A method for optimizing the cryopreservation of human blood disease samples and a method for establishing a PDX animal model using human blood samples

Abstract

The application provides a freezing optimization method of a human blood disease sample and a method for establishing a PDX animal model by using a human blood sample, and relates to the technical field of PDX model construction. The application specifically relates to a method for optimizing and improving long-term freezing of a human blood disease sample, and a method for optimizing and improving establishment of a blood disease PDX model, so that the success rate of establishing a PDX animal model by using a clinical blood disease sample after freezing and recovery is greatly improved. The method can be used for long-term storage of blood disease patient samples, and storage of rare and valuable samples such as drug-resistant and relapsed samples; meanwhile, the method can solve existing clinical problems such as long-distance patient sample transportation difficulties, and has short modeling time and high modeling rate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of PDX model construction, and particularly relates to a freezing optimization method of human blood disease samples and a method for establishing a PDX animal model by using human blood samples. BACKGROUND

[0002] Leukemia is a malignant tumor disease of the blood system, and its morbidity and mortality are high and unchangeable. Traditional chemotherapy treatment methods and some targeted drugs can improve the cure rate of some leukemias, but relapse and refractoriness are still the main problems of leukemia.

[0003] Patient-derived xenograft (PDX) animal model refers to transplanting tumor tissue from a patient into an immunodeficient animal to construct a humanized animal model. The model can well preserve the heterogeneity of the tumor, restore the tumor microenvironment in the patient's body, simulate the development and metastasis of the tumor, and thus truly reflect the response of the tumor to drugs. PDX is considered an important tool for selecting clinical treatment options, drug screening, and precision treatment.

[0004] The first PDX model of acute myeloid leukemia was established 40 years ago. In recent years, leukemia PDX models have been continuously developed and improved. By using immunodeficient mice with higher immunity, such as NSG mice, and improved immunodeficient mice expressing human cytokines, the success rate of establishing PDX models has been improved.

[0005] There are still many challenges in the establishment of leukemia PDX models, such as low success rate of PDX establishment and long modeling time. For example, when establishing a PDX mouse model, the immunodeficient mice are usually pretreated with 2-2.5Gy of X-rays 2-6h in advance, and the establishment of the PDX model can be completed 5-23 weeks after cell injection, but the success rate is only about 10% (see, D.J. Pearce et al, Blood 103:1166-73 (2006)).

[0006] Since the leukemia patient samples are from clinical hospitals, there can be reasons such as too late sample acquisition time or too far distance between the hospital and the animal platform, so that the PDX model cannot be established in time; it is also very important to freeze the scarce and precious leukemia patient samples. Similarly, there is also a strong need for freezing to achieve long-distance transportation of patient samples across provinces. However, the current preservation of human leukemia samples usually uses fetal bovine serum containing dimethyl sulfoxide for preservation, and the cell survival rate after resuscitation is only about 36%, and the vitality is low (see, for example, P. Bourgoin et al, Future Microbiology 16:955-966 (2021)). How to ensure the high resuscitation survival rate of long-term frozen human leukemia samples is an important scientific problem at present. SUMMARY

[0007] The purpose of the present application is to provide an optimized method for improving the freezing of human hematological disease samples and an optimized method for improving the establishment of PDX animal models. Using the present method, leukemia patient samples can be stored for a long time, and the leukemia cells after resuscitation can construct PDX animal models, with the characteristics of short modeling time and high modeling rate.

[0008] The present application provides an optimized method for freezing human hematological disease samples, comprising the following steps: mixing the human hematological disease sample with a cell freezing solution without dimethyl sulfoxide, placing it in a-80℃ environment for 12-16h, and then transferring it to liquid nitrogen for long-term storage.

[0009] Preferably, the volume ratio of the human hematological disease sample to the cell freezing solution without dimethyl sulfoxide is (2:1) to (1:4).

[0010] Preferably, the cell freezing solution without dimethyl sulfoxide includes a cell freezing solution with catalog number CPL-A1 purchased from Funakoshi, a cell freezing solution with catalog number 12648010 from Thermo Fisher, or a cell freezing solution with catalog number YC0100 from Shanghai Qipun Biotechnology Co., Ltd.

[0011] Preferably, the human hematological disease sample includes a whole blood sample of leukemia and leukemia-related hematological diseases without mononuclear cell separation.

