CN110623770B - Construction method of malignant schwannoma xenograft mouse model - Google Patents
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Abstract
The invention provides a method for constructing a malignant schwannoma xenograft mouse model, which comprises the steps of recovering a malignant schwannoma patient tumor tissue block stored in a liquid nitrogen environment, then transplanting the recovered patient tumor tissue block into an immunodeficiency mouse under an aseptic condition in a heterogenic subcutaneous way, wherein the heterogenic subcutaneous transplantation comprises the steps of selecting an immunodeficiency mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the recovered patient tumor tissue block into a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the patient tumor tissue block into the mouse under the skin, suturing an incision, and feeding the mouse to ensure the growth of the tumor in vivo. The invention also comprises the step of transplanting the tumor tissue blocks of the cryopreserved resuscitated patients into immunodeficient mice in a heterogenic and in-situ manner. The invention uses the xenograft mice to replace clinical patients to screen different combined drugs, thereby selecting the most suitable drugs, greatly improving the effectiveness and reducing the side effect of the drugs.
Description
Technical Field
The invention relates to the field of establishment of tumor transplantation mouse models, in particular to a method for establishing a malignant schwannomas xenograft mouse model.
Background
Malignant schwannoma, also known as neurosarcoma, is derived from the peripheral nerve sheath, consists of malignant neuroblasts, and is mostly malignant from neurofibroma or schwannoma. The malignant degree is high, lung metastasis is easy to occur, the disability rate and the death rate are extremely high, and the prognosis is poor. Malignant schwannoma imposes a heavy economic burden on society and even countries, and brings about a greater social problem. Because of the heterogeneity of malignant schwannoma, the basic research results based on malignant schwannoma cell line in the past can be rarely reproduced in patients with clinical malignant schwannoma, and the low disease incidence results in the delay of the diagnosis and treatment level of clinical malignant schwannoma.
The invention patent application CN201710822683.2 discloses human NF2 -/- A method for establishing a schwann cell line of vestibular nerve sheath tumor and a cell line thereof. The establishing method comprises the following steps: A. separating Schwann cells in tumor to obtain human NF2 -/- Performing primary culture on the primary Schwann cells; B. establishment of immunodeficient sciatic nerve human NF2 in mice -/- A primary Schwann cell transplantation tumor model; C. screening transplanted tumors with the diameter larger than 1cm from sciatic nerves of mice; D. separating Schwann cells, and performing primary cell culture; E. carrying out continuous subculture for more than 5 generations; F. obtaining NF2 from the screening -/- The cell morphology and the main biological characteristics of the primary Schwann cell and the primary transplantation tumor cell strain are consistent, and NF2 with infinite passage can be generated -/- Vestibular nerve Schwann cell.
Tumor models are important vehicles for tumor research. Researches show that the cells are continuously adapted to in-vitro environment in the passage process, the biological characteristics are not in accordance with those of in-vivo tumor cells, and the cell line xenograft model has greater consistency with the tumor of a patient, so that the failure rate of clinical tests of the antitumor drugs is high.
A human derived tumor xenograft (PDX) model is an in-vivo model, and a large amount of tumor tissues of a patient can be amplified by taking a mouse as a carrier by directly transplanting fresh tumor tissues of the patient to an immunodeficiency mouse after being treated and depending on the environment growth and gradual passage provided by the mouse. The PDX model has the following advantages: firstly, the model is equivalent to a model corresponding to a patient, can well reflect the difference of tumors among different individuals, and can better reflect the actual condition of clinical patients compared with the traditional model; secondly, the PDX model can be passaged, and the passaged tumor tissue can keep high consistency with the initial tumor tissue in gene copy number, gene mutation, expression mode and the like, which is equivalent to the amplification of a patient tumor sample, thereby providing good conditions for drug screening and patient disease mechanism research; and thirdly, the tumor blocks cultured by the PDX can be frozen and recovered, and a new PDX model can be still constructed, which is equivalent to the construction of a tumor living body library of different patients.
The existing technology is mainly to perform subcutaneous modeling after fresh malignant nerve sheath tumor tissues are subjected to a chopping treatment or a cell homogenization treatment.
