A method for constructing a liquid nitrogen-frozen tumor tissue PDX model and a reagent combination thereof

Abstract

The application provides a modified PDX model construction method for liquid nitrogen cryopreservation of cancer tumor tissue allograft and a reagent combination thereof, which comprises the following steps: a mouse pretreatment step, a step of using liquid nitrogen cryopreserved tumor tissue to make a mouse tumorigenic, a step of preparing a cryopreservation mixed suspension, a step of ex vivo post-treatment of tumor tissue, and a step of activating a thawed tumor mass during transplantation. The method can significantly shorten the tumorigenic days and improve the tumorigenic rate.

Description

Technical Field

[0001] This invention belongs to the field of cancer treatment technology, and in particular relates to a method for constructing a modified human-derived liquid nitrogen cryopreserved tumor xenograft (PDX) model and its reagent combination. Background Technology

[0002] Globally, cancer (malignant tumors) has become the leading cause of death threatening human health, with a mortality rate far exceeding that of other diseases (such as cardiovascular and cerebrovascular diseases). Therefore, medical researchers urgently need to conduct more in-depth research on cancer treatment methods. Due to ethical and moral reasons, oncology research conducted in humans is limited to analytical and observational studies, and clinical trials focusing on treatment are restricted in vivo. Therefore, preclinical mouse tumor models, as an indispensable intermediate experimental model system, undertake the task of effectively combining in vitro research with human research.

[0003] Traditional tumor xenograft models refer to cell line-derived tumor xenografts (CDTX) models, which are animal models created by injecting in vitro cultured tumor cell lines into immunodeficient mice. Single tumor cell lines, after repeated in vitro passages, adapt to the external culture environment. Furthermore, due to the lack of tumor-associated matrix and blood supply, the microenvironment of in vitro cell lines is completely different from that of primary tumors. This makes it impossible to predict genetic variations and tumor heterogeneity arising from the cell line, and to accurately determine the role of drugs in clinical trials. The average success rate of conversion from animal tumor-bearing models to clinical cancer patients is less than 8%, possibly because this model cannot faithfully reflect the carcinogenic process in humans. Highly anaplastic cancer cells cultured in vitro represent an extreme derivative of highly advanced cancer, independent of the primary tumor matrix, which is considered a key factor in tumor metastasis. Moreover, key genetic, molecular, immune, and cellular differences between humans and mice hinder this model from becoming an effective means of personalized cancer treatment.

[0004] To overcome these limitations, medical researchers urgently need more accurate and effective methods to predict and assess drug efficacy, and PDX models can partially solve these problems.

[0005] The most significant advantage of PDX models is their similarity to primary tumors. Researchers have established PDX models for various tumors, including gastric cancer, colorectal cancer, hepatocellular carcinoma, pancreatic cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, ovarian cancer, uterine sarcoma, prostate cancer, renal cell carcinoma, and melanoma. Analysis from histopathological and genetic perspectives has confirmed that transplanted tumors retain the pathological and histological characteristics of the primary tumor, replicating the stromal and stem cell characteristics of tumor cells and reflecting the genetic diversity of the patient's "in vivo primary" tumor. Genome-wide gene expression analysis shows that PDTX maintains the activity of most key genes and global pathways in primary tumors. Studies by Fichtne et al. have shown that in a non-small cell lung cancer (NSCLC) PDX model, hierarchical clustering of genome-wide gene expression profiles revealed that 9 out of 17 primary tumors were directly identical to the transplanted PDX model, with correlation coefficients ranging from 0.78 to 0.95. Importantly, 10 out of 17 pairs of primary and PDX tumors showed a correlation coefficient greater than 0.90, indicating a high degree of similarity between the primary tumors and the corresponding PDX models.

[0006] PDX models have good predictive value for clinical efficacy and are often used in new drug development, especially in patient screening for clinical trials of targeted drugs and in the study of predictive biomarkers. They are also frequently used to help patients with recurrent or advanced cancer to screen for sensitive drugs and to help predict patient efficacy, toxicity, and absorption. The construction of PDX models has become an indispensable technical means in precision medicine.