[0012] The application further provides an optimized method for establishing a PDX animal model by using a human blood disease sample, comprising the following steps: resuscitating a cryopreserved sample stored by using the optimized method for cryopreserving a human blood disease sample, mixing the resuscitated sample with cells in the bone marrow of an animal, and then injecting the mixture into an immunodeficient animal that has been pretreated by inhibiting the host hematopoietic system to obtain a human blood disease PDX animal model; or separating white blood cells from a fresh human blood disease sample, mixing the white blood cells with cells in the bone marrow of an animal, and then injecting the mixture into an immunodeficient animal that has been pretreated by inhibiting the host hematopoietic system to obtain a human blood disease PDX animal model.

[0013] Preferably, the mixing ratio of the resuscitated cells and the cells in the bone marrow of the animal is (1×10 4 ~ 1×10 7 ): (1×10 4 ~ 1×10 6 ).

[0014] Preferably, the pretreatment method for inhibiting the host hematopoietic system comprises busulfan pretreatment.

[0015] Preferably, the busulfan pretreatment comprises injecting busulfan, and the injection amount is 10 mg / kg to 100 mg / kg.

[0016] Preferably, the immunodeficient animal comprises an athymic nude mouse, an NCG, an NSG, an SCD mouse, a CBA / N mouse, an NOD / SCID mouse, and a Rag1 and IL-2Rγ gene double knockout mutant rat of an SDRG rat.

[0017] The application further provides an application of the optimized method for cryopreserving a human blood disease sample or the optimized method for establishing a PDX animal model by using a human blood disease sample in improving the success rate of modeling a PDX animal model of a human blood disease sample.

[0018] Beneficial effects: the present application provides an optimized method for cryopreservation of human blood disease samples, which can realize long-term preservation of original sample cells of leukemia and other blood-related disease patients by using cell cryopreservation solution without dimethyl sulfoxide, and can solve the problem of long-distance transportation of patient samples. The present application also improves the method for establishing a PDX animal model of human blood disease samples, and the cryopreserved samples can be used to establish a PDX model using the method. The method can not only solve the problem of long-term preservation of leukemia and other blood-related disease patient samples, but also can be used for long-distance transportation across provinces and internationally; it is proved by the examples that a small amount of cryopreserved and recovered leukemia and other blood-related disease patient sample cells can quickly establish a PDX animal model, and the modeling rate is more than 90%, and the modeling time period is generally about 8-12 weeks, and the leukemia and other blood-related disease PDX animal model can be successfully established. The PDX animal model provided by the present application can track the pathological characteristics and the change characteristics of tumor stem cells and the metastasis of the same patient at different time periods. It provides a technical basis for leukemia and other blood-related disease drug research and development and for formulating treatment plans for patients. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1 The results of establishing a mouse PDX model using a fresh leukemia sample of a patient are shown in the figure, wherein a is a flow chart of establishing a PDX model; b is a mouse peripheral blood detection figure, 1x10 6 After injecting human leukemia cells, the mouse tail vein blood was taken at 4 weeks, 8 weeks and 12 weeks, and after lysing red blood cells, ACEANovoCyte flow cytometry was used for detection; c is a PDX model flow analysis result figure, after 12 weeks, the mouse was euthanized, the cells were flushed out from the PDX mouse bone marrow, the mouse spleen was ground, and 1x10 6cells were detected using ACEA NovoCyte flow cytometer; mCD45, murine leukocytes; d is the comparison results of Giemsa staining of immunodeficient mice, patient samples and PDX models, respectively, Giemsa staining of peripheral blood of NCG mice, PDX mouse models and fresh patient samples, and the arrows respectively point to the patient sample leukemia cells, the peripheral blood leukocytes of immunodeficient mice, and the peripheral blood leukemia cells of PDX mouse models; e is the comparison results of hematoxylin-eosin staining of immunodeficient mice and PDX model livers, and the livers of NCG mice and 12-week PDX mouse models were fixed with formalin and then stained with hematoxylin-eosin, and the arrow points to the leukemia cells infiltrated into the liver;

[0021] Figure 2 Figure 4 is a flow chart of the optimized patient leukemia sample freezing method;

[0022] Figure 3 Figure 5 is a comparison results chart of white blood cell survival rate of frozen PDX spleen cells and fresh PDX spleen cells, and after the cells frozen for 12 months were recovered, 1x10 6 cells were detected using ACEA NovoCyte flow cytometer, and compared with fresh spleen cells from the same PDX mouse model, and 7-AAD was used to mark cell death and life;