Disclosure of Invention
However, homogenization of fresh malignant schwannoma tissue is considered to destroy the microenvironment of tumor tissue itself, and the consistency of tumor and surrounding stroma with patients cannot be well maintained. In addition, malignant schwannoma is derived from the nerve sheath, and compared with subcutaneous modeling, in-situ modeling can better simulate the tumorigenic environment. The method successfully establishes a tissue block transplantation mode, well maintains the stable relation between the tumor and the peripheral interstitium, successfully establishes subcutaneous modeling and nerve sheath in-situ modeling of malignant nerve sheath tumor, and maintains the consistency of the growth environment of the model tumor and the tumor of a patient.
Therefore, the invention firstly provides a method for constructing a malignant schwannoma xenograft mouse model, which comprises the steps of collecting a malignant schwannoma patient tumor tissue block, carrying out xenograft on the fresh patient tumor tissue block into an immunodeficiency mouse body under aseptic conditions, wherein the xenograft comprises selecting an immunodeficiency mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the collected fresh patient tumor tissue block into an aseptic incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the patient tumor tissue block into the mouse under the skin, suturing an incision, and feeding the mouse to ensure the tumor growth in the body.
The invention also provides a method for constructing a malignant schwannoma xenograft mouse model, which comprises the steps of collecting a malignant schwannoma patient tumor tissue block, transplanting a fresh patient tumor tissue block into an immunodeficiency mouse in a xenograft in situ under aseptic conditions, wherein the xenograft in situ comprises selecting an immunodeficiency mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the collected fresh patient tumor tissue block in a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the patient tumor tissue block into a nerve sheath of the mouse, suturing an incision, and feeding the mouse to ensure that the tumor in the mouse grows.
In a specific embodiment, the subcutaneous implantation comprises making a skin incision in the mouse axilla, implanting tumor tissue subcutaneously, and suturing the surgical incision; the orthotopic transplantation comprises making a skin incision at the axillary nerve sheath or sciatic nerve sheath of the mouse, separating the nerve sheath and opening, implanting a tumor tissue block of the patient into the nerve sheath, and then suturing the surgical incision.
In a specific embodiment, the tumor tissue mass of the malignant schwannoma patient implanted in the mouse has a unilateral length of less than or equal to 3mm, preferably less than or equal to 1mm.
In a specific embodiment, the weight of the mouse in subcutaneous transplantation or orthotopic transplantation is 8-20 g, preferably 12-15g, and the sterile incubation solution further comprises an antibiotic, preferably the sterile incubation solution comprises an antibiotic and a DMEM medium or an RPMI-1640 medium, and more preferably the antibiotic comprises penicillin and streptomycin.
In a specific embodiment, the immunodeficient mouse is a NOD/SCID mouse.
In a specific embodiment, the method further comprises observing the size of the malignant schwannoma in the mouse using a small animal living body imager, preferably also using the small animal living body imager to observe whether the tumor has metastasized to the internal organs of the mouse.
In a specific embodiment, the method further comprises feeding the mouse such that the mouse is dissected after the in vivo tumor growth, detaching the tumor body to obtain a P0 generation tumor body, xenotransplanting the P0 generation tumor body subcutaneously or xenotransplanting it in situ into the immunodeficient mouse to obtain a P1 generation tumor body, optionally xenotransplanting the P1 generation tumor body subcutaneously or xenotransplanting it in situ into the immunodeficient mouse to obtain a P2 generation tumor body, and so on for passage; preferably the method further comprises the step of programming the fraction of the P0 generation tumour volume and storing it in liquid nitrogen, and optionally the steps of programming the fractions of the P1 and P2 generation tumour volume and storing them in liquid nitrogen.
The invention also provides a method for constructing a malignant schwannoma xenograft mouse model, which comprises the steps of heterogeneously and subcutaneously transplanting or heterogeneously and orthotopically transplanting P0 generation malignant schwannoma tumor tissue, P1 generation malignant schwannoma tumor tissue or P2 generation malignant schwannoma tumor tissue in a fresh mouse model into an immunodeficient mouse under the aseptic condition, wherein the heterogeneously and subcutaneously transplanting or heterogeneously and orthotopically transplanting comprises selecting an immunodeficient mouse with the age of 2-6 weeks, the sex of the mouse is consistent with that of a patient, soaking the malignant schwanoma tumor tissue block into a sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, transplanting the malignant schwanoma tumor tissue block into the subcutaneous or nerve sheath of the mouse, suturing an incision, and raising the mouse to enable the tumor in vivo to grow.
The invention also provides application of the mouse model obtained by the method in screening drugs for preventing or treating malignant schwannoma.
