical efficacy study of anticancer drugs. According to the location of the transplantation, the transplant tumor model can be divided into an orthotopic transplant tumor model and an ectopic transplant tumor model. The orthotopic transplant tumor model can better simulate the original environment of the tumor, but it is not easy to continuously observe the growth of the tumor; the ectopic transplant tumor model is mostly formed by subcutaneously injecting a certain amount of tumor cells into the back of the experimental animal. The subcutaneous tumor formation operation is simple and easy to master, with a high success rate, and the change in tumor volume is easy to measure, and it is easy to give local intervention. It is a common method for tumor animal modeling.
[0003] Common heterotopic transplant tumor models generally use mice as test animals and are established by inoculating tumor cell suspensions or tumor cells into tumor masses. Both methods require the test animals to be injected with cell suspensions to form tumors. Although subcutaneous injection of cell suspensions into animals to form tumors is simple and easy in actual operation, its success rate is affected by many factors. In addition to factors such as the malignancy and status of the tumor cells themselves and the status of the test animals, the number of tumor cells is crucial.
[0004] At present, the construction of subcutaneous transplanted tumor models in animals, represented by mice, is mainly achieved by direct injection of tumor cell suspension or by using matrix gel (such as Matrigel). TM ) After mixing tumor cells and then injecting subcutaneously, the main disadvantages are as follows:
[0005] (1) Model construction by direct injection of cell suspension: Tumor cells grow in a dispersed manner in a two-dimensional environment and lack connections similar to those between tissues. They require a high concentration of tumor cell suspension to easily form a tumor, and the overall tumor formation rate is slow.
[0006] (2) Use Matrigel TM Matrigel model construction:
[0007] ① Researchers have extracted the soluble basement membrane component Matrigel from Engelbreth-Holm-Swarm (EHS) mouse sarcoma TMMatrigel contains laminin, IV collagen and nestin, as well as basement membrane polysaccharide, TGF-β, epidermal growth factor, insulin-like growth factor, tissue plasminogen and other factors, which can provide a favorable microenvironment for the invasion, migration and formation of luminal structures of tumor cells, and effectively improve the success rate of tumor formation in vivo. Although it can improve the success rate of tumor formation, cells in Matrigel TM Although cells can aggregate, they are not organized, which is quite different from the natural morphology of cells (Tome, Y, Uehara, F, Kanaya, F, Hoffman, R M. Comparison of "Dimensionality" of Cancer Cell Culture in Gelfoam Histoculture and Matrigel[M]. 2018.), which affects the growth and proliferation of cells. TM The ingredients are complex and contain multiple active factors, which can easily interfere with the experimental results (Polykandriotis, E, Arkudas, A, Horch, RE, Kneser, U, Mitchell, G. To Matrigel or Not to Matrigel [J]. American Journal of Pathology, 2008, 172 (5): 1441-1442.), and there are differences between different batches of products, affecting the repeatability of the experiment.
[0008] ②Matrigel TM It is expensive and needs to be stored in a -20℃ refrigerator. For long-term storage, it should be placed in a -80℃ refrigerator. The storage conditions are relatively harsh. When using it, it needs to be buried in a crushed ice box in advance, and the ice box is placed in a 4℃ refrigerator to melt overnight. The subsequent subpackaging process is all done on the ice box. All reagents and consumables that come into contact with the matrix gel need to be pre-cooled to ensure a low-temperature environment. The operation procedures and precautions are numerous, which increases the difficulty and cost of the experiment. Summary of the invention
[0009] The purpose of the present application includes, for example, providing a method for constructing a novel subcutaneous tumor transplantation animal model to at least solve some of the above problems.
[0010] The embodiments of the present application can be implemented as follows:
[0011] On the one hand, the present invention provides a novel method for constructing a subcutaneous tumor transplantation animal model, comprising:
[0012] (1) Collagen sponge inoculation: Place the collagen sponge into the inner core of the inoculation needle and puncture the animal subcutaneously to the inoculation position. Withdraw the needle and leave the inner core, and place the collagen sponge in place.
[0013] (2) Tumor cell suspension inoculation: Inject a single tumor cell suspension into a collagen sponge block to obtain a subcutaneous tumor transplant animal model.