[0023] Figure 4 Figure 6 is a results chart of establishing a passaging transplantation mouse model using PDX spleen cell samples frozen for one year, wherein a is a mouse peripheral blood detection chart, and after injection of 1x10 6 PDX spleen cells, blood was taken from the tail vein of the mouse at 4 weeks, 8 weeks and 12 weeks, and after lysis of red blood cells, the cells were detected using ACEA NovoCyte flow cytometer; b is a PDX model flow analysis results chart, and after 12 weeks, the mouse was euthanized, the cells were flushed out from the bone marrow of the PDX mouse, and the mouse spleen was ground, and 1x10 6 cells were detected using ACEA NovoCyte flow cytometer; c) is a comparison results chart of Giemsa staining of immunodeficient mice, patient samples and PDX models, respectively, Giemsa staining of peripheral blood of NCG mice, PDX mouse models and fresh patient samples, and the arrows respectively point to the patient sample leukemia cells, the peripheral blood leukocytes of immunodeficient mice, and the peripheral blood leukemia cells of PDX mouse models;

[0024] Figure 5 Figure 7 is a results chart of establishing a mouse PDX model using a patient leukemia sample frozen for one year, wherein a is a mouse peripheral blood detection chart, and 1x10 6After human leukemia cells, 4 weeks, 8 weeks, 12 weeks, mice tail vein blood, after lysis of red blood cells, using ACEA NovoCyte flow cytometer for detection; b is the PDX model flow analysis result figure; 12 weeks after euthanasia, the cells were flushed out from the PDX mouse bone marrow, the mouse spleen was ground, and 1x10 6 cells were detected using ACEA NovoCyte flow cytometer; c is the comparison result figure of Giemsa staining of immunodeficient mice, patient samples and PDX models; NCG mice, PDX mouse models and fresh patient samples were taken for Giemsa staining; the arrows respectively point to patient sample leukemia cells, immunodeficient mouse peripheral blood leukocytes, and PDX mouse model peripheral blood leukemia cells;

[0025] Figure 6 For comparison of PDX animal model results of directly frozen and thawed cells and leukocyte separated and frozen and thawed cells, a is a flow chart of using different freezing processes; b is a difference result figure of whole blood sample freezing method and leukocyte separation freezing method for establishing PDX model; 12 weeks later, the peripheral blood of mice was taken, and 1x10 6 cells were detected using ACEA NovoCyte flow cytometer;

[0026] Figure 7 For comparison of different mouse pretreatment methods before transplantation to establish PDX model flow chart. DETAILED DESCRIPTION

[0027] The present application provides an optimized method for freezing human hematological disease samples, the flow is as shown in Figure 2 , comprising the following steps: mixing the human hematological disease sample with a cell freezing solution containing no dimethyl sulfoxide, placing it in a-80℃ environment for 12-16h, and then transferring it to liquid nitrogen for long-term storage.

[0028] The human blood disease sample of the present application preferably includes samples of leukemia and leukemia-related blood diseases, more preferably samples of myeloid leukemia, lymphoid leukemia, and myelodysplastic syndrome, etc., and whole blood disease samples, whole spleen samples, or whole bone marrow samples derived from humans, and whole blood disease samples, whole spleen samples, or whole bone marrow samples taken from PDX animal models. The leukemia sample is used as an example in the examples, but it cannot be solely identified as the entire protection scope of the present application. The cell freezing solution without dimethyl sulfoxide of the present application is preferably a common cell freezing solution in the art, which only needs to meet the requirement of not containing dimethyl sulfoxide, and preferably includes the cell freezing solution with product number CPL-A1 purchased from Funakoshi, the cell freezing solution with product number 12648010 from Thermo Fisher, or the cell freezing solution with product number YC0100 from Shanghai Qipun Biotechnology Co., Ltd. To ensure the long-term preservation of patient samples and the cell viability after long-term preservation and recovery, the patient sample is mixed with the freezing solution immediately after being taken out of the body and placed in a freezing box, and then stored in a-80℃ refrigerator for freezing; if it cannot be frozen immediately, the patient sample should be stored in an EDTA-containing anticoagulant tube at 4℃, and should not be overnight. If coagulation, blood clots, etc. occur, it will affect the preservation of the patient sample and the cell viability after freezing and recovery. Using the optimized freezing method of the present application, the ex vivo sample does not need to be purified, and red blood cells do not need to be removed.