The invention also provides a method for constructing a malignant schwannoma xenograft mouse model, which is characterized by comprising the steps of recovering the malignant schwanoma patient tumor tissue block stored in a liquid nitrogen environment, then heterodermically transplanting the recovered malignant schwanoma patient tumor tissue block into an immunodeficient mouse under an aseptic condition, wherein the heterodermic transplantation comprises the steps of selecting an immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the recovered malignant schwanoma tissue block into sterile incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the tumor tissue block of the patient into the subcutaneous tissue of the mouse, suturing an incision, and feeding the mouse to ensure the tumor in the mouse to grow.
The invention also provides a method for constructing the malignant schwannoma xenograft mouse model, which is characterized by comprising the steps of resuscitating the malignant schwanoma patient tumor tissue block stored in a liquid nitrogen environment, then transplanting the resuscitated patient tumor tissue block into an immunodeficient mouse in a heterogenic and in-situ manner under an aseptic condition, wherein the heterogenic and in-situ transplantation comprises the steps of selecting the immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the resuscitated patient tumor tissue block into a sterile incubation liquid containing fetal calf serum, fixing the mouse after anaesthetizing, implanting the patient tumor tissue block into a nerve sheath of the mouse, suturing an incision, and feeding the mouse to ensure the tumor in vivo to grow.
In a specific embodiment, the method further comprises the processes of programmed cooling and liquid nitrogen preservation and rapid warming resuscitation of the collected tumor tissue mass of the malignant schwannomas patient.
In a specific embodiment, the tumor tissue block of the malignant schwannoma patient is preserved in DMEM containing DMSO, FBS and NEAA, more than three sections of the tumor tissue blocks are cooled to the temperature of liquid nitrogen in a program and preserved in the temperature of the liquid nitrogen, and the rapid heating resuscitation refers to resuscitation of the frozen tissue in a constant-temperature water bath at 36-38 ℃.
In a specific embodiment, the subcutaneous implantation comprises making a skin incision in the mouse axilla, implanting tumor tissue subcutaneously, and suturing the surgical incision; the orthotopic transplantation comprises making a skin incision at the axillary nerve sheath or sciatic nerve sheath of the mouse, separating the nerve sheath and opening, implanting a tumor tissue block of the patient into the nerve sheath, and then suturing the surgical incision.
In a specific embodiment, the weight of the mouse is 8-20 g, preferably 12-15g, when the mouse is transplanted subcutaneously or in situ, the sterile incubation solution further comprises antibiotics, preferably the sterile incubation solution comprises antibiotics and DMEM medium or RPMI-1640 medium, and more preferably the antibiotics comprise penicillin and streptomycin.
In a specific embodiment, the immunodeficient mouse is a NOD/SCID mouse, and the tumor tissue mass of the malignant schwannoma patient implanted in the mouse has a unilateral length of less than or equal to 3mm, preferably less than or equal to 1mm.
In a specific embodiment, the method further comprises observing the size of the malignant schwannoma in the mouse by using a small animal living body imager, and preferably observing whether the tumor is transferred to the internal organs of the mouse by using the small animal living body imager.
In a specific embodiment, the method further comprises raising the mouse such that the mouse is dissected after the growth of the tumor in vivo, and the tumor is detached to obtain P0 generation tumor, and the P0 generation tumor is xenografted subcutaneously or orthotopically into immunodeficient mice to obtain P1 generation tumor, and optionally the P1 generation tumor is xenografted subcutaneously or orthotopically into immunodeficient mice to obtain P2 generation tumor, and so on; preferably, the method further comprises the step of programming and storing the portion of the P0 generation tumor mass in liquid nitrogen, and optionally the portions of the P1 and P2 generation tumor masses in liquid nitrogen.
The invention also provides a method for constructing a malignant schwannoma xenograft mouse model, which comprises the following steps of carrying out xenogeneic subcutaneous transplantation or xenogeneic in-situ transplantation on P0 generation malignant schwannoma tumor tissues, P1 generation malignant schwannoma tumor tissues or P2 generation malignant schwannoma tumor tissues in a mouse model which is frozen and stored in a liquid nitrogen environment and revived under an aseptic condition, wherein the xenogeneic subcutaneous transplantation or the xenogeneic in-situ transplantation comprises selecting immunodeficient mice with the age of 2-6 weeks, keeping the sex of the mice consistent with that of patients, soaking malignant schwanoma tumor tissue blocks in sterile incubation liquid containing fetal calf serum, anaesthetizing the mice for fixation, implanting the malignant schwanoma tumor tissue blocks into the subcutaneous tissues or the nerve sheaths of the mice, suturing incisions, and breeding the mice to enable the tumors to grow in the mice.