[0007] PDX models are built using fresh tumor tissue, requiring only a small sample size to successfully establish a PDX model. Furthermore, the first-generation model can be used for the next generation, rapidly scaling up the sample size. This provides a new and convincing research method for tumors where obtaining tumor tissue is difficult, such as pancreatic acinar cell carcinoma, small cell lung cancer, and liver metastases from colorectal cancer. The high consistency between PDX models and primary tumors represents a significant breakthrough in translational medicine. Implantation of human primary tumors subcutaneously or in vivo in immunodeficient mice relies primarily on the number and viability of tumor cells that survive in the allogeneic body. Reports indicate that the overall success rate (i.e., transplantation success rate) of fresh tumor tissue in PDX models for liver cancer, breast cancer, and colorectal cancer is approximately 29.5%-33.8%, 17%-20%, and 36.4%-47%, respectively. From the day of transplantation, the average time for the tumor tissue to grow to a volume of 200 mm3 was approximately 67 days, 80 days, and 55 days, respectively (Hidalgo et al. Cancer Discovery, 2014.; Owonikoko et al. Journal of Translational Medicine, 2016.; Izumchenko et al. Annals of Oncology, 2017.; Hidalgo et al. Cancer Discovery, 2014.; Owonikoko et al. Journal of Translational Medicine, 2016.).

[0008] Given the poor quality of life of patients with tumor recurrence and advanced cancer, it is necessary to "race against time" to prolong the patient's survival and improve their quality of life by fighting tumor growth and metastasis. Therefore, improving the success rate of PDX models and shortening the time to tumor formation has become an urgent problem to be solved in the clinical treatment of tumors. Summary of the Invention

[0009] Since the monitoring and treatment of most malignant tumors is a lifelong process, the choice of subsequent treatment becomes more cautious when patients experience tumor progression, recurrence, or metastasis after surgery. If a pre-selected alternative is chosen for the patient, the likelihood of receiving a new treatment plan further decreases if the tumor continues to develop, and the tumor progression further affects the patient's overall condition, leading to treatment failure. Therefore, by reconstructing a PDX model of the primary tumor and comparing its tissue characteristics, tumor microenvironment, genetic information, and other factors, preliminary screening of alternative treatment plans can be conducted in a short period of time, and then applied to the patient's treatment in a timely manner, which is expected to improve the effectiveness of the patient's treatment.

[0010] Although theoretically, a small amount of tissue can be pre-frozen in liquid nitrogen and used to construct tumors in living animals, the use of liquid nitrogen to freeze tumor tissue and construct tumors in living animals is not currently being carried out because the cancer cells in liquid nitrogen-frozen tissue have less activity and fewer live cells than in fresh tissue, making it less likely to form tumors under the skin of mice.

[0011] To address the shortcomings of existing technologies, this invention provides a method for constructing PDX models of tumor tissue cryopreserved in liquid nitrogen. This method is simple to operate, low in cost, has a high success rate, and produces objective and stable results.

[0012] The technical solution of the present invention is as follows:

[0013] In a first aspect, the present invention provides a method for constructing a PDX model from tumor tissue cryopreserved in liquid nitrogen, the method comprising any one or an optional combination of the following three steps:

[0014] 1) Mouse pretreatment procedures and procedures for tumor formation in mice using liquid nitrogen cryopreservation of tumor tissue;

[0015] 2) Preparation of cryopreserved mixed suspensions and post-ex vivo processing of tumor tissue;

[0016] 3) The thawed tumor fragments are activated before transplantation. Further, the pretreatment involves injecting heparin into the mice before inoculation.

[0017] Furthermore, the pretreatment involves injecting mice with heparin and Matrigel matrix gel prior to inoculation.

[0018] Furthermore, the pretreatment involves subcutaneous injection of heparin into the peritoneal cavity of mice and subcutaneous application of Matrigel matrix gel to the inoculation site prior to inoculation.

[0019] According to an embodiment of the present invention, the specific operation of the pretreatment is as follows: 2-3 hours before inoculation, the inoculation site near the lymph node in the right axilla of the mouse is gently wiped with a cotton ball containing 75% medical alcohol. Heparin injection solution (total volume of 200µL) is injected subcutaneously into the peritoneum at a dose of 1mg / kg body weight using a 1mL sterile syringe. 50µL of Matrigel matrix gel (2-fold diluted and pre-stored on ice) is slowly injected subcutaneously into the inoculation site using a 1mL sterile syringe.

[0020] In a second aspect of the invention, the method includes the preparation of a cryopreserved mixed suspension and post-ex vivo processing of tumor tissue.