[0029] The human blood disease sample and the cell freezing solution without dimethyl sulfoxide are mixed in the present application, and the volume ratio of the human blood disease sample to the cell freezing solution without dimethyl sulfoxide is preferably (2:1) to (1:4). The mixture is preferably placed in a freezing box, and then stored in liquid nitrogen after being placed in a-80℃ refrigerator overnight (12-16 hours). Using this freezing method, leukemia and other blood-related disease patient sample cells can be preserved for a long time. Using the freezing method of the present application, the human blood disease sample can be preserved for more than 1 year.

[0030] The present application also provides an optimized method for establishing a PDX animal model using a human blood disease sample recovered after freezing, which comprises the following steps: recovering the frozen sample preserved for a long time by the above-mentioned method for freezing the human blood disease sample, mixing with intramedullary cells, and injecting into immunodeficient mice to obtain a human blood disease PDX animal model.

[0031] Or, after separating white blood cells from a fresh human blood disease sample, mixing with animal intramedullary cells, and injecting into an immunodeficient animal pretreated to suppress the host hematopoietic system, a human blood disease PDX animal model is obtained.

[0032] The present application firstly recovers the cryopreserved sample, preferably including melting the long-term preserved cryopreserved sample in a 37℃ environment, cleaning with cell culture medium after melting, resuspending with serum-containing cell culture medium, and culturing the resuspended cells. The melting is preferably in a 37℃ water bath, and the cryopreserved liquid is cleaned with cell culture medium and resuspended with serum-containing cell culture medium, and the proportion of viable cells after cryopreservation and recovery is above 95%. The cell culture medium is preferably DMEM medium.

[0033] The mixing ratio of the recovered cells to the bone marrow cells is preferably (1×10 4 ~ 1×10 7 ): (1×10 4 ~ 1×10 6 ), and the bone marrow cells of the animal are preferably derived from the bone marrow cells of immunodeficient mice. In the embodiments of the present application, 100 μL of 1×10 4 ~ 1×10 7 resuspended sample cells are mixed with 100 μL of 1×10 4 ~ 1×10 6 immunodeficient mouse bone marrow cells, and then injected into immunodeficient mice. The immunodeficient mice of the present application preferably include but are not limited to athymic nude mice, NCG, NSG, SCID mice, CBA / N mice, NOD / SCID mice, SDRG rats (Rag1 and IL-2Rγ gene double knockout), etc. and preferably also include treatment with busulfan (10 mg / kg ~ 100 mg / kg) before injection. The injection of the present application preferably includes tail vein injection, and the total number of patient cells injected is 1×10 4 ~ 1×10 7 cells. The present application monitors every 4 weeks after injection, and according to the differences in the sample itself and the differences in the amount of injected cells, a PDX animal model of leukemia and other blood-related diseases can be successfully established in about 12 weeks.

[0034] The present application also provides an optimized method for cryopreserving the above-mentioned human blood disease sample or an optimized method for establishing a PDX animal model using the above-mentioned human blood disease sample, and the application of the optimized method in improving the success rate of establishing a PDX animal model of a human blood disease sample.

[0035] The present application is based on the optimization of the existing blood sample cryopreservation method, and the PDX modeling of the blood sample cryopreserved and recovered by the technology, and the success rate of modeling is high, which can reach 90%, and the modeling time is short, which can cause PDX leukemia in 8-12 weeks (Table 1).

[0036] Table 1 Summary of PDX model established by cryopreserved cells

[0037]

[0038] To further illustrate the present application, a method for establishing a PDX animal model after cryopreservation and recovery of a human blood disease sample is described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.

[0039] The present application takes the acute myeloid leukemia (AML) M2 subtype as an example to establish a leukemia PDX model, and uses cryopreserved and recovered cells to establish a PDX model.

[0040] In the present application, the reagents used, unless otherwise specified, are common reagents in the art, which can be obtained by commercial means.