The feeding of the mice is specifically feeding the mice in an SPF-level environment.
The invention has at least the following beneficial effects:
the invention utilizes the PDX model with better homogeneity with the primary tumor of the patient, can deeply discuss the pathogenesis of malignant schwannoma, can be well repeated in the body of the patient based on the result developed by PDX, can greatly expand limited malignant schwannoma samples in the body of the clinical patient, and uses a xenograft mouse to replace the clinical patient to carry out different combined drug screening, thereby selecting the most suitable drug, greatly improving the effectiveness and reducing the side effect of the drug.
The PDX model establishment and application of the invention can hopefully improve the effectiveness of clinical malignant nerve sheath tumor treatment and the prognosis of patients, prevent the patients from disability and even death to bring great medical social burden, reduce treatment cost and government financial expenditure, have great practical significance in these aspects, and have great economic benefits.
In addition, the research on the malignant nerve sheath tumor is lagged, the animal model which can be used for research per se is few, and the difference with the clinic is large, the in-situ implanted PDX model can better reflect the actual condition of a patient, and provides a better animal model for the basic research and drug screening of the malignant nerve sheath tumor. That is, in situ modeling is more realistic than subcutaneous modeling, the screened drug regimen can be better used on the patient, and in situ modeling is theoretically more likely to have tumor metastasis than subcutaneous modeling.
Drawings
Fig. 1 is a photograph of a pathology report for a patient.
FIG. 2 is a photograph of a tumor tissue mass in an immunodeficient mouse with PDX modeling.
FIG. 3 is a photograph of the success of primary cell culture on fresh patient tumor tissue blocks.
Detailed Description
Approved by the ethical committee of hospitals, patients sign informed consent before sample collection, collected tissues are subjected to histopathological examination, tumor tissue blocks of malignant schwannoma patients are confirmed through pathology, stored in sterile physiological saline and placed on ice. Dividing the tissue block into 3 parts, and directly treating (see below for details) the tissue block under aseptic conditions in part 1 to perform xenografting into immunodeficient mice; part 2 was placed in DMEM (a medium containing various amino acids and glucose) supplemented with 10% DMSO (dimethyl sulfoxide), 90% FBS (fetal bovine serum) and 1% NEAA (non-essential amino acids), gradient-frozen to-80 degrees, and placed in liquid nitrogen for storage; and the part 3 is put into liquid nitrogen for quick freezing and then is put into the liquid nitrogen for preservation. Part 1 is used for modeling, part two is used for resuscitation and inoculation, and part three is used for gene detection of submission.
Examples
Subcutaneous inoculation: selecting 10 NOD/SCID mice with the age of 4 weeks, namely the NOD/SCID mice with non-obese diabetic/severe combined immunityDefective mice, with sex consistent with patient sex (to avoid some unknown hormonal disturbances), weighing approximately 12-15g, fresh tumor tissue (or malignant schwannomas patient tumor tissue frozen and resuscitated with liquid nitrogen) were soaked in sterile FBS + penicillin and streptomycin incubations (final penicillin concentration 100U/ml, final streptomycin concentration 100U/ml) and cut into 1 x 1mm pieces with sterile scissors. Mice were anesthetized and fixed, skin incisions were made near the right underarm, tumor tissue blocks were implanted subcutaneously (P0), and surgical incisions were sutured. Mice were observed daily for tumor growth. Tumors grow to diameters exceeding 5 mm. Tumor volume was calculated by measuring tumor volume 2 times per week with vernier caliper and recording tumor length (a) and width (b) (V = a b) 2 /2). Meanwhile, a small animal living body imager is adopted to observe the viscera transfer condition, and the volume V of the tumor body is increased to 1500mm 3 When the time is about 42 days later, the animal is dissected, the tumor body is stripped and weighed, part of tumor mass is inoculated and passed, and the rest is placed into liquid nitrogen for preservation.