[0021] Furthermore, the processing method involves treating the excised tumor tissue with a mixed suspension of lactoferrin (preferably human lactoferrin), matrigel gel, buffer solution (preferably sterile PBS solution), and animal serum (preferably fetal bovine serum), followed by cryopreservation.

[0022] Furthermore, the mass ratio of lactoferrin, Matrigel gel, sterile PBS solution, and fetal bovine serum in the mixed suspension is (1-2):(5-10):(20-50):(2.5-5), for example, the mass ratio of lactoferrin, Matrigel gel, sterile PBS solution, and fetal bovine serum in the mixed suspension is 1:10:50:5, 1:5:20:2.5, 1:8:30:4, 1.5:6:25:3, 1.5:7:35:4, 2:6:20:2.5, 2:9:45:5.

[0023] According to a preferred embodiment of the present invention, an anti-apoptotic component is added to the mixed suspension.

[0024] The anti-apoptotic component may be selected from components known in the prior art that have anti-apoptotic activity, such as components or combinations thereof from the following group: gold tricarboxylic acid (ATA), zinc ions, antioxidants, peptides and cytokines with anti-apoptotic activity.

[0025] According to one embodiment of the present invention, the method for post-ex vivo processing of tumor tissue is as follows:

[0026] In a sterile operating room, the mucosa, blood clots, or growths surrounding the tumor tissue were removed on ice. The tissue was rinsed with ice-cold PBS solution containing penicillin-streptomycin (1:100) for 30 seconds and then soaked for 30 seconds, repeated three times. The tissue block was then cut into small pieces of 0.5cm × 0.5cm × 0.5cm using a sterile scalpel and placed in a mixed suspension of lactoferrin, matrigel gel, sterile PBS solution, and fetal bovine serum (mass ratio 1:10:50:5). The tissue was then cryopreserved using a cryo-80°C freezer-liquid nitrogen procedure.

[0027] In a third aspect of the invention, the improvement of the method over the prior art includes activating the thawed tumor mass during transplantation.

[0028] Furthermore, the activation involves treatment with a mixed suspension of lactoferrin and matrigel matrix gel.

[0029] According to one embodiment of the present invention, the formulation of the activating agent during transplantation is as follows:

[0030] Mix 0.5-2 parts lactoferrin (by weight), 5-15 parts Matrigel (by volume), and 80-95 parts buffer solution, preferably sterile PBS solution (by volume);

[0031] The preferred formulation is: 0.8-1.2 parts lactoferrin (mass), 8-12 parts Matrigel matrix gel (volume), and 86-92 parts buffer solution, preferably sterile PBS solution (volume), mixed together.

[0032] A further optimized formulation is: 1 part lactoferrin (by mass), 10 parts Matrigel matrix gel (by volume), and 89 parts sterile PBS solution (by volume) mixed and treated.

[0033] Preferably, an anti-apoptotic component is added to the mixed suspension;

[0034] The preferred formulation is as follows: the activation is a mixture of 1 part lactoferrin (mass), 10 parts Matrigel matrix gel (volume), 5 parts anti-apoptotic component and 84 parts sterile PBS solution (volume).

[0035] The activation reagent is prepared on ice and used immediately in a sterile operating table. When transplanting tumors, the tumors are immersed in the reagent for 120 seconds, and then removed for inoculation of the animals.

[0036] The anti-apoptotic component may be selected from components known in the prior art that have anti-apoptotic activity, such as components or combinations thereof from the following group: gold tricarboxylic acid (ATA), zinc ions, antioxidants, peptides and cytokines with anti-apoptotic activity.

[0037] According to a preferred embodiment of the present invention, the second and third aspects described above are used in combination.

[0038] According to a specific embodiment of the present invention, the cancer is liver cancer, breast cancer, and colon cancer.

[0039] According to a specific embodiment of the present invention, the animal used to construct the PDX model is a gene-deficient mouse (BALb / c nude mouse).

[0040] This invention is not limited to these three types of cancer, but also includes a variety of tumor tissues (gastric cancer, lung cancer, prostate cancer, melanoma, etc.); it is not limited to using gene-deficient mice (BALb / c nude mice or SCID mice), but also includes other types of gene-deficient mice and a variety of small mammals.