[0041] Table 2 HBSS+:(1000mL)

[0042] Solution composition Volume Final concentration 10x HBSS 100ml 1x Fetal bovine serum 20ml 2％ 1 M HEPES pH = 7.2 10ml 10 mM Antibiotics 10ml 1％ H2O 900ml

[0043] Table 3 10x ACK lysis buffer

[0044]

[0045]

[0046] Example 1, using fresh human leukemia samples to establish a PDX mouse

[0047] In order to optimize the construction technology of leukemia PDX model, we first used fresh human leukemia samples. And in order to improve the success rate of fresh patient sample transplantation, we used the chemotherapy drug busulfan to pretreat part of the hematopoietic stem cells of the recipient mice, instead of using the commonly used X-ray irradiation or γ-ray irradiation. The commonly used X-ray irradiation or γ-ray irradiation causes greater damage to the mouse body, which is not conducive to the support function of the recipient mice.

[0048] 1.1. Pretreatment of clinical human leukemia samples before transplantation

[0049] 1) Take the leukemia patient's bone marrow or peripheral blood sample (2-3 mL) from the anticoagulation tube, mix and dilute it with PBS at room temperature (20-22℃) at a ratio of 1:1-1:3;

[0050] 2) Take 2-5 mL of Ficoll solution in a tube, and then gently add the diluted sample to the Ficoll solution;

[0051] 3) Room temperature (20-22°C) by density gradient centrifugation method, 400g, 10min, after centrifugation, the white ring layer was transferred to a new tube, and 2-5 mL of washing solution was added for washing;

[0052] 4) Room temperature centrifugation 450g, 5min, discard the supernatant, use 1 mL HBSS+resuspend.

[0053] 1.2. Leukemia allogeneic transplantation experiment

[0054] 1) The immunodeficient NCG mice were pretreated two days in advance, and 25mg / kg busulfan was injected every day, a total of twice.

[0055] 2) 100μL 1×10 6 Patient leukemia cells were mixed with 100μL 2×10 5 carrier immunodeficient mouse bone marrow cells, and were transplanted into the recipient mice by tail vein injection.

[0056] 1.3. Detection of leukemia occurrence of PDX model

[0057] 1) 20μL of peripheral blood of the recipient mice was taken every four weeks for flow detection;

[0058] 2) 10×ACK lysis buffer was diluted with sterile water to 1×ACK lysis buffer at room temperature, the peripheral blood was mixed with 1×ACK lysis buffer, and was continuously inverted and mixed for 10min for complete lysis;

[0059] 3) Room temperature centrifugation 450g, 10min, discard the supernatant, add 1mL pre-cooled HBSS+resuspend, filter the cells through a cell filter (70μm);

[0060] 4) 4°C centrifugation 450g, 10min, discard the supernatant, add 200μL pre-cooled HBSS+resuspend, add antibody, avoid light, incubate at 4°C for 10min;

[0061] 5) 4°C centrifugation 450g, 5min, discard the supernatant, add 200μL pre-cooled HBSS+clear, 4°C centrifugation 450g, 5min, discard the supernatant, add 200μL pre-cooled HBSS+resuspend, add dead and live staining solution, avoid light, incubate at 4°C for 10min, and use a flow cytometer for detection.

[0062] 1.4. Analysis of experimental results of Example 1

[0063] At 8 weeks after transplantation, 1.9% human CD45+ cells were detected in the peripheral blood of the PDX leukemia mouse model, and 37.8% human cells were detected in the peripheral blood after 12 weeks Figure 1Middle b). The mice were euthanized, and bone marrow, spleen and peripheral blood cells were collected for cytobiological analysis. Flow cytometry analysis found that there were 40.3% human CD45+ leukocytes in the peripheral blood of the PDX mouse model. The human CD45+ cells did not express human B cell lineage antigen CD19 and T cell antigen CD3, but included 79.1% CD33+CD13- myeloid leukemia cells with higher differentiation degree and 4.2% CD33+CD13+ myeloid leukemia cells with lower differentiation degree. Similar situation also existed in the bone marrow and spleen. There were 74.1% and 77.1% human CD45+ leukocytes in the bone marrow and spleen, respectively. These human CD45+ leukocytes did not express B cell lineage antigens CD19 and CD3. Among them, 82.6% and 85.0% were CD33+CD13- myeloid leukemia cells with higher differentiation degree, and 12.6% and 11.5% were CD33+CD13+ myeloid leukemia cells with lower differentiation degree Figure 1 Middle c).