In-situ nerve sheath implantation: selecting 10 NOD/SCID mice with 4 weeks of age, keeping the sex consistent with the sex of a patient, weighing about 12-15g, soaking fresh tumor tissue (or tumor tissue preserved by liquid nitrogen and recovered) in sterile incubation solution of pure FBS + penicillin and streptomycin (the final concentration of penicillin is 100U/ml and the final concentration of streptomycin is 100U/ml), cutting into 1 x 1mm small blocks by using sterile scissors, fixing the mice after anesthesia, making skin incisions at axillary nerve sheaths or sciatic nerve sheaths, separating nerve sheaths and opening the nerves, implanting the tumor tissue blocks of the patient into sheaths (P0), ensuring that the tension is not too large, and suturing surgical incisions. The postoperative mice were placed on a 37 ℃ hotbed until resuscitated, and penicillin was routinely administered intraperitoneally to prevent postoperative infection. Mice were observed daily for tumor growth. Swelling skin of the part to be implanted, observing the size of malignant nerve sheath tumor and visceral metastasis condition (whether the tumor is transferred to the viscera of a mouse) by using a small animal living body imager every 3 weeks, and increasing the volume of the body to be implanted to 1500mm 3 When the animal is in use or after 42 days, dissecting the animal, stripping tumor body, weighing, inoculating partial tumor mass for passage, and preserving the rest in liquid nitrogen. The process of preserving tissue mass or whole tumor body in liquid nitrogen includes programmed cooling, specifically 4 deg.C, -20 deg.C, -80 deg.C (special freezing refrigerator) and liquid nitrogenThese several temperature gradients.
Passage of tumor tissue of malignant schwannoma xenografted mice: inoculating well-growing P0 generation xenograft to tumor-forming mice, and increasing the tumor volume to 1500mm 3 At the moment or 42 days, the mice are sacrificed, the tumors are completely stripped, the mice are inoculated on the axillary skin, the axillary nerve sheath, the sciatic nerve sheath and the like (P1) of 10 NOD/SCID mice as before, the growth condition of the tumors of the mice is observed every day, the length and the width of the tumors are measured by a vernier caliper as before, the volume of the tumors is calculated, the visceral metastasis condition is observed by a living body imaging instrument of the mice, the animals are dissected after 42 days, the tumors are stripped and weighed, part of the tumors are passaged by analogy (P2, P3 and the like), and the rest of the tumors are cooled in a gradient manner and are placed in liquid nitrogen for storage.
Resuscitating and inoculating primary and passage tumor tissues of malignant nerve sheath tumor: p0, P1 and P2 frozen in liquid nitrogen are taken to replace malignant schwannoma tumor tissues, quickly reheated to normal temperature, and respectively inoculated in the armpit and the nerve sheath of 10 NOD/SCID mice with the same sex.
Because malignant nerve sheath tumor tissue of a patient is a precious research material, the tumor tissue of tumor-bearing mice needs to be passaged so as to generate more tumor-bearing mice for experiments. The reason why the tumor tissue is frozen and thawed is that too large tumor masses cannot be used in each modeling experiment, and the tumor masses left for seed storage, namely frozen and thawed, can be used in the next experiment. Tumor tissues are passaged by P0, P1, P2 and the like, and theoretically, the tumor tissues are not changed, so that the experimental effects of each generation can be considered to be the same. And when in-situ modeling or subcutaneous modeling is carried out, as long as the generation of the tumor P0 is successful, subsequent passages and resuscitation of P1, P2 and the like are very simple and easy and can be completely successful.
1 patient in this subject group has been collected, and a PDX model was established by in situ and subcutaneous neoplasia to obtain corresponding cases of neoplasia. The patient has recurrence after malignant schwannoma of poplar, male, 28 years old and right thigh. "left popliteal vascular fibromyxoma resection" in 2010, "L3/4 right paraspinal schwannoma resection" in 11 months in 2015, "right femoral schwanoma resection" in 2 months in 2017, internal fixed soft tissue repair surgery in resection reconstruction of right femoral malignant schwanoma section in 12 months in 2017, and "right hip amputation" in 4 months in 2018.
Fig. 1 is a photograph of a pathology report for a patient. As can be seen in FIG. 1, the pathology of this patient was diagnosed with malignant schwannoma.
FIG. 2 is a photograph of a tumor tissue mass in an immunodeficient mouse with PDX modeling. Wherein the immunodeficient mouse is a full body white hair NOD/Scid mouse. Specifically, subcutaneous tumor formation in the axilla of the patient was successful, and in-situ tumor formation in the axilla nerve sheath was successful.