[0014] In some embodiments, the method for processing the collagen sponge block comprises: cutting the collagen sponge into blocks of appropriate size according to the volume of the tumor cell suspension to be inoculated; preferably, the volume of the tumor cell suspension is 50 to 100 μL, and the volume of the corresponding cut collagen sponge block is 16 to 32 mm 3 .
[0015] In some embodiments, the method for preparing the single tumor cell suspension includes: taking tumor cells in the logarithmic growth phase to clean the surface, adding an appropriate amount of trypsin digestion solution to digest and collect the suspension cells, blowing the cell surface with pre-cooled PBS or serum-free culture medium and preparing a single tumor cell suspension of a certain concentration.
[0016] In some embodiments, the tumor cells include homologous tumor cells and xenograft tumor cells; preferably, the homologous tumor cells include but are not limited to: S-180 sarcoma cells, Walker-256 cancer cells, mouse forestomach FC cells, Lewis lung cancer cells, mouse B16 melanoma cells, and the xenograft tumor cells include but are not limited to: lung cancer cells, breast cancer cells, colorectal cancer cells, liver cancer cells, melanoma cells.
[0017] In some embodiments, the collagen sponge block inoculation method includes: folding the collagen sponge block in half and placing it into the hollow trocar of the inoculation trocar, inserting the needle into the flank of the animal and passing it subcutaneously to the inoculation position, pushing the inner core, and placing the collagen sponge block in place.
[0018] In some embodiments, the method for constructing the tumor cell subcutaneous animal model further comprises:
[0019] Model observation and treatment: Regularly observe the tumor formation of animals, measure the tumor size, record the tumor growth curve, and remove the tumor mass after the tumor size reaches the ethical endpoint to observe the morphological structure of the tumor tissue.
[0020] In some embodiments, the animal is a mouse, preferably a C57 mouse.
[0021] On the other hand, the present application also provides an application of a novel method for constructing a subcutaneous tumor transplantation animal model, including but not limited to:
[0022] (1) Study the pathogenesis of tumors;
[0023] (2) Research on tumor treatment methods;
[0024] (3) Screening drugs or vaccines for the prevention and / or treatment of tumors;
[0025] (4) Testing the safety and effectiveness of drugs or vaccines used to prevent and / or treat tumors.
[0026] The method for constructing a novel subcutaneous transplanted tumor animal model provided in the embodiments of the present application uses a collagen sponge scaffold as a carrier, and then injects a tumor cell suspension into a collagen sponge to construct an animal model, thereby increasing the tumor formation rate and shortening the tumor formation time. At the same time, the natural morphology of tumor cells can be maintained, the repeatability of the experiment can be improved, and the experimental cost can be reduced, which is conducive to the experimental testing of various tumor biological mechanism studies, drug development and treatment, and strategy evaluation. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.
[0028] Figure 1 The scanning electron micrographs of the collagen sponge block inoculated with a single tumor cell cultured in vitro in Example 1 at different magnifications are shown.
[0029] Figure 2 Schematic diagram of the method for constructing the new subcutaneous tumor transplantation animal model in Example 1.
[0030] Figure 3 Figure 1 is a graph showing the growth curve of tumor cells in each group of mice in Example 1, wherein Figure a is a graph showing the number of days of tumor formation in mice injected directly with high and low concentrations of Hepa1-6 tumor cell suspensions (TL-2W + ,TH-2W + ), and Figure b shows the number of days of tumor formation in mice inoculated with high and low concentrations of Hepa1-6 tumor cell suspensions through collagen sponges (ST-L-2W + 、ST-H-2W + ).
[0031] Figure 4 Figure 1 is a graph showing the growth curve of tumor cells in each group of mice in Example 1, wherein Figure a is a graph showing the tumor volume of mice directly injected with a low concentration of Hepa1-6 tumor cell suspension (TL-2W + ), Figure b is a graph of the tumor volume of mice inoculated with a low concentration of Hepa1-6 tumor cell suspension via collagen sponge (ST-L-2W +), Figure c is the tumor volume of mice directly injected with high concentration of Hepa1-6 tumor cell suspension (TH-2W + ), Figure d is a graph of the tumor volume of mice inoculated with a high concentration of Hepa1-6 tumor cell suspension via collagen sponge (ST-H-2W + ).