[0064] To further determine that the human cells are leukemia cells, Giemsa staining was performed on leukemia patient samples, peripheral blood cells of NCG mice and peripheral blood cells of PDX mouse models, and morphological comparative analysis was carried out. Observation found that the host mouse leukocytes in the peripheral blood of NCG mice were small. The leukemia cells in the leukemia patient samples were large, and had an irregular, large nucleus. A large number of leukemia cells with this morphological feature were also observed in the peripheral blood of the PDX mouse model Figure 1 Middle d). At the same time, hematoxylin-eosin staining analysis was performed on the livers of NCG mice and PDX mouse models, and we also observed that in the PDX mouse model, leukemia cells infiltrated and invaded the perivascular tissue of the liver Figure 1 Middle e). Based on the above results, we established a high-efficiency PDX leukemia model. This technology ensures the PDX reconstruction efficiency of fresh or cryopreserved clinical leukemia samples.

[0065] Example 2, optimization of leukemia sample cryopreservation technology

[0066] Clinical leukemia samples are more fragile than leukemia cell lines. Therefore, improving the cell survival rate of clinical leukemia samples after cryopreservation and recovery is beneficial to improving the success rate of PDX transplantation. Therefore, we tried to use a cell cryopreservation solution without dimethyl sulfoxide (which has cytotoxicity) to improve the cell survival rate of clinical samples after cryopreservation and recovery.

[0067] 2.1. Cryopreservation and recovery of patient samples before transplantation

[0068] 1) Use Figure 2The shown freezing method, human leukemia PDX mouse spleen samples (containing a large number of human leukemia cells) are mixed with Funakoshi freezing solution without dimethyl sulfoxide at a ratio of 3:10, then placed in a gradient freezing box and placed in a -80°C refrigerator overnight, and then moved into liquid nitrogen for long-term storage.

[0069] 2) Take 10 mL of cell culture medium and heat in a 37°C water bath.

[0070] 3) The human leukemia PDX mouse spleen sample stored for 1 year is taken out from the liquid nitrogen and placed in a 37°C water bath for thawing.

[0071] 4) After thawing, the cells are transferred into cell culture medium (DMEM), centrifuged at 450g for 10 min, the supernatant is discarded, and the cells are resuspended in 200 μL of cell culture medium containing FBS.

[0072] 2.2. Analysis of experimental results of Example 2

[0073] In order to detect the effect of the new freezing technology, we resuscitated the PDX mouse spleen cells that had been frozen for one year and compared them with fresh PDX mouse spleen cells. As shown in Figure 3 the proportion of 7-AAD negative live cells after resuscitation of the cells stored for one year was 92.8%. Compared with the fresh PDX mouse spleen cells with a live cell proportion of 95.4%, the live cell proportions of the two were basically the same. More importantly, after freezing and resuscitation, the proportion of human CD45+ cells was 90.6%, indicating that the new freezing technology did not cause loss of human cells.

[0074] Example 3, establishment of a passaged transplantation mouse using frozen spleen cells of human leukemia PDX

[0075] Based on the optimized PDX transplantation technology and cell freezing technology in the above two examples, we expect to obtain better PDX transplantation effect of clinically frozen leukemia samples. Therefore, we first use the frozen PDX leukemia cells which are easier to obtain to complete the integration test.

[0076] 3.1. Freezing and resuscitation of human leukemia PDX mouse spleen cells before transplantation

[0077] The freezing process is carried out as in Example 2. The human leukemia PDX mouse spleen sample is frozen for 1 year and used for PDX passaging construction.

[0078] 3.2. Perform leukemia allogeneic transplantation experiment and detect human leukemia occurrence

[0079] The transplantation process is carried out as in Example 1.2 and 1.3. In this example, 5 x 10 6Splenocytes from human leukemia PDX mice were transplanted into immunodeficient mice pretreated with busulfan.

[0080] 3.3. Analysis of experimental results of Example 3

[0081] After transplantation, the human leukemia reconstitution in peripheral blood of mice was detected every 4 weeks. Flow cytometry detection found that 4.3% of human CD45+ cells could be detected in the peripheral blood of PDX mouse models after 8 weeks, and 92.5% of human CD45+ cells could be detected in the peripheral blood after 12 weeks Figure 4 , a and b). These human CD45+ cells did not express human lymphoid antigens CD19 and CD3; 80.2% were highly differentiated CD33+CD13- mature myeloid leukemia cells, and 10.0% were less differentiated CD33+CD13+ myeloid leukemia cells Figure 4 , b). Giemsa staining further confirmed leukemia cells similar to the original patient sample, and the leukemia cell morphological characteristics of the PDX mouse model established with the fresh sample were similar Figure 4 , c).