[0041] The fourth invention provides reagents or reagent combinations for constructing PDX models from tumor tissue cryopreserved in liquid nitrogen, the reagents or reagent combinations comprising:

[0042] The tumor tissue suspension for post-ex vivo processing and cryopreservation consists of a mixture of lactoferrin, matrigel gel, buffer solution, and animal serum.

[0043] and / or

[0044] The activation agent used during transplantation consists of lactoferrin, matrigel matrix gel, and buffer solution.

[0045] The buffer solution is preferably sterile PBS solution, and the animal serum is preferably fetal bovine serum.

[0046] The reagent combination for constructing PDX models of tumor tissues using liquid nitrogen cryopreservation according to the present invention further contains an anti-apoptotic component.

[0047] The anti-apoptotic component may be selected from the following groups or combinations thereof: gold tricarboxylic acid (ATA), zinc ions, antioxidants, peptides with anti-apoptotic activity, and cytokines.

[0048] Beneficial effects

[0049] 1) The method for constructing a PDX model of tumor tissue cryopreservation in liquid nitrogen in this invention includes steps such as processing and cryopreservation of patient tumor tissue after ex vivo, pre-transplant resuscitation, and subcutaneous treatment in mice. The PDX model constructed by this method has a significantly higher subcutaneous tumor formation rate (greater than 75%) than the tumor formation rate of the PDX model constructed by the control group of existing methods (ranging from 16.7% to 25%), and can significantly shorten the reported average tumor formation time (14-24 days vs. 29-41 days). It is applicable to different types of tumors (this invention uses three clinical tissues: liver cancer, breast cancer, and colon cancer).

[0050] 2) The liquid nitrogen cryopreservation method for constructing PDX from tumor tissues in this invention, especially in liver cancer, breast cancer and colon cancer tissue models, can significantly shorten the cycle of constructing PDX from liquid nitrogen cryopreserved tumor tissues, increase the probability of tumor formation in subcutaneous xenografts in gene-deficient mice, and be beneficial for screening sensitive drugs and formulating effective therapies for patients with cancer recurrence and metastasis in clinical applications. Attached Figure Description

[0051] Figure 1 This demonstrates the effect of intraperitoneal subcutaneous injection of heparin and dorsal injection of matrigel matrix gel on the tumorigenicity of hepatocellular carcinoma, breast cancer, and colon cancer tissues cryopreserved in liquid nitrogen, as shown in Example 1, by constructing a BALb / c nude mouse PDX model.

[0052] Figure 2 The results of Example 2 show the effect of constructing a BALb / c nude mouse PDX model, treating the tissues in vitro with cryopreservation solution, and then freezing them, on the tumor formation time of liver cancer, breast cancer, and colon cancer tissues frozen in liquid nitrogen.

[0053] Figure 3 Example 3 illustrates the effect of treating cryopreserved tumor tissue with a BALb / c nude mouse PDX model and using a resuscitation and activation reagent on the tumorigenicity of hepatocellular carcinoma, breast cancer, and colon cancer tissues cryopreserved in liquid nitrogen. Detailed Implementation

[0054] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0055] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0056] Example 1: The effect of mouse pretreatment on increasing tumor formation rate

[0057] To evaluate the effects of intraperitoneal subcutaneous heparin injection and dorsal inoculation site injection of Matrigel on the tumorigenicity of liquid nitrogen-crystallized liver cancer, breast cancer, and colon cancer tissues, 72 male BALb / c nude mice (20-22 g) were used to construct a PDX model. The mice were divided into two groups. One group received routine treatment as the control group, where the inoculation sites on the back of the mice were gently wiped with cotton balls containing 75% medical alcohol, and the thawed cancer tissue was injected subcutaneously into the inoculation sites using a cannula. The other group received improved treatment, where the inoculation sites on the back of the mice were gently wiped with cotton balls containing 75% medical alcohol, and then heparin (total volume 200 µL) was injected subcutaneously into the dorsal inoculation sites at a dose of 1 mg / kg body weight using a 1 mL sterile syringe. Simultaneously, 100 µL of Matrigel (pre-preserved on ice) was slowly injected subcutaneously into the dorsal inoculation sites using a 1 mL sterile syringe. Each group was further divided into three subgroups (liver cancer, breast cancer, and colon cancer), with 12 nude mice in each group. Human tumor tissue was immediately transferred to a sterile operating table after ex vivo. On ice, the surrounding mucosa, blood clots, or growths were removed. The tissue was rinsed with ice-cold PBS solution for 15 seconds and soaked for 30 seconds, repeated three times. Then, using a sterile scalpel, the tissue block was cut into 0.5cm × 0.5cm × 0.5cm pieces and quickly placed into cryovials (containing 1mL of cryopreservation solution) and stored in liquid nitrogen. During thawing, the liquid nitrogen-frozen cancer tissue was placed in a 37°C water bath and rapidly shaken until completely thawed. Each small piece was then inserted into a 0.75cm diameter cannula and injected into the back of nude mice, one injection per mouse (one tumor piece per mouse). Tumor formation was observed daily from the first day of inoculation until day sixty. The number of mice with tumor formation was counted, and the tumor formation rate (%) for each group was calculated as: (Number of mice with tumor formation / 12 mice) × 100%.