[0032] Figure 5 This is a histomorphological diagram of the stained tissue pathological sections of the subcutaneous transplanted tumor cells in animals in Example 1 (HE staining diagram, 400×). DETAILED DESCRIPTION
[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The components of the embodiments of the present application described and shown in the drawings here can be arranged and designed in various different configurations.
[0034] It should be noted that, in the absence of conflict, the features in the embodiments of the present application may be combined with each other.
[0035] Collagen is the main component of the extracellular matrix and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. Collagen's tissue support function makes it an excellent tissue engineering scaffold material with a long history of use. It has many excellent biological carrier properties such as degradability, biocompatibility, hemostasis, promotion of tissue repair, anti-adhesion, and drug carrier. In recent years, it has been widely used in the field of surgery and has formed a variety of product forms with mature processes.
[0036] Collagen sponge is formed by freeze-drying collagen solution. It is a major product form based on collagen biomaterials. It has good functions and characteristics, as well as unique advantages such as easy processing, sterilization and preservation. It has extensive scientific research and clinical application value. Collagen sponge has a single component, stable properties, good biocompatibility, low immunogenicity, and is degradable. It minimizes research interference factors and improves the repeatability of experiments.
[0037] The inventors inoculated a Hepa1-6 single tumor cell suspension into a collagen sponge in a culture dish, and then observed the adhesion of the tumor cells on the collagen sponge using a scanning electron microscope at different magnifications. Figure 1 shown.
[0038] Depend on Figure 1It can be seen that collagen sponge provides a good three-dimensional growth environment for tumor cells, enabling cells to attach and grow better, thereby increasing the tumor formation rate of animal models.
[0039] Based on this situation, the inventors used collagen sponge as a carrier to hold the cell suspension, implanted it into animal receptors, and constructed an animal cell subcutaneous transplant tumor model, thereby reducing the cost and difficulty of constructing an animal tumor model and establishing a measured path for various tumor biological mechanism studies, drug development and treatment, and strategy evaluation research.
[0040] It should be noted that due to the biological characteristics of the collagen sponge carrier, it can accommodate a variety of homologous tumor cells or xenograft tumor cells, wherein the homologous tumor cells include but are not limited to: S-180 sarcoma cells, Walker-256 cancer cells, mouse forestomach FC cells, Lewis lung cancer cells, mouse B16 melanoma cells, and xenograft tumor cells include but are not limited to: lung cancer cells, breast cancer cells, colorectal cancer cells, liver cancer cells, melanoma cells. In addition, each tumor cell can be implanted into conventional experimental animals such as mice and rabbits using collagen sponges as carriers.
[0041] This article only uses mice as test animals and liver cancer cell Hepa1-6 cells as reagents to construct a typical subcutaneous tumor transplantation animal model. Taking this as a representative, the construction method of the subcutaneous tumor transplantation animal model based on collagen sponge as a carrier is explained and the modeling effect is evaluated.
[0042] Example 1: Construction of a novel subcutaneous tumor transplantation animal model
[0043] (1) Preparation of collagen sponge
[0044] Select commercially available medical sterile collagen sponge (2 mm thick) and cut it into blocks of 2 mm*4 mm*4 mm in size under sterile conditions.
[0045] (2) Preparation of single tumor cell suspension
[0046] ① Washing: Take Hepa1-6 cells in the logarithmic growth phase, remove the high-glucose DMEM containing fetal bovine serum in the culture bottle, add PBS to rinse twice, and remove the residual culture medium;
[0047] ②Digestion: After absorbing the PBS used for washing as much as possible, add an appropriate amount of trypsin, shake to make the trypsin evenly cover the bottom of the bottle, and place it in an incubator at 37℃ for digestion; take it out for observation every 1 minute during digestion, and cells can be seen to shrink significantly under the microscope. When some cells begin to fall off by tapping the culture bottle, immediately add serum-containing culture medium to terminate digestion and blow off the cells attached to the wall;
[0048] ③ Collection: Transfer the cell suspension to a centrifuge tube and centrifuge at 1000 rpm for 3 min to collect the cells; discard the supernatant after centrifugation, wash the cell pellet with pre-cooled PBS or serum-free medium, centrifuge at 1000 rpm for 3 min to collect the cells, repeat twice; resuspend the cells with pre-cooled PBS or serum-free medium to prepare a concentration of 1×10 6 / mL (low concentration, L) and 2×10 6 / mL (high concentration, H) single tumor cell suspension was placed on ice for later use.