[0082] Example 4, PDX transplantation mice established using human leukemia cryopreserved samples

[0083] Based on the good PDX transplantation technology and cryopreservation technology in Examples 1 and 2, and the good PDX model established after cell cryopreservation and recovery in Example 3, we carried out experiments using an acute myeloid leukemia bone marrow sample cryopreserved for 1 year in this example.

[0084] 4.1. Cryopreservation and recovery of human leukemia before transplantation

[0085] The cryopreservation process was carried out as in Example 2. The acute myeloid leukemia bone marrow sample was cryopreserved for 1 year and used for PDX construction.

[0086] 4.2. Carrying out leukemia allogeneic transplantation experiments and detecting human leukemia occurrence

[0087] The transplantation process was carried out as in 1.2 and 1.3 of Example 1. After injection of 1x10 6 human leukemia cells recovered by tail vein into immunodeficient mice pretreated with busulfan.

[0088] 4.3. Analysis of experimental results of Example 4

[0089] After transplantation, the human leukemia reconstitution in peripheral blood of mice was detected every 4 weeks. Detection found that 1.3% of human CD45+ cells could be detected in the peripheral blood of PDX mouse models after 8 weeks, and 7.1% of human CD45+ cells could be detected in the peripheral blood after 12 weeks Figure 5(a and b). These human CD45+ cells do not express human lymphoid antigens CD19 and CD3; 80.2% are highly differentiated CD33+CD13- mature myeloid leukemia cells, and 10.0% are less differentiated CD33+CD13+ myeloid leukemia cells. Figure 5 (b) Giemsa staining further identified leukemia cells similar to those in the original patient sample, and morphologically similar to those of leukemia cells in a PDX mouse model established from fresh samples. Figure 5 (c). In summary, since human leukemia cells cryopreserved for one year using the cryopreservation method of the present invention undergo a 12-week leukemia development process similar to that of fresh leukemia cells, the PDX reconstitution activity of leukemia cells cryopreserved using the cryopreservation method of the present invention remains essentially unchanged.

[0090] Example 5: Comparison of the effects of different cryopreservation methods on the construction of PDX transplant mice from human leukemia samples.

[0091] This embodiment is for comparative study. Figure 6 The impact of Method 1 (direct cryopreservation of raw tissue samples without leukocyte separation) and Method 2 (separation and subsequent cryopreservation of leukemia cells) on the PDX transplantation rate of leukemia cells.

[0092] 5.1. Pre-transplant cryopreservation and resuscitation of human leukemia cells

[0093] 1) Adopt Figure 2 The cryopreservation method shown involves mixing human hematologic bone marrow aspiration samples with Funakoshi cryopreservation solution at a ratio of 3:10 (Note: data was extracted from Example 4 for comparison), or mixing human hematologic dialysis-enriched leukocyte samples (rich in leukemia cells) with Funakoshi cryopreservation solution at a ratio of 3:10, then placing them in a gradient cryopreservation box and storing them overnight at -80°C, before transferring them to liquid nitrogen for long-term preservation.

[0094] 2) Take 10 mL of cell culture medium and heat it in a water bath at 37°C.

[0095] 3) After removing the human blood disease samples that have been stored for 1 year from liquid nitrogen, place them in a 37°C water bath to thaw.

[0096] 4) After thawing, transfer the cells to cell culture medium (DMEM), centrifuge at 450g for 10min, discard the supernatant, and resuspend the cells in 200μL of cell culture medium containing FBS.

[0097] 5.2. Conduct leukemia allogeneic transplantation experiments and detect the occurrence of human leukemia.

[0098] The transplant procedure was the same as in sections 1.2 and 1.3 of Example 1. 1×10⁻⁶ resuscitation solution was administered via tail vein injection. 6Whole bone marrow derived leukemia cells or leukemia cells enriched by hemodialysis were transplanted into immunodeficient mice pretreated with busulfan.