FIG. 3 is a photograph of the success of primary cell culture on fresh patient tumor tissue blocks. The primary cells can be used for in vitro experiments of malignant schwannomas, but animal modeling is not performed by the cells in the invention, and in-situ modeling and subcutaneous modeling in an immunodeficiency mouse are performed by tissue blocks.
The PDX has far-reaching in-situ modeling significance, the tumor obtained by in-situ modeling has better homogeneity with the primary tumor of a patient, the pathogenesis of malignant nerve sheath tumor can be deeply discussed after successful modeling, the research and development results can be better repeated in the body of the patient based on the PDX, meanwhile, a large number of limited malignant nerve sheath tumor samples in the body of a clinical patient can be amplified, and a xenografted mouse is used for replacing the clinical patient to screen different combined drugs, so that the most suitable drug is selected, the effectiveness is greatly improved, and the side effect of the drug is reduced. Therefore, the in-situ modeling and application can hopefully improve the effectiveness of clinical malignant nerve sheath tumor treatment and the prognosis of patients, prevent the patients from being disabled and even dying to bring great medical and social burden, reduce the treatment cost and the government financial expenditure, have great practical significance in the aspects and have great economic benefit.
The foregoing is a further detailed description of the present invention in connection with specific preferred embodiments thereof, and it is not intended to limit the invention to the specific embodiments thereof. For those skilled in the art to which the invention pertains, several simple deductions and substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (7)
1. A method for constructing a malignant schwannoma xenograft mouse model is characterized by comprising the steps of recovering a malignant schwannoma patient tumor tissue block stored in a liquid nitrogen environment, transplanting the recovered malignant schwannoma tissue block into an immunodeficient mouse NOD/SCID mouse in a heterogenous and in-situ manner under an aseptic condition, wherein the heterogenous and in-situ transplantation comprises the steps of selecting an immunodeficient mouse with the age of 2-6 weeks, keeping the sex of the mouse consistent with the sex of the patient, soaking the recovered malignant schwannoma tissue block of the patient in an aseptic incubation liquid containing fetal calf serum, fixing the mouse after anesthesia, implanting the tumor tissue block of the patient into an axillary nerve sheath of the mouse, suturing an incision, and feeding the mouse to enable the tumor in the mouse to grow; the unilateral length of the tumor tissue block of the malignant schwannoma patient implanted in the mouse is less than or equal to 1mm; the heterogeneous in-situ transplantation comprises the steps of firstly making a skin incision at an axillary nerve sheath of a mouse, separating the axillary nerve sheath and opening the axillary nerve sheath, implanting a tumor tissue block of a patient into the axillary nerve sheath, then suturing an operation incision, feeding the mouse to enable the mouse to dissect the mouse after the tumor in the mouse grows, and stripping the tumor to obtain a P0 generation tumor body.
2. The method of claim 1, further comprising programmed cooling and liquid nitrogen preservation of the collected malignant schwannoma patient tumor tissue mass and rapid warming resuscitation.
3. The method according to claim 2, wherein the tumor tissue mass of the patient with malignant schwannomas is preserved in DMEM containing DMSO, FBS and NEAA, the temperature is reduced to liquid nitrogen temperature by three or more steps, and the tumor tissue mass is preserved in the liquid nitrogen temperature, and the rapid heating resuscitation refers to resuscitation by placing frozen tissues in a constant temperature water bath at 36-38 ℃.
4. The method as claimed in claim 1, wherein the weight of the mouse is 8 to 20g when the mouse is transplanted in a xenogenic orthotopic manner, the sterile incubation solution contains antibiotics and DMEM medium or RPMI-1640 medium, and the antibiotics comprise penicillin and streptomycin.
5. The method of claim 1, wherein the tumor volume in the mouse is increased to 1500mm 3 Mice were dissected at the time or 42 days later, tumor bodies were dissected and weighed, and a portion of the tumor mass was inoculated for passage.
6. The method of claim 1, further comprising observing the size of the malignant schwannoma in the mouse using a small animal in vivo imager.
7. The method of claim 1, further comprising xenogeneic transplantation of the tumor P0 generation into immunodeficient mice to obtain tumor P1 generation, xenogeneic transplantation of the tumor P1 generation into immunodeficient mice to obtain tumor P2 generation, and so on.
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