[0049] (3) Preparation of test animals
[0050] Forty male C57 mice weighing 18-22 g were divided into four groups, with 10 mice in each group.
[0051] (4) Collagen sponge and single tumor cell suspension inoculation
[0052] ① Such as Figure 2 As shown, the prepared collagen sponge block was folded in half and placed in the hollow trocar of the inoculation trocar; two groups of mice were treated in the same way: the needle was inserted into the flank of the mouse (the insertion site was disinfected with iodine), passed subcutaneously to the back side of the armpit, the needle core of the inoculation trocar was pushed to place the collagen sponge block in place, and then the needle was withdrawn.
[0053] ②Blow the prepared single tumor cell suspension evenly, draw high and low concentration cell suspensions respectively with a 1mL syringe, and inject 100μL of high or low concentration cell suspension subcutaneously onto the collagen sponge block for each group of mice. Pay attention to the slow speed during the injection process to ensure that the collagen sponge fully absorbs the single tumor cell suspension, forming a low concentration single tumor cell suspension + collagen sponge protein group and a high concentration single tumor cell suspension + collagen sponge protein group respectively.
[0054] ③ The other two groups of mice followed the same operation, and each group was directly injected subcutaneously with 100 μL of high or low concentration cell suspension to form a low concentration single tumor cell suspension group and a high concentration single tumor cell suspension group.
[0055] ④ Each group of mice was housed in separate cages, and various indicators of the mice were observed and the tumor formation status of the mice was recorded.
[0056] (5) Model evaluation
[0057] ① Observation of tumor surface
[0058] Starting from the first day of inoculation, when tumor mass formation could be felt, the length L and width W of the mouse tumor were measured with an electronic vernier caliper every 3 days, and the mouse body weight was weighed. The ellipsoid tumor volume was calculated using the formula (V = (π / 6) × L × W 2 ) Calculate tumor volume and draw a tumor change trend chart ( Figure 3 ,4 ), the growth time of mice was not restricted, and mice were euthanized when the tumor diameter L or tumor width W exceeded 20 mm. The number of mice with tumors in each group was recorded, and the tumor formation status of mice in each group was statistically analyzed. The statistical results are shown in Table 1.
[0059] Table 1: Tumor formation in mice in each group
[0060]
[0061] As shown in Table 1, the success rate of tumor formation in mice loaded with collagen sponges with different concentrations of single tumor cell suspensions was significantly higher than that in mice directly injected with single tumor cell suspensions. Figure 3 , 4 It can be seen that without limiting the growth time of mice, the tumors of mice loaded with different concentrations of tumor cell suspensions in collagen sponges reached the ethical endpoint volume faster. This shows that compared with the traditional cell suspension injection method, the collagen sponge scaffold can accelerate the growth rate of tumor cells, shorten the tumor formation time, and significantly improve the tumor formation rate of animal models.
[0062] ②Pathological section examination
[0063] Tissue samples were taken from some tumor-forming mice from the high-concentration single tumor cell group and the high-concentration single tumor cell + collagen sponge group for pathological examination.
[0064] The tumor mass was removed and quickly immersed in 10% neutral buffered formaldehyde solution for fixation for 24 hours to ensure the stability of the tissue structure. Subsequently, the tissue was dehydrated (gradient ethanol), transparent (xylene) and paraffin embedded in turn to make paraffin sections (thickness is generally 4-6μm). The sections were first dewaxed with xylene, hydrated to water with gradient ethanol, and then immersed in hematoxylin staining solution to stain the cell nucleus for 3-5 minutes, rapidly differentiated with differentiation solution (1% hydrochloric acid alcohol), and then blued with weak alkaline water (such as tap water) to make the cell nucleus appear clear blue. After that, the sections were immersed in eosin staining solution to stain the cytoplasm for 1-2 minutes, dehydrated with 95% ethanol, transparentized with xylene, and finally sealed with neutral gum to observe the morphological structure of the tumor tissue. Figure 5 shown.