[0058] Experimental results are as follows Figure 1As shown, in the liver cancer model, the tumor rate of the improved treatment group increased to 50.1% compared with the control group (16.7%); in the breast cancer model, the tumor rate of the improved treatment group increased to 41.9% compared with the control group (8.3%); and in the colon cancer model, the tumor rate of the improved treatment group increased to 66.7% compared with the tumor rate of the control group (25.2%).

[0059] Example 2: The effect of using cryopreservation solution on in vitro tissue treatment and cryopreservation to shorten the tumor formation time of liver cancer, breast cancer, and colon cancer under liquid nitrogen cryopreservation.

[0060] To evaluate the effect of cryopreservation on the time to tumor formation in liver cancer, breast cancer, and colon cancer tissues after in vitro tissue treatment and cryopreservation, a PDX model was constructed using 72 male BALb / c nude mice weighing 20-22 grams. The mice were divided into two groups: one group received conventional treatment as a control group, i.e., tumor tissue was immersed in conventional cell cryopreservation solution (20% DMSO + 80% fetal bovine serum FBS); the other group received in vitro tissue treatment plus a specially prepared cryopreservation reagent as an improvement treatment group. Each group was further divided into three subgroups (liver cancer, breast cancer, and colon cancer), with 12 mice in each subgroup. Immediately after in vitro treatment, the tumor tissues were transferred to a sterile operating table. The surrounding mucosa, blood clots, or other growths were removed on ice. The tissues were then rinsed with ice-cold PBS solution for 15 seconds and immersed for 30 seconds, repeated three times. Then, using a sterile scalpel, the tissue blocks were cut into small pieces of 0.5cm × 0.5cm × 0.5cm and placed in standard cell cryopreservation solution. The improved treatment group used a combination of lactoferrin, Matrigel gel, sterile PBS solution, and fetal bovine serum in a mass ratio of 1:10:50:5 as cryopreservation reagent. The cryopreservation tubes containing the tissue and cryopreservation reagent were then cryopreserved in the following order: cryopreservation box – -80°C freezer – liquid nitrogen. After 72 hours, the cryopreservation box in liquid nitrogen was removed and rapidly thawed in a 37°C water bath. The skin of nude mice was disinfected with 75% ethanol. The thawed tumor tissue pieces were individually inserted into a 0.75cm diameter cannula and injected into the back of the nude mice, one injection per mouse, i.e., one tumor piece per mouse. Starting from the first day of tumor inoculation, tumor formation was observed daily, and the length and width of the tumor were measured using calipers. The tumor volume (mm³) was calculated using the formula... 3 = 0.5 × length (mm) × width (mm) 2 Calculate tumor volume until the tumor volume reaches 200 mm. 3 The number of days is the tumor formation time (days).

[0061] Experimental results are as follows Figure 2As shown, in the colon cancer model, compared with the control group (33.3±5.2 days), the time to tumor formation after improvement treatment was shortened to 18.3±3.1 days; in the breast cancer model, compared with the control group (41.3±7.5 days), the time to tumor formation after improvement treatment was shortened to 23.7±4.5 days; and in the liver cancer model, compared with the control group (29.2±6.4 days), the time to tumor formation after improvement treatment was shortened to 14.4±3.8 days. In all three cancer models, the improvement treatment significantly shortened the time to subcutaneous tumor formation in nude mice. The symbol * indicates the difference compared with the control group without any treatment. P<0.05, indicating a statistically significant difference.