[0099] 5.3. Analysis of experimental results of Example 5

[0100] Peripheral blood cells were extracted from two different PDX recipient mice at 12 weeks, respectively, and flow cytometry was performed to detect the reconstitution of human leukemia cells. We found that 7.1% human CD45+ cells were detected in the peripheral blood of PDX recipient mice injected with whole bone marrow frozen samples of leukemia patients, but no human cells were detected in the peripheral blood of PDX mice models injected with frozen cells of leukemia patients derived from hemodialysis separated white blood cells Figure 6 b), meaning that the use of frozen cells separated from white blood cells failed to construct a PDX model. Note: PDX mouse data of whole bone marrow frozen human leukemia cells is from Example 4.

[0101] Example 6, Comparison of success rate of PDX transplantation mice of human leukemia cells established by different pretreatment of recipient mice

[0102] This example is to study the effect of Figure 7 (1) busulfan pretreatment and (2) common X-ray pretreatment on the success rate of PDX mouse model construction.

[0103] 6.1. Perform leukemia allogeneic transplantation experiments according to Figure 7

[0104] 1) Immunodeficient NCG mice were pretreated two days in advance, 25 mg / kg busulfan was injected every day, a total of 2 times; or 2Gy X-ray irradiation was used 2 hours in advance.

[0105] 2) Take 100 μL 1x10 5 - 1x10 7 Human leukemia cells were mixed with 100 μL 1x10 5 carrier immunodeficient mouse bone marrow cells, and transplanted into recipient mice by tail vein injection.

[0106] 6.2. Perform leukemia allogeneic transplantation experiments and detect human leukemia

[0107] The transplantation procedure was carried out as in 1.2, 1.3 of Example 1. Different patient peripheral blood or bone marrow sample cells were injected into immunodeficient mice pretreated with busulfan by tail vein injection. 20 μL of peripheral blood of the recipient mice was taken every 4 weeks for flow cytometry detection.

[0108] 6.3. Analysis of experimental results of Example 6

[0109] ​Compared with the traditional and commonly used X-ray pretreatment, the PDX model built by using the immunodeficient mice pretreated with busulfan has a higher success rate (Table 4).

[0110] Table 4 Comparison of PDX animal models built by different pretreatment methods

[0111]

[0112] Although the above embodiments have made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiments without creativity, which all belong to the protection scope of the present application.

Claims

1. An optimized method for establishing a PDX animal model using human blood disease samples, characterized in that, The method comprises the following steps: using a human blood disease sample, freezing; freezing the human blood disease sample mixed with a cell freezing solution without dimethyl sulfoxide; the human blood disease sample is a human whole blood disease sample, without the need for purification of an ex vivo sample, and without the need for removal of red blood cells; the human blood disease sample is selected from bone marrow, spleen and peripheral blood.

2. The optimization method of claim 1, wherein, The volume ratio of the human blood disease sample to the cell freezing solution without dimethyl sulfoxide is (2:1) to (1:4).

3. The optimization method of claim 1 or 2, wherein, The cell freezing solution without dimethyl sulfoxide comprises a cell freezing solution with a product number of CPL-A1 purchased from Funakoshi, a cell freezing solution with a product number of 12648010 from Thermo Fisher, or a cell freezing solution with a product number of YC0100 from Shanghai Qipun Biotechnology Co., Ltd.

4. The optimization method of claim 1, wherein, The human blood disease sample comprises a whole blood sample of leukemia and leukemia-related blood diseases without mononuclear cell separation.

5. The optimization method of claim 1, wherein, The ratio of the number of recovered leukemia cells to the number of cells in the bone marrow of the animal can be (1 x 10 4 ~ 1 x 10 7 ): (1 x 10 4 ~ 1 x 10 6 ).

6. The optimization method of claim 1, wherein, The pretreatment method for inhibiting the host hematopoietic system comprises busulfan pretreatment.

7. The optimization method of claim 6, wherein, The busulfan pretreatment comprises injection of busulfan at an injection amount of 10 mg / kg to 100 mg / kg.

8. The optimization method of claim 1, wherein, The immunodeficient animal comprises an athymic nude mouse, an SCID mouse, an NCG, an NSG, a CBA / N mouse, an NOD / SCID mouse, and an SDRG rat (Rag1 and IL-2Rγ gene double knockout mutant rat).

9. Use of the optimization method according to any one of claims 1 to 8 in improving the success rate of modeling of a human blood disease sample PDX animal model.

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