[0065] Depend on Figure 5 It can be seen that when the tumor of mice in the collagen sponge group carrying tumor cell suspension reached the ethical endpoint, the collagen sponge on the pathological tissue had been completely degraded, and there was no obvious immune cell aggregation and no inflammatory response when it continued to exist. This shows that the collagen sponge, as a single tumor cell carrier, has no effect on the subject and is conducive to the natural growth and proliferation of tumor cells.
[0066] In summary, this application uses collagen sponge to carry single tumor cell suspension and inoculate it into animal recipients to successfully construct an animal subcutaneous transplant tumor model. Compared with the model construction method of directly injecting cell suspension, the tumor formation probability and tumor formation rate are significantly improved. In addition, collagen sponge is used as a tumor carrier and has no interference with the natural production of tumor cells.
[0067] The successful construction of the above-mentioned animal subcutaneous transplant tumor model will greatly reduce the cost and difficulty of constructing tumor animal models, and provide strong experimental model support for tumor biological mechanism research, drug development and treatment, and strategy evaluation.
[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. A novel method for constructing a subcutaneous tumor transplantation animal model, characterized in that: include: (1) Collagen sponge inoculation: Place the collagen sponge into the inner core of the inoculation needle and puncture the animal subcutaneously to the inoculation position. Withdraw the needle and leave the inner core, and place the collagen sponge in place. (2) Tumor cell suspension inoculation: Inject a single tumor cell suspension into a collagen sponge block to obtain a subcutaneous tumor transplant animal model.
2. The method for constructing an animal model according to claim 1, characterized in that: The method for processing the collagen sponge block comprises: cutting the collagen sponge into blocks of appropriate size according to the volume of the tumor cell suspension to be inoculated.
3. The method for constructing an animal model according to claim 2, characterized in that: The volume of the tumor cell suspension is 50-100 μL, and the volume of the corresponding cut collagen sponge block is 16-32 mm 3 .
4. The method for constructing an animal model according to claim 1, characterized in that: The method for preparing the single tumor cell suspension comprises: washing the surface of tumor cells in the logarithmic growth phase, adding an appropriate amount of trypsin digestion solution to digest and collect the suspension cells, washing the cell surface with pre-cooled PBS or serum-free culture medium and preparing a single tumor cell suspension of a certain concentration.
5. The method for constructing an animal model according to claim 1, characterized in that: The tumor cells include syngeneic tumor cells and xenograft tumor cells.
6. The method for constructing an animal model according to claim 5, characterized in that: The homologous tumor cells include: S-180 sarcoma cells, Walker-256 cancer cells, mouse forestomach cancer FC cells, Lewis lung cancer cells, mouse B16 melanoma cells, and the xenotransplanted tumor cells include: lung cancer cells, breast cancer cells, colorectal cancer cells, liver cancer cells, and melanoma cells.
7. The method for constructing an animal model according to claim 1, characterized in that: The collagen sponge block inoculation method comprises: folding the collagen sponge block in half and placing it into the hollow trocar of the inoculation trocar, inserting the needle into the animal's flank and passing it subcutaneously to the inoculation position, pushing the inner core, and placing the collagen sponge block in place.
8. The method for constructing an animal model according to claim 1, characterized in that: The animals selected are mice.
9. A subcutaneous tumor transplantation animal model constructed by the method for constructing an animal model according to any one of claims 1 to 8.
10. Use of the method for constructing an animal model according to any one of claims 1 to 8 or the subcutaneous tumor transplantation animal model according to claim 9 in any of the following aspects: (1) Study the pathogenesis of tumors; (2) Research on tumor treatment methods; (3) Screening drugs or vaccines for the prevention and / or treatment of tumors; (4) Testing the safety and effectiveness of drugs or vaccines used to prevent and / or treat tumors.