[0062] Example 3: Effect of tissue resuscitation and activation treatment on improving the tumorigenesis rate of liver cancer, breast cancer, and colon cancer preserved in liquid nitrogen.

[0063] To evaluate the effect of tissue activation reagent treatment on the tumorigenicity of hepatocellular carcinoma, breast cancer, and colon cancer tissues cryopreserved in liquid nitrogen, a PDX model was constructed using 72 male BALb / c nude mice weighing 20-22 grams. These mice were divided into two groups: one group received the routine treatment reported in the literature (control group), and the other group received tissue activation reagent treatment (improved treatment group). Each group was further divided into three subgroups (hepatocellular carcinoma, breast cancer, and colon cancer), with 12 mice in each subgroup. Tumor tissues were immediately placed in a 37°C activation reagent after being removed from liquid nitrogen. The activation reagent for the control group consisted of 20% fetal bovine serum + 80% DMEM medium, while the activation reagent for the improved treatment group consisted of 1 part lactoferrin (mass) + 10 parts Matrigel (volume) + 5 parts anti-apoptotic component (mass) + 84 parts sterile PBS solution (volume) (mass unit g, volume unit mL). Then, the nude mice were disinfected with 75% ethanol. Small pieces of tumor tissue were individually inserted into a 0.75cm diameter cannula and rapidly injected into the right axillary lymph node of each mouse. One mouse received one injection, resulting in one tumor piece per mouse. From the day of tumor inoculation until day 50, the formation of subcutaneous tumors in the mice was observed. The tumor formation rate (%) for each group was calculated as: (Number of mice with tumor formation / 12 mice) × 100%.

[0064] Experimental results are as follows Figure 3 As shown, in the liver cancer model, the tumorigenesis rate of the improved treatment group was 40.7% compared with the control group (16.6%); in the breast cancer model, the tumorigenesis rate of the improved treatment group was 50.4% compared with the control group (8.3%); and in the colon cancer model, the tumorigenesis rate of the improved treatment group was 75.2% compared with the control group (25.2%). In all three cancer tissue models, the improved treatment significantly increased the subcutaneous tumorigenesis rate in nude mice.

[0065] The specific embodiments of the present invention have been described above by way of example. However, the scope of protection of the present invention is not limited to the above exemplary embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a PDX model from tumor tissue cryopreserved in liquid nitrogen, characterized in that, The method includes the following steps: 1) Mouse pretreatment procedures and procedures for tumor formation in mice using liquid nitrogen cryopreservation of tumor tissue; 2) Preparation of cryopreserved mixed suspensions and post-ex vivo processing of tumor tissue; 3) Activate the thawed tumor mass during transplantation; The preprocessing is selected from any one of method AC: Method A: Inject heparin into mice before inoculation; Method B: Mice were injected with heparin and Matrigel matrix gel before inoculation; Method C: Before inoculation, mice were injected subcutaneously into the peritoneal cavity with heparin and subcutaneously into the inoculation site with Matrigel matrix gel; Step 2) describes the preparation of the cryopreservation mixed suspension and the post-ex vivo treatment of tumor tissue, which includes treating the ex vivo tumor tissue with a mixed suspension of lactoferrin, matrigel gel, buffer solution and animal serum, and then cryopreserving it. In step 3), the thawed tumor mass is activated by an activation reagent prepared from a mixture of lactoferrin and Matrigel matrix gel during transplantation.

2. The method for constructing a PDX model from tumor tissue cryopreserved in liquid nitrogen according to claim 1, characterized in that, The specific pretreatment procedure is as follows: 2-3 hours before inoculation, gently wipe the inoculation site near the lymph node in the right axilla of the mouse with a cotton ball containing 75% medical alcohol, inject heparin solution subcutaneously into the peritoneum at a dose of 1 mg / kg body weight using a 1 mL sterile syringe, and slowly inject 50 µL of Matrigel matrix gel subcutaneously into the inoculation site using a 1 mL sterile syringe.

3. The method for constructing a PDX model from tumor tissue cryopreserved in liquid nitrogen according to claim 1, characterized in that, The lactoferrin is human lactoferrin, the buffer solution is sterile PBS solution, and the animal serum is fetal bovine serum.

4. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to claim 3, characterized in that, The mass ratio of lactoferrin, matrigel gel, sterile PBS solution and fetal bovine serum in the mixed suspension is (1-2):(5-10):(20-50):(2.5-5).

5. The method for constructing a PDX model from tumor tissue cryopreserved in liquid nitrogen according to claim 1, characterized in that, An anti-apoptotic component is added to the mixed suspension; the anti-apoptotic component may be selected from the following components or combinations thereof: gold tricarboxylic acid, zinc ions, antioxidants, peptides with anti-apoptotic activity, and cytokines.

6. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to any one of claims 1-5, characterized in that, The following are the methods for processing tumor tissue after ex vivo: Inside a sterile operating table, the mucosa, blood clots, or growths surrounding the tumor tissue were removed on ice. The tissue was rinsed for 30 seconds and soaked for 30 seconds with ice-cold PBS solution containing penicillin-streptomycin bispecific antibodies, repeated 3 times. Then, the tissue block was cut into small pieces of 0.5cm × 0.5cm × 0.5cm using a sterile scalpel and placed in a mixed suspension of lactoferrin, matrigel gel, sterile PBS solution, and fetal bovine serum. The mass ratio of lactoferrin, matrigel gel, sterile PBS solution, and fetal bovine serum was 1:10:50:

5. The tissue was then cryopreserved using a cryo-80°C freezer-liquid nitrogen procedure.

7. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to any one of claims 1-5, characterized in that, The activation reagent is prepared on ice and used immediately in a sterile operating table. When transplanting tumors, the tumors are soaked in the activation reagent for 120 seconds, and then removed for inoculation of the animals.

8. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to claim 7, characterized in that, The activation reagent formulation for transplantation is shown below: Mix 0.5-2 parts by weight of lactoferrin, 5-15 parts by volume of Matrigel matrix gel, and 80-95 parts by volume of sterile PBS solution. The parts by weight are in grams and the parts by volume are in mL.

9. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to claim 8, characterized in that, The activation reagent formulation for transplantation is as follows: 0.8-1.2 parts by weight of lactoferrin, 8-12 parts by volume of Matrigel matrix gel, and 86-92 parts by volume of sterile PBS solution are mixed and treated. The parts by weight are in g and the parts by volume are in mL.

10. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to claim 8, characterized in that, The activation reagent formula for transplantation is: 1 part by weight of lactoferrin, 10 parts by volume of Matrigel matrix gel, and 89 parts by volume of sterile PBS solution mixed together. The parts by weight are in g and the parts by volume are in mL.

11. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to any one of claims 1-6, characterized in that, Anti-apoptotic components are added to the activating reagent suspension during transplantation.

12. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to claim 11, characterized in that, The activation reagent formulation for transplantation is as follows: the activation is a mixture of 1 part by mass of lactoferrin, 10 parts by volume of Matrigel matrix gel, 5 parts by mass of anti-apoptotic component and 84 parts by volume of sterile PBS solution. The mass unit is g and the volume unit is mL.

13. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to any one of claims 1-6, characterized in that, The tumors mentioned are liver cancer, breast cancer, colon cancer, stomach cancer, lung cancer, prostate cancer, and melanoma.

14. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to any one of claims 1-6, characterized in that, The animals used to construct the PDX model included genetically defective mice and various small mammals.

15. The method for constructing a PDX model by cryopreserving tumor tissue in liquid nitrogen according to any one of claims 1-6, characterized in that, The animal used to construct the PDX model was the BALb / c nude mouse, which was a gene defect.

16. A reagent combination for constructing PDX models from tumor tissue cryopreserved in liquid nitrogen, characterized in that, The reagent combination includes: The tumor tissue suspension for post-ex vivo processing and cryopreservation consists of a mixture of lactoferrin, matrigel gel, buffer solution, and animal serum. The activation agent used during transplantation consists of lactoferrin, Matrigel matrix gel, and buffer solution. The buffer solution is sterile PBS solution, and the animal serum is fetal bovine serum.

17. The reagent combination for constructing PDX models of tumor tissue by liquid nitrogen cryopreservation according to claim 16, characterized in that, The activating reagent in the reagent combination further contains an anti-apoptotic component; the anti-apoptotic component may be selected from the following components or combinations thereof: gold tricarboxylic acid, zinc ions, antioxidants, peptides and cytokines with anti-apoptotic activity.

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